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  • 1.
    Almlöf, Martin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Andér, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Energetics of codon-anticodon recognition on the small ribosomal subunit2007In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 1, p. 200-209Article in journal (Refereed)
    Abstract [en]

    Recent crystal structures of the small ribosomal subunit have made it possible to examine the detailed energetics of codon recognition on the ribosome by computational methods. The binding of cognate and near-cognate anticodon stem loops to the ribosome decoding center, with mRNA containing the Phe UUU and UUC codons, are analyzed here using explicit solvent molecular dynamics simulations together with the linear interaction energy (LIE) method. The calculated binding free energies are in excellent agreement with experimental binding constants and reproduce the relative effects of mismatches in the first and second codon position versus a mismatch at the wobble position. The simulations further predict that the Leu2 anticodon stem loop is about 10 times more stable than the Ser stem loop in complex with the Phe UUU codon. It is also found that the ribosome significantly enhances the intrinsic stability differences of codon-anticodon complexes in aqueous solution. Structural analysis of the simulations confirms the previously suggested importance of the universally conserved nucleotides A1492, A1493, and G530 in the decoding process.

  • 2.
    Andér, Martin
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Does glutamine methylation affect the intrinsic conformation of the universally conserved GGQ motif in ribosomal release factors?2009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 15, p. 3483-3489Article in journal (Refereed)
    Abstract [en]

    The GGQ motif is the only universally conserved feature of ribosomal class 1 release factors. Mutational experiments and structural studies have suggested that the glutamine residue of the GGQ motif Q 185 in human eRF1 numbering) is critical for catalysis of the termination   reaction on the ribosome. Furthermore, it has been established that Q185 is NE methylated in prokaryotes as well as eukaryotes, and that methylation significantly enhances the catalytic activity. It is, however, not known whether this methylation affects the intrinsic   structure of the free release factor, which could be important for its interaction with the ribosome. In this work, we report molecular dynamics simulations, starting from 25 different NMR structures of human eRF1, in addressing this problem. The results show that there is   no such structural effect on the free release factor caused by the NE methylation of Q185, suggesting that its role is intimately associated with the ribosome environment.

  • 3. Bai, Shi
    et al.
    Jain, Mahendra K.
    Berg, Otto G.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Contiguous binding of decylsulfate on the interface-binding surface of pancreatic phospholipase A22008In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 9, p. 2899-2907Article in journal (Refereed)
    Abstract [en]

    Pig pancreatic IB phospholipase A(2) (PLA2) forms three distinguishable premicellar E-i(#) (i = 1, 2, and 3) complexes at successively higher decylsulfate concentrations. The Hill coefficient for E-1(#) is n(1) = 1.6, and n(2) and n(3) for E-2(#) and E-3(#) are about 8 each. Saturation-transfer difference nuclear magnetic resonance (NMR) and other complementary results with PLA2 show that decylsulfate molecules in E-2(#) and E-3(#) are contiguously and cooperatively clustered on the interface-binding surface or i-face that makes contact with the substrate interface. In these complexes, the saturation-transfer difference NMR signatures of H-1 in decylsulfate are different. The decylsulfate epitope for the successive E, complexes increasingly resembles the micellar complex formed by the binding of PLA2 to preformed micelles. Contiguous cooperative amphiphile binding is predominantly driven by the hydrophobic effect with a modest electrostatic shielding of the sulfate head group in contact with PLA2. The formation of the complexes is also associated with structural change in the enzyme. Calcium affinity of E-2(#) appears to be modestly lower than that of the free enzyme and Ell. Binding of decylsulfate to the i-face does not require the catalytic calcium required for the substrate binding to the active site and for the chemical step. These results show that E-i(#) complexes are useful to structurally characterize the cooperative sequential and contiguous binding of amphiphiles on the i-face. We suggest that the allosteric changes associated with the formation of discrete E-i(#) complexes are surrogates for the catalytic and allosteric states of the interface activated PLA2.

  • 4. Balogh, Larissa M.
    et al.
    Le Trong, Isolde
    Kripps, A
    Tars, Kaspars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Stenkamp, E
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Atkins, William M.
    Structural Analysis of a Glutathione Transferase A1-1 Mutant Tailored for High Catalytic Efficiency with Toxic Alkenals2009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 32, p. 7698-7704Article in journal (Refereed)
    Abstract [en]

    The specificity of human glutathione transferase (GST) A1-1 is drastically altered to favor alkenal substrates in the GIMFhelix mutant designed to mimic first-sphere interactions utilized by GSTA4-4. This redesign serves as a model for improving our understanding of the structural determinants that contribute to the distinct specificities of alpha class GSTs. Herein we report the first crystal structures of GIMFhelix, both in complex with GSH and in apo form at 1.98 and 2.38 angstrom resolution. In contrast to the preorganized hydrophobic binding pocket that accommodates alkenals in GSTA4-4, GSTA1-1 includes a dynamic alpha 9 helix that undergoes a ligand-dependent localization to complete the active site. Comparisons of the GIMFhelix structures with previously reported structures show a striking similarity with the GSTA4-4 active site obtained within an essentially GSTA1-1 scaffold and reveal the 0 helix assumes a similar localized structure regardless of active site occupancy in a manner resembling that of GSTA4-4. However, Are cannot fully account for all the structural elements important in GSTA4-4 within the mutant's active site. The contribution of Phe10 to the Tyr212-Phe10-Phe220 network prevents complete C-terminal Closure and demonstrates that the presence of Phe10 within the context of a GSTA4-4-like active site may ultimately hinder Phe220, a key C-terminal residue, from effectively contributing to the active site. In total, these results illustrate the remaining structural differences presumably reflected in the previously reported catalytic efficiencies of GIMFhelix and GSTA4-4 and emphasize the F10P mutation as being necessary to completely accomplish the transformation to a highly specific GST from the more promiscuous GSTA1-1 enzyme.

  • 5. Balogh, Larissa M.
    et al.
    Le Trong, Isolde
    Kripps, Kimberly A.
    Shireman, Laura M.
    Stenkamp, Ronald E.
    Zhang, Wei
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Atkins, William M.
    Substrate Specificity Combined with Stereopromiscuity in Glutathione Transferase A4-4-Dependent Metabolism of 4-Hydroxynonenal2010In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, no 7, p. 1541-1548Article in journal (Refereed)
    Abstract [en]

    Conjugation to glutathione (GSH) by glutathione transferase A4-4 (GSTA4-4) is a major route of elimination for the lipid peroxidation product 4-hydroxynonenal (HNE), a toxic compound that contributes to numerous diseases. Both enantiomers of HNE are presumed to be toxic, and GSTA4-4 has negligible stereoselectivity toward them, despite its high catalytic chemospecificity for alkenals. In contrast to the highly flexible, and substrate promiscuous, GSTA1-1 isoform that has poor catalytic efficiency with HNE, GSTA4-4 has been postulated to be a rigid template that is preorganized for HNE metabolism. However, the combination of high substrate chemoselectivity and low substrate stereoselectivity is intriguing. The mechanism by which GSTA4-4 achieves this combination is important, because it must metabolize both enantiomers of HNE to efficiently detoxify the biologically formed mixture. The crystal structures of GSTA4-4 and ail engineered variant of GSTA1-1 with high catalytic efficiency toward HNE, cocrystallized with a GSH-HNE conjugate analogue, demonstrate that GSTA4-4 undergoes no enantiospecific induced fit; instead, the active site residue Arg15 is ideally located to interact with the 4-hydroxyl group of either HNE enantiomer. The results reveal an evolutionary strategy for achieving biologically useful stereopromiscuity toward a toxic racemate, concomitant with high catalytic efficiency and substrate specificity toward ail endogenously formed toxin.

  • 6.
    Berg, Otto G.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Yu, Bao-Zhu
    Jain, Mahendra K.
    Thermodynamic Reciprocity of the Inhibitor Binding to the Active Site and the Interface Binding Region of IB Phospholipase A22009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 14, p. 3209-3218Article in journal (Refereed)
    Abstract [en]

    Interfacial activation of pig pancreatic IB phospholipase A(2) (PLA2) is modeled in terms of the three discrete premicellar complexes (E-i(#), i = 1, 2, or 3) consecutively formed by the cooperative binding of a monodisperse amphiphile to the i-face (the interface binding region of the enzyme) without or with an occupied active site. Monodisperse PCU, the sn-2-amide analogue of the zwitterionic substrate, is a competitive inhibitor. PCU cooperatively binds to the i-face to form premicellar complexes ((E) over tilde (i), i = 1 or 2) and also binds to the active site of the premicellar complexes in the presence of calcium. In the (E) over tilde I-i complex formed in the presence of PCU and calcium, one inhibitor molecule is bound to the active site and a number of others are bound to the i-face. The properties of the (E) over tilde (i) complexes with PCU are qualitatively similar to those of E-i(#) formed with decylsulfate. Decylsulfate binds to the i-face but does not bind to the active site in the presence of calcium, nor does it interfere with the binding of PCU to the active site in the premicellar complexes. Due to the strong coupling between binding at the i-face and at the active site, it is difficult to estimate the primary binding constants for each site in these complexes. A model is developed that incorporates the above boundary conditions in relation to a detailed balance between the complexes. A key result is that a modest effect on cooperative amphiphile binding corresponds to a large change in the affinity of the inhibitor for the active site. We suggest that besides the binding to the active site, PCU also binds to another site and that full activation requires additional amphiphiles on the i-face. Thus, the activation of the inhibitor binding to the active site of the E-2(#) complex or, equivalently, the shift in the E-1(#) to E-2(#) equilibrium by the inhibitor is analogous to the allosteric activation of the substrate binding to the enzyme bound to the interface.

  • 7.
    Berg, Otto
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Yu, Bao-Zhu
    Apitz-Castro, Rafael J.
    Jain, Mahendra K.
    Phosphatidylinositol-specific phospholipase C forms different complexes with monodisperse and micellar phosphatidylcholine2004In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 7, p. 2080-2090Article in journal (Refereed)
    Abstract [en]

    Phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus cereus forms a premicellar complex E# with monodisperse diheptanoylphosphatidylcholine (DC7PC) that is distinguishable from the E* complex formed with micelles. Results are interpreted with the assumption that in both cases amphiphiles bind to the interfacial binding surface (i-face) of PI-PLC but not to the active site. Isothermal calorimetry and fluorescence titration results for the binding of monodisperse DC7PC give an apparent dissociation constant of K2 = 0.2 mM with Hill coefficient of 2. The gel-permeation, spectroscopic, and probe partitioning behaviors of E# are distinct from those of the E* complex. The aggregation and partitioning behaviors suggest that the acyl chains in E# but not in E* remain exposed to the aqueous phase. The free (E) and complexed (E# and E*) forms of PI-PLC, each with distinct spectroscopic signatures, readily equilibrate with changing DC7PC concentration. The underlying equilibria are modeled and their significance for the states of the PI-PLC under monomer kinetic conditions is discussed to suggest that the Michaelis−Menten complex formed with monodisperse DC7PC is likely to be E#S or its aggregate rather than the classical monodisperse ES complex.

  • 8.
    Berg, Otto
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Yu, Bao-Zhu
    Chang, Cherry
    Koehler, Karl A.
    Jain, Mahendra K.
    Cooperative binding of monodisperse anionic amphiphiles to the i-Face: Phospholipase A2-paradigm for interfacial binding2004In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 25, p. 7999-8013Article in journal (Refereed)
    Abstract [en]

    Equilibrium parameters for the binding of monodisperse alkyl sulfate along the i-face (the interface binding surface) of pig pancreatic IB phospholipase A2 (PLA2) to form the premicellar complexes (Ei#) are characterized to discern the short-range specific interactions. Typically, Ei# complexes are reversible on dilution. The triphasic binding isotherm, monitored as the fluorescence emission from the single tryptophan of PLA2, is interpreted as a cooperative equilibrium for the sequential formation of three premicellar complexes (Ei#, i = 1, 2, 3). In the presence of calcium, the dissociation constant K1 for the E1# complex of PLA2 with decyl sulfate (CMC = 4500 μM) is 70 μM with a Hill coefficient n1 = 2.1 ± 0.2; K2 for E2# is 750 μM with n2 = 8 ± 1, and K3 for E3# is 4000 μM with an n3 value of about 12. Controls show that (a) self-aggregation of decyl sulfate alone is not significant below the CMC; (b) occupancy of the active site is not necessary for the formation of Ei#; (c) Ki and ni do not change significantly due to the absence of calcium, possibly because alkyl sulfate does not bind to the active site of PLA2; (d) the Ei# complexes show a significant propensity for aggregation; and (e) PLA2 is not denatured in Ei#. The results are interpreted to elaborate the model for atomic level interactions along the i-face: The chain length dependence of the fit parameters suggests that short-range specific anion binding of the headgroup is accompanied by desolvation of the i-face of Ei#. We suggest that allosteric activation of PLA2 results from such specific interactions of the amphiplies and the desolvation of the i-face. The significance of these primary interfacial binding events and the coexistence of the E* and Ei# aggregates is discussed.

  • 9.
    Bjelic, Sinisa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Brandsdal, Bjørn O
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structural Molecular Biology.
    Cold adaptation of enzyme reaction rates2008In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 47, no 38, p. 10049-10057Article in journal (Refereed)
    Abstract [en]

    A major issue for organisms living at extreme temperatures is to preserve both stability and activity of their enzymes. Cold-adapted enzymes generally have a reduced thermal stability, to counteract freezing, and show a lower enthalpy and a more negative entropy of activation compared to mesophilic and thermophilic homologues. Such a balance of thermodynamic activation parameters can make the reaction rate decrease more linearly, rather than exponentially, as the temperature is lowered, but the structural basis for rate optimization toward low working temperatures remains unclear. In order to computationally address this problem, it is clear that reaction simulations rather than standard molecular dynamics calculations are needed. We have thus carried out extensive computer simulations of the keto-enol(ate) isomerization steps in differently adapted citrate synthases to explore the structure-function relationships behind catalytic rate adaptation to different temperatures. The calculations reproduce the absolute rates of the psychrophilic and mesophilic enzymes at 300 K, as well as the lower enthalpy and more negative entropy of activation of the cold-adapted enzyme, where the latter simulation result is obtained from high-precision Arrhenius plots. The overall catalytic effect originates from electrostatic stabilization of the transition state and enolate and the reduction of reorganization free energy. The simulations, however, show psychrophilic, mesophilic, and hyperthermophilic citrate synthases to have increasingly stronger electrostatic stabilization of the transition state, while the energetic penalty in terms of internal protein interactions follows the reverse order with the cold-adapted enzyme having the most favorable energy term. The lower activation enthalpy and more negative activation entropy observed for cold-adapted enzymes are found to be associated with a decreased protein stiffness. The origin of this effect is, however, not localized to the active site but to other regions of the protein structure.

  • 10.
    Boukharta, Lars
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Keränen, Henrik
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Stary-Weinzinger, Anna
    Wallin, Göran
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    de Groot, Bert L.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Computer Simulations of Structure-Activity Relationships for hERG Channel Blockers2011In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 50, no 27, p. 6146-6156Article in journal (Refereed)
    Abstract [en]

    The hERG potassium channel is of major pharmaceutical importance, and its blockade by various compounds, potentially causing serious cardiac side effects, is a major problem in drug development. Despite the large amounts of existing biochemical data on blockade of hERG by drugs and druglike compounds, relatively little is known regarding the structural basis of binding of blockers to the channel. Here, we have used a recently developed homology model of hERG to conduct molecular docking experiments with a series of channel blockers, followed by molecular dynamics simulations of the complexes and evaluation of binding free energies with the linear interaction energy method. The calculations yield a remarkably good agreement with experimental binding affinities and allow for a rationalization of three-dimensional structure-activity relationships in terms of a number of key interactions. Two main interaction regions of the channel are thus identified with implications for further mutagenesis experiments and design of new compounds.

  • 11. Cajal, Yolanda
    et al.
    Berg, Otto
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Evolution, Genomics and Systematics, Molecular Evolution.
    Jain, Mahendra K.
    Origins of delays in monolayer kinetics: Phospholipase A2 paradigm2004In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 43, no 28, p. 9256-9264Article in journal (Refereed)
    Abstract [en]

    The interfacial kinetic paradigm is adopted to model the kinetic behavior of pig pancreatic phospholipase A2 (PLA2) at the monolayer interface. A short delay of about a minute to the onset of the steady state is observed under all monolayer reaction progress conditions, including the PLA2-catalyzed hydrolysis of didecanoylphosphatidyl-choline (PC10) and -glycerol (PG10) monolayers as analyzed in this paper. This delay is independent of enzyme concentration and surface pressure and is attributed to the equilibration time by stationary diffusion of the enzyme added to the stirred subphase to the monolayer through the intervening unstirred aqueous layer. The longer delays of up to several hours, seen with the PC10 monolayers at >15 mN/m, are influenced by surface pressure as well as enzyme concentration. Virtually all features of the monolayer reaction progress are consistent with the assumption that the product accumulates in the substrate monolayer, although the products alone do not spread as a compressible monolayer. These results rule out models that invoke slow “activation” of PLA2 on the monolayer. The observed steady-state rate on monolayers after the delays is <1% of the rate observed with micellar or vesicles substrates of comparable substrate. Together these results suggest that the monolayer steady-state rate includes contributions from steps other than those of the interfacial turnover cycle. Additional considerations that provide understanding of the pre-steady-state behaviors and other nonideal effects at the surface are also discussed.

  • 12.
    Carlsson, Gunilla H.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Nicholls, Peter
    Svistunenko, Dimitri
    Berglund, Gunnar I.
    Hajdu, Janos
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Complexes of horseradish peroxidase with formate, acetate and carbon monoxide2005In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 44, no 2, p. 635-642Article in journal (Refereed)
    Abstract [en]

    Carbon monoxide, formate, and acetate interact with horseradish peroxidase (HRP) by binding to subsites within the active site. These ligands also bind to catalases, but their interactions are different in the two types of enzymes. Formate (notionally the “hydrated” form of carbon monoxide) is oxidized to carbon dioxide by compound I in catalase, while no such reaction is reported to occur in HRP, and the CO complex of ferrocatalase can only be obtained indirectly. Here we describe high-resolution crystal structures for HRP in its complexes with carbon monoxide and with formate, and compare these with the previously determined HRP−acetate structure [Berglund, G. I., et al. (2002) Nature 417, 463−468]. A multicrystal X-ray data collection strategy preserved the correct oxidation state of the iron during the experiments. Absorption spectra of the crystals and electron paramagnetic resonance data for the acetate and formate complexes in solution correlate electronic states with the structural results. Formate in ferric HRP and CO in ferrous HRP bind directly to the heme iron with iron−ligand distances of 2.3 and 1.8 Å, respectively. CO does not bind to the ferric iron in the crystal. Acetate bound to ferric HRP stacks parallel with the heme plane with its carboxylate group 3.6 Å from the heme iron, and without an intervening solvent molecule between the iron and acetate. The positions of the oxygen atoms in the bound ligands outline a potential access route for hydrogen peroxide to the iron. We propose that interactions in this channel ensure deprotonation of the proximal oxygen before binding to the heme iron.

  • 13. Carpenter, Katharine A.
    et al.
    Schmidt, Ralf
    von Mentzer, Bengt
    Haglund, Ulla
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences.
    Roberts, Edward
    Walpole, Chris
    Turn structures in CGRP C-terminal analogues promote stable arrangements of key residue side chains2001In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 40, no 28, p. 8317-8325Article in journal (Refereed)
    Abstract [en]

    The 37-amino acid calcitonin gene-related peptide (CGRP) is a potent endogenous vasodilator thought to be implicated in the genesis of migraine attack. CGRP antagonists may thus have therapeutic value for the treatment of migraine. The CGRP C-terminally derived peptide [D(31),P(34),F(35)]CGRP(27-37)-NH(2) was recently identified as a high-affinity hCGRP(1) receptor selective antagonist. Reasonable CGRP(1) affinity has also been demonstrated for several related analogues, including [D(31),A(34),F(35)]CGRP(27-37)-NH(2). In the study presented here, conformational and structural features in CGRP(27-37)-NH(2) analogues that are important for hCGRP(1) receptor binding were explored. Structure-activity studies carried out on [D(31),P(34),F(35)]CGRP(27-37)-NH(2) resulted in [D(31),P(34),F(35)]CGRP(30-37)-NH(2), the shortest reported CGRP C-terminal peptide analogue exhibiting reasonable hCGRP(1) receptor affinity (K(i) = 29.6 nM). Further removal of T(30) from the peptide's N-terminus greatly reduced receptor affinity from the nanomolar to micromolar range. Additional residues deemed critical for hCGRP(1) receptor binding were identified from an alanine scan of [A(34),F(35)]CGRP(28-37)-NH(2) and included V(32) and F(37). Replacement of the C-terminal amide in this same peptide with a carboxyl, furthermore, resulted in a greater than 50-fold reduction in hCGRP(1) affinity, thus suggesting a direct role for the amide moiety in receptor binding. The conformational properties of two classes of CGRP(27-37)-NH(2) peptides, [D(31),X(34),F(35)]CGRP(27-37)-NH(2) (X is A or P), were examined by NMR spectroscopy and molecular modeling. A beta-turn centered on P(29) was a notable feature consistently observed among active peptides in both series. This turn led to exposure of the critical T(30) residue to the surrounding environment. Peptides in the A(34) series were additionally characterized by a stable C-terminal helical turn that resulted in the three important residues (T(30), V(32), and F(37)) adopting consistent interspatial positions with respect to one another. Peptides in the P(34) series were comparatively more flexible at the C-terminus, although a large proportion of the [D(31),P(34),F(35)]CGRP(27-37)-NH(2) calculated conformers contained a gamma-turn centered on P(34). These results collectively suggest that turn structures at both the C-terminus and N-terminus of CGRP(27-37)-NH(2) analogues may help to appropriately orient critical residues (T(30), V(32), and F(37)) for hCGRP(1) receptor binding.

  • 14.
    Chen, Guiying
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Han, Guangye
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Göransson, Erik
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Physical Chemistry.
    Mamedov, Fikret
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Styring, Stenbjörn
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - Ångström, Molecular Biomimetics.
    Stability of the S(3) and S(2) State Intermediates in Photosystem II Directly Probed by EPR Spectroscopy2012In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 1, p. 138-148Article in journal (Refereed)
    Abstract [en]

    The stability of the S(3) and S(2) states of the oxygen evolving complex in photosystem II (PSII) was directly probed by EPR spectroscopy in PSII membrane preparations from spinach in the presence of the exogenous electron acceptor PpBQ at 1, 10, and 20 degrees C. The decay of the S(3) state was followed in samples exposed to two flashes by measuring the split S(3) EPR signal induced by near-infrared illumination at 5 K. The decay of the S(2) state was followed in samples exposed to one flash by measuring the S(2) state multiline EPR signal. During the decay of the S(3) state, the S(2) state multiline EPR signal first increased and then decreased in amplitude. This shows that the decay of the S(3) state to the S(1) state occurs via the S(2) state. The decay of the S(3) state was biexponential with a fast kinetic phase with a few seconds decay half-time. This occurred in 10-20% of the PSII centers. The slow kinetic phase ranged from a decay half-time of 700 s (at 1 degrees C) to similar to 100 s (at 20 degrees C) in the remaining 80-90% of the centers. The decay of the S(2) state was also biphasic and showed quite similar kinetics to the decay of the S(3) state. Our experiments show that the auxiliary electron donor Y(D) was oxidized during the entire experiment. Thus, the reduced form of Y(D) does not participate to the fast decay of the S(2) and S(3) states we describe here. Instead, we suggest that the decay of the S(3) and S(2) states reflects electron transfer from the acceptor side of PSII to the donor side of PSII starting in the corresponding S state. It is proposed that this exists in equilibrium with Y(Z) according to S(3)Y(2) double left right arrow S(2)Y(Z)(.) in the case of the S(3) state decay and S(2)Y(Z) double left right arrow S(1)Y(Z)(.) in the case of the S(2) state decay. Two kinetic models are discussed, both developed with the assumption that the slow decay of the S(3) and S(2) states occurs in PSII centers where Y(Z) is also a fast donor to P(680)(+) working in the nanosecond time regime and that the fast decay of the S(3) and S(2) states occurs in centers where Y(Z) reduces P(680)(+) with slower microsecond kinetics. Our measurements also demonstrate that the split S(3) EPR signal can be used as a direct probe to the S(3) state and that it can provide important information about the redox properties of the S(3) state.

  • 15.
    Chi, Celestine N.
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ETH.
    Haq, S. Raza
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Rinaldo, S.
    Dogan, Jakob
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Cutruzzolà, F.
    Engström, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Gianni, S.
    Lundström, P.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Interactions outside the boundaries of the canonical binding groove of a pdz domain influence ligand binding2012In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 44, p. 8971-8979Article in journal (Refereed)
    Abstract [en]

    The postsynaptic density protein-95/discs large/zonula occludens-1 (PDZ) domain is a protein-protein interaction module with a shallow binding groove where protein ligands bind. However, interactions that are not part of this canonical binding groove are likely to modulate peptide binding. We have investigated such interactions beyond the binding groove for PDZ3 from PSD-95 and a peptide derived from the C-terminus of the natural ligand CRIPT. We found via nuclear magnetic resonance experiments that up to eight residues of the peptide ligand interact with the PDZ domain, showing that the interaction surface extends far outside of the binding groove as defined by the crystal structure. PDZ3 contains an extra structural element, a C-terminal helix (α3), which is known to affect affinity. Deletion of this helix resulted in the loss of several intermolecular nuclear Overhauser enhancements from peptide residues outside of the binding pocket, suggesting that α3 forms part of the extra binding surface in wild-type PDZ3. Site-directed mutagenesis, isothermal titration calorimetry, and fluorescence intensity experiments confirmed the importance of both α3 and the N-terminal part of the peptide for the affinity. Our data suggest a general mechanism in which different binding surfaces outside of the PDZ binding groove could provide sites for specific interactions.

  • 16.
    Chi N, Celestine
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. ETH.
    Bach, Anders
    Engström, Åke
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Wang, Huiqun
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Strømgaard, Kristian
    Gianni, Stefano
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    A sequential binding mechanism in a PDZ domain2009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 30, p. 7089-7097Article in journal (Refereed)
    Abstract [en]

    Conformational selection and induced fit are two well-known mechanisms of allosteric protein-ligand interaction. Some proteins, like ubiquitin, have recently been found to exist in multiple conformations at equilibrium, suggesting that the conformational selection may be a general mechanism of interaction, in particular for single-domain proteins. Here, we found that the PDZ2 domain of SAP97 binds its ligand via a sequential (induced fit) mechanism. We performed binding experiments using SAP97 PDZ2 and peptide ligands and observed biphasic kinetics with the stopped-flow technique, indicating that ligand binding involves at least a two-step process. By using an ultrarapid continuous-flow mixer, we then detected a hyperbolic dependence of binding rate constants on peptide concentration, corroborating the two-step binding mechanism. Furthermore, we found a similar dependence of the rate constants on both PDZ and peptide concentration, demonstrating that the PDZ2-peptide interaction involves a precomplex, which then undergoes a conformational change, and thereby follows an induced fit mechanism.

  • 17. Dahl, Goran
    et al.
    Gutierrez Arenas, Omar
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Public Health and Caring Sciences, Centre for Research Ethics and Bioethics.
    Danielson, Helena
    Hepatitis C virus NS3 protease is activated by low concentrations of protease inhibitors2009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995Article in journal (Refereed)
  • 18.
    Dahl, Göran
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Gutiérrez Arenas, Omar
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Danielson, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Hepatitis C Virus NS3 Protease Is Activated by Low Concentrations of  Protease Inhibitors2009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 48, p. 11592-11602Article in journal (Refereed)
    Abstract [en]

    The nonstructural protein 3 (NS3) of hepatitis C virus (HCV) is a   bifunctional enzyme with a protease and a helicase functionality   located in each of the two domains of the single peptide chain. There   is little experimental evidence for a functional role of this   unexpected arrangement since artificial single domain forms of both   enzymes are catalytically competent. We have observed that low   concentrations of certain protease inhibitors activate the protease of   full-length NS3 from HCV genotype 1a with up to 100%, depending on the   preincubation time and the inhibitor used. The activation was reduced,   but not eliminated, by increased ionic strength, lowered glycerol   concentration, or lowered pH. In all cases, it was at the expense of a   significant loss of activity. Activation was not seen with the   artificial protease domain of genotype 1b NS3 fused with a fragment of   the NS4A cofactor. This truncated and covalently modified enzyme form   was much less active and exhibited fundamentally different catalytic   properties to the full-length NS3 protease without (he fused cofactor.   The most plausible explanation for the activation was found to involve   a slow transition between two enzyme conformations, which differed in   their catalytic ability and affinity for inhibitors. Equations derived   based on this assumption resulted in better fits to the experimental   data than the equation for simple competitive inhibition. The mechanism   may involve an inhibitor-induced stabilization of the helicase domain   in a conformation that enhances the protease activity, or all improved   alignment of the catalytic triad in the protease. The proposed mnemonic   mechanism and derived equations are viable for both these explanations   and can serve as a basic framework for future studies of enzymes   activated by inhibitors or other ligands.

  • 19.
    Danelius, Emma
    et al.
    University of Gothenburg, SE-41296 Gothenburg, Sweden.
    Andersson, Hanna
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry. University of Gothenburg, SE-41296 Gothenburg, Sweden.
    Jarvoll, Patrik
    University of Gothenburg, SE-41296 Gothenburg, Sweden.
    Lood, Kajsa
    University of Gothenburg, SE-41296 Gothenburg, Sweden.
    Gräfenstein, Jürgen
    University of Gothenburg, SE-41296 Gothenburg, Sweden.
    Erdélyi, Máté
    University of Gothenburg, SE-41296 Gothenburg, Sweden.
    Halogen Bonding: A Powerful Tool for Modulation of Peptide Conformation2017In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Biochemistry, ISSN 0006-2960, Vol. 56, no 25, p. 3265-3272Article in journal (Refereed)
    Abstract [en]

    Halogen bonding is a weak chemical force that has so far mostly found applications in crystal engineering. Despite its potential for use in drug discovery, as a new molecular tool in the direction of molecular recognition events, it has rarely been assessed in biopolymers. Motivated by this fact, we have developed a peptide model system that permits the quantitative evaluation of weak forces in a biologically relevant proteinlike environment and have applied it for the assessment of a halogen bond formed between two amino acid side chains. The influence of a single weak force is measured by detection of the extent to which it modulates the conformation of a cooperatively folding system. We have optimized the amino acid sequence of the model peptide on analogues with a hydrogen bond-forming site as a model for the intramolecular halogen bond to be studied, demonstrating the ability of the technique to provide information about any type of weak secondary interaction. A combined solution nuclear magnetic resonance spectroscopic and computational investigation demonstrates that an interstrand halogen bond is capable of conformational stabilization of a β-hairpin foldamer comparable to an analogous hydrogen bond. This is the first report of incorporation of a conformation-stabilizing halogen bond into a peptide/protein system, and the first quantification of a chlorine-centered halogen bond in a biologically relevant system in solution.

  • 20.
    Danielson, U. Helena
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Jiang, Fanyi Y
    Hansson, Lars O.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Probing the kinetic mechanism and coenzyme specificity of glutathione reductase from the cyanobacterium Anabaena PCC 7120 by redesign of the pyridine-nucleotide-binding site1999In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 38, no 29, p. 9254-9263Article in journal (Refereed)
    Abstract [en]

    Glutathione reductase from the cyanobacterium Anabaena PCC 7120 contains a pyridine-nucleotide-binding motif differing from that of the enzyme from other sources and an insertion of 10 amino acid residues. Homology modeling was used to obtain a model of the enzyme structure. It revealed that in the Anabaena enzyme Lys(203) replaces Arg, found to interact with the 2'-phosphate of NADP(H) in the enzyme from other sources, and that it has an extra loop near the entrance of the pyridine-nucleotide-binding site. The steady-state and preequilibrium kinetic properties were characterized for the wild-type enzyme, a K203R, and a loop deletion mutant. All enzyme forms had higher catalytic efficiency with NADPH than with NADH, although the difference was less than for glutathione reductase from other sources. The specificity was most pronounced in the formation of the charge-transfer complex between the pyridine nucleotide and oxidized enzyme-bound FAD, as compared to later steps in the reaction. Unexpectedly, by replacing Lys(203) with Arg, the specificity for NADPH was diminished in the complete redox reaction. Ser(174) appears to interact with the 2'-phosphate of NADPH and introduction of arginine instead of lysine, therefore, has little effect on the interaction with this coenzyme. However, the efficiency in forming the charge-transfer complex between the pyridine nucleotide and oxidized enzyme-bound FAD was increased in the K203R mutant using NADPH but not with NADH. The lack of affinity toward 2',5'-ADP-Sepharose by the wild-type enzyme was not changed by replacing Lys(203) with Arg but deletion of the loop resulted in an enzyme that bound to the immobilized ligand. Removal of the loop increased the efficiency of the enzyme in the reductive half-reaction with both pyridine-nucleotides as well as in the overall catalytic mechanism.

  • 21.
    Dogan, Jakob
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Jonasson, Josefin
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Andersson, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Binding Rate Constants Reveal Distinct Features of Disordered Protein Domains2015In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 54, no 30, p. 4741-4750Article in journal (Refereed)
    Abstract [en]

    Intrinsically disordered proteins (IDPs) are abundant in the proteome and involved in key cellular functions. However, experimental data about the binding kinetics of IDPs as a function of different environmental conditions are scarce. We have performed an extensive characterization of the ionic strength dependence of the interaction between the molten globular nuclear co-activator binding domain (NCBD) of CREB binding protein and five different protein ligands, including the intrinsically disordered activation domain of p160 transcriptional co-activators (SRC1, TIF2, ACTR), the p53 transactivation domain, and the folded pointed domain (PNT) of transcription factor ETS-2. Direct comparisons of the binding rate constants under identical conditions show that the association rate constant, k(on), for interactions between NCBD and disordered protein domains is high at low salt concentrations (90-350 x 10(6) M-1 s(-1) at 4 degrees C) but is reduced significantly (10-30-fold) with an increasing ionic strength and reaches a plateau around physiological ionic strength. In contrast, the k(on) for the interaction between NCBD and the folded PNT domain is only 7 x 10(6) M-1 s(-1) (4 degrees C and low salt) and displays weak ionic strength dependence, which could reflect a distinctly different association that relies less on electrostatic interactions. Furthermore, the basal rate constant (in the absence of electrostatic interactions) is high for the NCBD interactions, exceeding those typically observed for folded proteins. One likely interpretation is that disordered proteins have a large number of possible collisions leading to a productive on-pathway encounter complex, while folded proteins are more restricted in terms of orientation. Our results highlight the importance of electrostatic interactions in binding involving IDPs and emphasize the significance of including ionic strength as a factor in studies that compare the binding properties of IDPs to those of ordered proteins.

  • 22.
    Dogan, Jakob
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology. Stockholm Univ, Dept Biochem & Biophys, SE-10691 Stockholm, Sweden..
    Toto, Angelo
    Univ Cambridge, Dept Chem, Lensfield Rd, Cambridge CB2 1EW, England..
    Andersson, Eva
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Gianni, Stefano
    Univ Rome, Inst Pasteur, Fdn Cenci Bolognetti, I-00185 Rome, Italy.;Univ Rome, Ist Biol & Patol Mol, CNR, Dipartimento Sci Biochim A Rossi Fanelli Sapienza, I-00185 Rome, Italy.;Univ Cambridge, Dept Chem, Lensfield Rd, Cambridge CB2 1EW, England..
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Activation Barrier-Limited Folding and Conformational Sampling of a Dynamic Protein Domain2016In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 55, no 37, p. 5289-5295Article in journal (Refereed)
    Abstract [en]

    Folding reaction mechanisms of globular protein domains have been extensively studied by both experiment and simulation and found to be highly concerted chemical reactions in which numerous noncovalent bonds form in an apparent two-state fashion. However, less is known regarding intrinsically disordered proteins because their folding can usually be studied only in conjunction with binding to a ligand. We have investigated by kinetics the folding mechanism of such a disordered protein domain, the nuclear coactivator-binding domain (NCBD) from CREB-binding protein. While a previous computational study suggested that NCBD folds without an activation free energy barrier, our experimental data. demonstrate that NCBD, despite its highly dynamic structure, displays relatively slow folding (similar to 10 ms at 277 K) consistent with a barrier-limited process. Furthermore, the folding kinetics corroborate previous nuclear magnetic resonance data showing that NCBD exists in two folded conformations and one more denatured conformation at equilibrium and, thus, that the folding mechanism is a three-state mechanism. The refolding kinetics is limited by unfolding of the less populated folded conformation, suggesting that the major route for interconversion between the two folded states is via the denatured State. Because the two folded conformations have been suggested to bind distinct ligands, our results have mechanistic implications for conformational sampling in protein protein interactions.

  • 23. Domínguez, José L
    et al.
    Christopeit, Tony
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Villaverde, M Carmen
    Gossas, Thomas
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Otero, José M
    Nyström, Susanne
    Baraznenok, Vera
    Lindström, Erik
    Danielson, U Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Sussman, Fredy
    Effect of the Protonation State of the Titratable Residues on the Inhibitor Affinity to BACE-12010In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, no 34, p. 7255-7263Article in journal (Refereed)
    Abstract [en]

    BACE-1 is one of the aspartic proteases involved in the cleavage of beta amyloid peptide, an initial step in the formation of amyloid plaques whose toxicity induces neuron death in Alzheimer's disease patients. One of the central issues in the search of novel BACE-1 inhibitors is the optimum pH for the binding of inhibitors to the enzyme. It is known that the enzyme has optimal catalytic activity at acidic pH, while cell active inhibitors may bind optimally at higher pH. In this work we determine the effect of the pH on the affinities of a set of inhibitors, with a variety of chemical motifs, for the ectodomain region of BACE-1 by a surface plasmon resonance (SPR) biosensor based assay. In order to understand the molecular interactions that underlie the diverse optimum pH for the binding of the various inhibitors as observed experimentally, we have calculated the titration curves for a set of BACE-1 ligand complexes. The results indicate that the pK(a) values of the titratable residues of the protein depend on the nature of the ligand involved, in disagreement with previous work. The enzyme-inhibitor structures with the resulting protonation states at pH values 4.5 and 7.4 served as the starting point for the prediction of the pH-dependent binding ranking. Our calculations reproduced the entire affinity ranking observed upon pH increase and most of the binding trends among inhibitors, especially at low pH. Finally, our cell-based assays indicate a possible correlation between high inhibitor affinity at both acidic and neutral pH values, with optimal cell response, a result that may open new venues for the search of potent BACE-1 inhibitors that are active at the cellular level.

  • 24.
    Dourado, Daniel F. A. R.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational and Systems Biology.
    Fernandes, Pedro Alexandrino
    Ramos, Maria Joao
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Mechanism of Glutathione Transferase P1-1-Catalyzed Activation of the Prodrug Canfosfamide (TLK286, TELCYTA)2013In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, no 45, p. 8069-8078Article in journal (Refereed)
    Abstract [en]

    Canfosfamide (TLK286, TELCYTA) is a prodrug that upon activation by glutathione transferase P1-1 (GST P1-1) yields an anticancer alkylating agent and a glutathione derivative. The rationale underlying the use of TLK286 in chemotherapy is that tumor cells overexpressing GST P1-1 will be locally exposed to the released alkylating agent with limited collateral toxicity to the surrounding normal tissues. TLK286 has demonstrated clinical effects in phase II and III clinical trials for the treatment of malignancies, such as ovarian cancer, nonsmall cell lung cancer, and breast cancer, as a single agent and in combination with other chemotherapeutic agents. In spite of these promising results, the detailed mechanism of GST P1-1 activation of the prodrug has not been elucidated. Here, we propose a mechanism for the TLK286 activation by GST P1-1 on the basis of density functional theory (DFT) and on potential of mean force (PMF) calculations. A catalytic water molecule is instrumental to the activation by forming a network of intermolecular interactions between the active-site Tyr7 hydroxyl and the sulfone and COO- groups of TLK286. The results obtained are consistent with the available experimental kinetic data and provide an atomistic understanding of the TLK286 activation mechanism.

  • 25. Erdelyi, Mate
    Application of the Halogen Bond in Protein Systems.2017In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 56, no 22, p. 2759-2761Article, review/survey (Refereed)
  • 26. Estrada, S
    et al.
    Pavlova, A
    Björk, I
    The contribution of N-terminal region residues of cystatin A (stefin A) to the affinity and kinetics of inhibition of papain, cathepsin B, and cathepsin L.1999In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 38, no 22, p. 7339-45Article in journal (Refereed)
    Abstract [en]

    The affinity and kinetics of binding of three N-terminally truncated variants of the cysteine proteinase inhibitor cystatin A to cysteine proteinases were characterized. Deletion of Met-1 only minimally altered the inhibitory properties of the protein. However, deletion also of Ile-2 resulted in reduced affinities of 900-, >/=3-, and 200-fold for papain and cathepsins L and B, respectively. Further truncation of Pro-3 substantially increased the inhibition constants to approximately 0.5 microM for papain and cathepsin L and to 60 microM for cathepsin B, reflecting additionally 2 x 10(3)-, 2 x 10(4)-, and 400-fold decreased affinities, respectively. The reductions in affinity shown by the latter mutant indicate that the N-terminal region contributes about 40% of the total free energy of binding of cystatin A to cysteine proteinases. Moreover, Pro-3 and to a lesser extent Ile-2 are the residues responsible for this binding energy. The reduced affinities for papain and cathepsin L were due only to higher dissociation rate constants, whereas both lower association and higher dissociation rate constants contributed to the decreased affinity for cathepsin B. These differential effects indicate that the N-terminal portion of cystatin A primarily functions by stabilizing the complexes with enzymes having easily accessible active-site clefts, e.g., papain and cathepsin L. In contrast, the N-terminal region is required also for an initial binding of cystatin A to cathepsin B, presumably by promoting the displacement of the occluding loop and allowing facile interaction of the rest of the inhibiting wedge with the active-site cleft of the enzyme.

  • 27.
    Fabini, Edoardo
    et al.
    Alma Mater Studiorum Univ Bologna, Dept Pharm & Biotechnol, Bologna, Italy;CNR, Natl Res Council, Inst Organ Synth & Photoreact ISOF, Bologna, Italy.
    Talibov, Vladimir O
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Mihalic, Filip
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC.
    Naldi, Marina
    Alma Mater Studiorum Univ Bologna, Dept Pharm & Biotechnol, Bologna, Italy;St Orsola Marcello Malpighi Hosp, Ctr Appl Biomed Res CRBA, Bologna, Italy.
    Bartolini, Manuela
    Alma Mater Studiorum Univ Bologna, Dept Pharm & Biotechnol, Bologna, Italy.
    Bertucci, Carlo
    Alma Mater Studiorum Univ Bologna, Dept Pharm & Biotechnol, Bologna, Italy.
    Del Rio, Alberto
    CNR, Natl Res Council, Inst Organ Synth & Photoreact ISOF, Bologna, Italy;Innovamol Consulting Srl, Modena, Italy.
    Danielson, U. Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Unveiling the Biochemistry of the Epigenetic Regulator SMYD32019In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 58, no 35, p. 3634-3645Article in journal (Refereed)
    Abstract [en]

    SET and MYND domain-containing protein 3 (SMYD3) is a lysine methyltransferase that plays a central role in a variety of cancer diseases, exerting its pro-oncogenic activity by methylation of key proteins, of both nuclear and cytoplasmic nature. However, the role of SMYD3 in the initiation and progression of cancer is not yet fully understood and further biochemical characterization is required to support the discovery of therapeutics targeting this enzyme. We have therefore developed robust protocols for production, handling, and crystallization of SMYD3 and biophysical and biochemical assays for clarification of SMYD3 biochemistry and identification of useful lead compounds. Specifically, a time-resolved biosensor assay was developed for kinetic characterization of SMYD3 interactions. Functional differences in SMYD3 interactions with its natural small molecule ligands SAM and SAH were revealed, with SAM forming a very stable complex. A variety of peptides mimicking putative substrates of SMYD3 were explored in order to expose structural features important for recognition. The interaction between SMYD3 and some peptides was influenced by SAM. A nonradioactive SMYD3 activity assay using liquid chromatography-mass spectrometry (LC-MS) analysis explored substrate features of importance also for methylation. Methylation was notable only toward MAP kinase kinase kinase 2 (MAP3K2_K-260)-mimicking peptides, although binary and tertiary complexes were detected also with other peptides. The analysis supported a random bi-bi mechanistic model for SMYD3 methyltransferase catalysis. Our work unveiled complexities in SMYD3 biochemistry and resulted in procedures suitable for further studies and identification of novel starting points for design of effective and specific leads for this potential oncology target.

  • 28.
    Feierberg, Isabella
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    The catalytic power of ketosteroid isomerase investigated by computer simulation2002In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 41, no 52, p. 15728-15735Article in journal (Refereed)
    Abstract [en]

    Ketosteroid isomerase (KSI) catalyzes the isomerization of Delta(5)-3-ketosteroids and Delta(4)-3-ketosteroids at very high rates. Here we examine the principles underlying the catalytic efficiency of KSI by computer simulations using the empirical valence bond method in combination with molecular dynamics free energy perturbation simulations. The simulations reproduce available kinetic and structural data very well and allow us to examine several features of the catalytic mechanism in detail. It is found that about 60% of the rate enhancement is due to stabilization of the negatively charged dienolate intermediate by hydrogen bonding. The critical H-bond between Tyr16 and the intermediate is found to be a normal ionic H-bond with the preferred proton location on the tyrosine residue. The remaining 40% of the catalytic effect originates from a reduction of the reorganization energy of the reaction. The possibility of an active site water molecule occupying the empty cavity adjacent to the catalytic base (Asp40) is also addressed. The existence of such a water molecule could explain how the enzyme manages to maintain a low pK(a) for the general base residue.

  • 29. Gadjieva, Rena
    et al.
    Mamedov, Fikret
    Renger, Gernot
    Styring, Stenbjörn
    Interconversion of low and high potential forms of cytochrome b559 in tris-washed photosystem II membranes under aerobic/anaerobic conditions1999In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Biochem, Vol. 38, p. 10578-10584Article in journal (Refereed)
  • 30. Gagnon, Keith T.
    et al.
    Pendergraff, Hannah M.
    Deleavey, Glen F.
    Swayze, Eric E.
    Potier, Pierre
    Randolph, John
    Roesch, Eric B.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Bioorganic Chemistry.
    Damha, Masad J.
    Bennett, C. Frank
    Montaillier, Christophe
    Lemaitre, Marc
    Corey, David R.
    Allele-Selective Inhibition of Mutant Huntingtin Expression with Antisense Oligonucleotides Targeting the Expanded CAG Repeat2010In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, no 47, p. 10166-10178Article in journal (Refereed)
    Abstract [en]

    Huntington's disease (HD) is a currently incurable neurodegenerative disease caused by the expansion of a CAG trinucleotide repeat within the huntingtin (HTT) gene. Therapeutic approaches include selectively inhibiting the expression of the mutated HTT allele while conserving function of the normal allele. We have evaluated a series of antisense oligonucleotides (ASOs) targeted to the expanded CAG repeat within HTT mRNA for their ability to selectively inhibit expression of mutant HTT protein. Several ASOs incorporating a variety of modifications, including bridged nucleic acids and phosphorothioate internucleotide linkages, exhibited allele-selective silencing in patient-derived fibroblasts. Allele-selective ASOs did not affect the expression of other CAG repeat-containing genes and selectivity was observed in cell lines containing minimal CAG repeat lengths representative of most HD patients. Allele-selective ASOs left HTT mRNA intact and did not support ribonuclease H activity in vitro. We observed cooperative binding of multiple ASO molecules to CAG repeat-containing HTT mRNA transcripts in vitro. These results are consistent with a mechanism involving inhibition at the level of translation. ASOs targeted to the CAG repeat of HTT provide a starting point for the development of oligonucleotide-based therapeutics that can inhibit gene expression with allelic discrimination in patients with HD.

  • 31. Geitmann, Matthis
    et al.
    Retra, Kim
    de Kloe, Gerdien E
    Homan, Evert
    Smit, August B
    de Esch, Iwan J P
    Danielson, Helena
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry, Biochemistry.
    Interaction kinetic and structural dynamic analysis of ligand binding to acetylcholine-binding protein2010In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 49, no 37, p. 8143-8154Article in journal (Refereed)
    Abstract [en]

    The mechanism of agonist interactions with Cys-loop ligand-gated ion channels has been studied using the acetylcholine-binding protein (AChBP) from Lymnaea stagnalis as a model protein, and acetylcholine, nicotine, epibatidine and a series of substituted quinuclidines as ligands. A biosensor-based assay for direct interaction studies of immobilized AChBP and small molecule ligands was developed. It allowed the characterization of the interaction kinetics of the ligands and the structural dynamics of the protein. The interactions with AChBP were very sensitive to variations in the experimental conditions and showed several types of complexities. These could be resolved into two types of ligand-induced secondary effects with different kinetics, representing fast and slow conformational changes. The data could be rationalized in a mechanistic model and a structural interpretation of the interaction was obtained by molecular modelling involving induced-fit and loop flexibility simulations. The data suggests that AChBP exhibits ligand-induced structural dynamics, as expected for the ligand gating mechanism of Cys-loop receptors. It shows that the formation of the initial encounter complex between AChBP and ligands is very rapid, in accordance with the functional characteristics required of neurotransmission. These developed procedures will enable further exploration of the mechanism of Cys-loop receptor function and the identification of specific ligands suitable for pharmacological use.

  • 32. Gianni, Stefano
    et al.
    Brunori, Maurizio
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Oliveberg, Mikael
    Zhang, Mingjie
    Distinguishing between smooth and rough free energy barriers in protein folding2009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 49, p. 11825-11830Article in journal (Refereed)
    Abstract [en]

    Analysis of curved chevron plots is a powerful tool in investigating protein folding pathways, as the curvatures can be used to gain information about both early and late folding events. When and if accumulation of low-energy intermediates can be ruled out, two different models have classically been applied to describe curved chevron plots, namely , (i) Hammond effects along smooth barrier profiles and (ii) changes in the rate-limiting step between two discrete transition states. The two models lead to very similar numerical solutions, which are generally indistinguishable. This is not surprising, since the smooth barrier assumption approximates barrier profiles with a more complex topology involving multiple local maxima that are too close, or too broad, to yield clear-cut kinks in the chevron data. In this work, we have reconstructed the transition state shifts as a function of protein stability over a wide stability range for three small globular proteins, to screen for fingerprints more sensitive for different barrier profiles. We show that such an analysis represents a valuable test for the discrimination between the two different scenarios.

  • 33.
    Gustafsson, Ann
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Pettersson, Pär L.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Grehn, Leif
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Jemth, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry.
    Role of the glutamyl alpha-carboxylate of the substrate glutathione in the catalytic mechanism of human glutathione transferase A1-12001In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 40, no 51, p. 15835-15845Article in journal (Refereed)
    Abstract [en]

    The Glu alpha-carboxylate of glutathione contributes to the catalytic function of the glutathione transferases. The catalytic efficiency of human glutathione transferase A1-1 (GST A1-1) in the conjugation reaction with 1-chloro-2,4-dinitrobenzene is reduced 15 000-fold if the decarboxylated analogue of glutathione, dGSH (GABA-Cys-Gly), is used as an alternative thiol substrate. The decrease is partially due to an inability of the enzyme to promote ionization of dGSH. The pK(a) value of the thiol group of the natural substrate glutathione decreases from 9.2 to 6.7 upon binding to GST A1-1. However, the lack of the Glu alpha-carboxylate in dGSH raised the pK(a) value of the thiol in the enzymatic reaction to that of the nonenzymatic reaction. Furthermore, K(M)(dGSH) was 100-fold higher than K(M)(GSH). The active-site residue Thr68 forms a hydrogen bond to the Glu alpha-carboxylate of glutathione. Introduction of a carboxylate into GST A1-1 by a T68E mutation increased the catalytic efficiency with dGSH 10-fold and reduced the pK(a) value of the active site bound dGSH by approximately 1 pH unit. The altered pK(a) value is consistent with a catalytic mechanism where the carboxylate contributes to ionization of the glutathione thiol group. With Delta(5)-androstene-3,17-dione as substrate the efficiency of the enzyme is decreased 24 000-fold while with 4-nitrocinnamaldehyde (NCA) the decrease is less than 150-fold. In the latter reaction NCA accepts a proton and, unlike the other reactions studied, may not be dependent on the Glu alpha-carboxylate for deprotonation of the thiol group. An additional function of the Glu alpha-carboxylate may be productive orientation of glutathione within the active site.

  • 34.
    Gutiérrez-de-Terán, Hugo
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Nervall, Martin
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Ersmark, Karolina
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Liu, Peng
    Janka, Linda K.
    Dunn, Ben M.
    Hallberg, Anders
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Medicinal Chemistry.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Inhibitor binding to the Plasmepsin IV aspartic protease from Plasmodium falciparum2006In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 45, no 35, p. 10529-10541Article in journal (Refereed)
    Abstract [en]

    Plasmepsin IV (Plm IV) is one of the aspartic proteases present in the food vacuole of the malaria parasite Plasmodium falciparum involved in host hemoglobin degradation by the parasite. Using a series of previously synthesized plasmepsin inhibitors [Ersmark, K., et al. (2005) J. Med. Chem. 48, 6090-106], we report here experimental data and theoretical analysis of their inhibitory activity toward Plm IV. All compounds share a 1,2-dihydroxyethylene unit as the transition state mimic. They possess symmetric P1 and P1' side chains and either a diacylhydrazine, a five-membered oxadiazole ring, or a retroamide at the P2 and P2' positions. Experimental binding affinities are compared to those predicted by the linear interaction energy (LIE) method and an empirical scoring function, using both a crystal structure and a homology model for the enzyme. Molecular dynamics (MD) simulations of the modeled complexes allow a rational interpretation of the structural determinants for inhibitor binding. A ligand bearing a P2 and P2' symmetric oxadiazole which is devoid of amide bonds is identified both experimentally and theoretically as the most potent inhibitor of Plm IV. For the P2 and P2' asymmetric compounds, the results are consistent with earlier predictions regarding the mode of binding of this class of inhibitors to Plm II. Theoretical estimation of selectivity for some compounds is also reported. Significant features of the Plm IV binding pocket are discussed in comparison to related enzymes, and the results obtained here should be helpful for further optimization of inhibitors.

  • 35.
    Hamnevik, Emil
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Maurer, Dirk
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Enugala, Thilak Reddy
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Chu, Thao
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Löfgren, Robin
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Dobritzsch, Doreen
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Directed Evolution of Alcohol Dehydrogenase for Improved Stereoselective Redox Transformations of 1-Phenylethane-1,2-Diol and Its Corresponding Acyloin2018In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 57, p. 1059-1062Article in journal (Refereed)
    Abstract [en]

    Laboratory evolution of alcohol dehydrogenase produced enzyme variants with improved turnover numbers with a vicinal 1,2-diol and its corresponding hydroxyketone. Crystal structure and transient kinetics analysis aids in rationalizing the new functions of these variants.

  • 36. Hansson, Lars O.
    et al.
    Widersten, Mikael
    Mannervik, Bengt
    Mechanism-based phage display selection of active-site mutants of human glutathione transferase A1-1 catalyzing SNAr reactions1997In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 36, p. 11252-11260Article in journal (Refereed)
  • 37.
    Havelius, Kajsa G. V.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
    Styring, Stenbjörn
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Photochemistry and Molecular Science, Molecular Biomimetics.
    pH Dependent Competition between YZ and YD in Photosystem II Probed by Illumination at 5 K2007In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 26, p. 7865-7874Article in journal (Refereed)
    Abstract [en]

    The photosystem II (PSII) reaction center contains two redox active tyrosines, YZ and YD, situated on the D1 and D2 proteins, respectively. By illumination at 5 K, oxidation of YZ in oxygen-evolving PSII can be observed as induction of the Split S1 EPR signal from YZ* in magnetic interaction with the CaMn4 cluster, whereas oxidation of YD can be observed as the formation of the free radical EPR signal from YD*. We have followed the light induced induction at 5 K of the Split S1 signal between pH 4-8.5. The formation of the signal, that is, the oxidation of YZ, is pH independent and efficient between pH 5.5 and 8.5. At low pH, the split signal formation decreases with pKa approximately 4.7-4.9. In samples with chemically pre-reduced YD, the pH dependent competition between YZ and YD was studied. Only YZ was oxidized below pH 7.2, but at pH above 7.2, the oxidation of YD became possible, and the formation of the Split S1 signal diminished. The onset of YD oxidation occurred with pKa approximately 8.0, while the Split S1 signal decreased with pKa approximately 7.9 demonstrating that the two tyrosines compete in this pH interval. The results reflect the formation and breaking of hydrogen bonds between YZ and D1-His190 (HisZ) and YD and D2-His190 (HisD), respectively. The oxidation of respective tyrosine at 5 K demands that the hydrogen bond is well-defined; otherwise, the low-temperature oxidation is not possible. The results are discussed in the framework of recent literature data and with respect to the different oxidation kinetics of YZ and YD.

  • 38.
    Hemming, Joanna M.
    et al.
    Univ London, Dept Biol Sci, Birkbeck Coll, London WC1E 7HX, England.;Univ London, Inst Struct & Mol Biol, Birkbeck Coll, London WC1E 7HX, England..
    Hughes, Brian R.
    Univ London, Dept Biol Sci, Birkbeck Coll, London WC1E 7HX, England.;Univ London, Inst Struct & Mol Biol, Birkbeck Coll, London WC1E 7HX, England..
    Rennie, Adrian R.
    Uppsala University, Disciplinary Domain of Science and Technology, Physics, Department of Physics and Astronomy, Materials Physics.
    Tomas, Salvador
    Univ London, Dept Biol Sci, Birkbeck Coll, London WC1E 7HX, England.;Univ London, Inst Struct & Mol Biol, Birkbeck Coll, London WC1E 7HX, England..
    Campbell, Richard A.
    Inst Max Von Laue Paul Langevin, C520156, F-38042 Grenoble 9, France..
    Hughes, Arwel V.
    STFC Rutherford Appleton Lab, ISIS Pulsed Neutron & Muon Source, Didcot OX11 0QX, Oxon, England..
    Arnold, Thomas
    Diamond Light Source, Didcot OX11 0DE, Oxon, England..
    Botchway, Stanley W.
    STFC Rutherford Appleton Lab, STFC Lasers Sci Facil, Cent Laser Facil, Didcot OX11 0FA, Oxon, England..
    Thompson, Katherine C.
    Univ London, Dept Biol Sci, Birkbeck Coll, London WC1E 7HX, England.;Univ London, Inst Struct & Mol Biol, Birkbeck Coll, London WC1E 7HX, England..
    Environmental Pollutant Ozone Causes Damage to Lung Surfactant Protein B (SP-B)2015In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 54, no 33, p. 5185-5197Article in journal (Refereed)
    Abstract [en]

    Lung surfactant protein B (SP-B) is an essential protein found in the surfactant fluid at the air water interface of the lung. Exposure to the air pollutant ozone could potentially damage SP-B and lead to respiratory distress. We have studied two peptides, one consisting of the N-terminus of SP-B [SP-B(1-25)] and the other a construct of the N- and C-termini of SP-B [SP-B-(1-25,B-63-78)], called SMB. Exposure to dilute levels of ozone (similar to 2 ppm) of monolayers of each peptide at the air water interface leads to a rapid reaction, which is evident from an increase in the surface tension. Fluorescence experiments revealed that this increase in surface tension is accompanied by a loss of fluorescence from the tryptophan residue at the interface. Neutron and X-ray reflectivity experiments show that, in contrast to suggestions in the literature, the peptides are not solubilized upon oxidation but rather remain at the interface with little change in their hydration. Analysis of the product material reveals that no cleavage of the peptides occurs, but a more hydrophobic product is slowly formed together with an increased level of oligomerization. We attributed this to partial unfolding of the peptides. Experiments conducted in the presence of phospholipids reveal that the presence of the lipids does not prevent oxidation of the peptides. Our results strongly suggest that exposure to low levels of ozone gas will damage SP-B, leading to a change in its structure. The implication is that the oxidized protein will be impaired in its ability to interact at the air water interface with negatively charged phosphoglycerol lipids, thus compromising what is thought to be its main biological function.

  • 39.
    Hjelm, Rebecka
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Schedin-Weiss, Sophia
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    High affinity interaction between a synthetic, highly negatively charged pentasaccharide and alpha- or beta-antithrombin is predominantly due to nonionic interactions2007In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 11, p. 3378-3384Article in journal (Refereed)
    Abstract [en]

    Idraparinux is a synthetic O-sulfated, O-methylated pentasaccharide that binds tightly to antithrombin (AT) and thereby specifically and efficiently induces the inactivation of the procoagulant protease, factor Xa. In this study, the affinity and kinetics of the interaction of this high-affinity pentasaccharide with α- and β-AT were compared with those of a synthetic pentasaccharide comprising the natural AT-binding sequence of heparin. Dissociation equilibrium constants, Kd, for the interactions of Idraparinux with α- and β-AT were approximately 0.4 and 0.1 nM, respectively, corresponding to an over 100-fold enhancement in affinity compared with that of the normal pentasaccharide. This large enhancement was due to a 400-fold tighter conformationally activated complex formed in the second binding step, whereas the encounter complex established in the first step was 4-fold weaker. The high-affinity and normal pentasaccharides both made a total of four ionic interactions with AT, although the high-affinity saccharide only established one ionic interaction in the first binding step and was compensated by three in the second step, whereas the normal pentasaccharide established two ionic interactions in each step. In contrast, the affinities of the nonionic interactions (Kd 450 and 90 nM for the binding to α- and β-AT, respectively) were considerably higher than those for the normal pentasaccharide and the highest of all AT−saccharide interactions reported so far. The nonionic contribution to the total free energy of the high-affinity pentasaccharide binding to AT thus amounted to 70%. These findings show that nonionic interactions can play a predominant role in the binding of highly charged saccharide ligands to proteins and can be successfully exploited in the design of such biologically active ligands.

  • 40.
    Honcharenko, Dmytro
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Barman, Jharna
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Varghese, Oommen P.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Chattopadhyaya, Jyoti
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Bioorganic Chemistry.
    Comparison of the RNase H Cleavage Kinetics and Blood Serum Stability of the North-Conformationally Constrained and 2‘-Alkoxy Modified Oligonucleotides2007In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 19, p. 5635-5646Article in journal (Refereed)
    Abstract [en]

    The RNase H cleavage potential of the RNA strand basepaired with the complementary antisense oligonucleotides (AONs) containing NorthEast conformationally constrained 1‘,2‘-methylene-bridged (azetidine-T and oxetane-T) nucleosides, North-constrained 2‘,4‘-ethylene-bridged (aza-ENA-T) nucleoside, and 2‘-alkoxy modified nucleosides (2‘-O-Me-T and 2‘-O-MOE-T modifications) have been evaluated and compared under identical conditions. When compared to the native AON, the aza-ENA-T modified AON/RNA hybrid duplexes showed an increase of melting temperature (ΔTm = 2.5−4 °C per modification), depending on the positions of the modified residues. The azetidine-T modified AONs showed a drop of 4−5.5 °C per modification with respect to the native AON/RNA hybrid, whereas the isosequential oxetane-T modified counterpart, showed a drop of 5−6 °C per modification. The 2‘-O-Me-T and 2‘-O-MOE-T modifications, on the other hand, showed an increased of Tm by 0.5 °C per modification in their AON/RNA hybrids. All of the partially modified AON/RNA hybrid duplexes were found to be good substrates for the RNase H mediated cleavage. The Km and Vmax values obtained from the RNA concentration-dependent kinetics of RNase H promoted cleavage reaction for all AON/RNA duplexes with identical modification site were compared with those of the reference native AON/RNA hybrid duplex. The catalytic activities (Kcat) of RNase H were found to be greater (1.4−2.6-fold) for all modified AON/RNA hybrids compared to those for the native AON/RNA duplex. However, the RNase H binding affinity (1/Km) showed a decrease (1.7−8.3-fold) for all modified AON/RNA hybrids. This resulted in less effective (1.1−3.2-fold) enzyme activity (Kcat/Km) for all modified AON/RNA duplexes with respect to the native counterpart. A stretch of five to seven nucleotides in the RNA strand (from the site of modifications in the complementary modified AON strand) was found to be resistant to RNase H digestion (giving a footprint) in the modified AON/RNA duplex. Thus, (i) the AON modification with azetidine-T created a resistant region of five to six nucleotides, (ii) modification with 2‘-O-Me-T created a resistant stretch of six nucleotides, (iii) modification with aza-ENA-T created a resistant region of five to seven nucleotide residues, whereas (iv) modification with 2‘-O-MOE-T created a resistant stretch of seven nucleotide residues. This shows the variable effect of the microstructure perturbation in the modified AON/RNA heteroduplex depending upon the chemical nature as well as the site of modifications in the AON strand. On the other hand, the enhanced blood serum as well as the 3‘-exonuclease stability (using snake venom phosphodiesterase, SVPDE) showed the effect of the tight conformational constraint in the AON with aza-ENA-T modifications in that the 3‘-exonuclease preferentially hydrolyzed the 3‘-phosphodiester bond one nucleotide away (n + 1) from the modification site (n) compared to all other modified AONs, which were 3‘-exonuclease cleaved at the 3‘-phosphodiester of the modification site (n). The aza-ENA-T modification in the AONs made the 5‘-residual oligonucleotides (including the n + 1 nucleotide) highly resistant in the blood serum (remaining after 48 h) compared to the native AON (fully degraded in 2 h). On the other hand, the 5‘-residual oligonucleotides (including the n nucleotide) in azetidine-T, 2‘-O-Me-T, and 2‘-O-MOE-T modified AONs were more stable compared to that of the native counterpart but more easily degradable than that of aza-ENA-T containing AONs.

  • 41.
    Isaksen, Geir Villy
    et al.
    Univ Tromso, Fac Sci & Technol, Dept Chem, Ctr Theoret & Computat Chem, N-9037 Tromso, Norway..
    Hopmann, Kathrin Helen
    Univ Tromso, Fac Sci & Technol, Dept Chem, Ctr Theoret & Computat Chem, N-9037 Tromso, Norway..
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Brandsdal, Björn Olav
    Univ Tromso, Fac Sci & Technol, Dept Chem, Ctr Theoret & Computat Chem, N-9037 Tromso, Norway..
    Computer Simulations Reveal Substrate Specificity of Glycosidic Bond Cleavage in Native and Mutant Human Purine Nucleoside Phosphorylase2016In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 55, no 14, p. 2153-2162Article in journal (Refereed)
    Abstract [en]

    Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine ribonucleosides and 2'-deoxyribonucleosides, yielding the purine base and (2'deoxy)ribose 1-phosphate as products. While this enzyme has been extensively studied, several questions with respect to the catalytic mechanism have remained largely unanswered. The role of the phosphate and key amino acid residues in the catalytic reaction as well as the purine ring protonation state is elucidated using density functional theory calculations and extensive empirical valence bond (EVB) simulations. Free energy surfaces for adenosine, inosine, and guanosine are fitted to ab initio data and yield quantitative agreement with experimental data when the surfaces are used to model the corresponding enzymatic reactions. The cognate substrates 6-aminopurines (inosine and guanosine) interact with PNP through extensive hydrogen bonding, but the substrate specificity is found to be a direct result of the electrostatic preorganization energy along the reaction coordinate. Asn243 has previously been identified as a key residue providing substrate specificity. Mutation of Asn243 to Asp has dramatic effects on the substrate specificity, making 6-amino- and 6-oxopurines equally good as substrates. The principal effect of this particular mutation is the change in the electrostatic preorganization energy between the native enzyme and the Asn243Asp mutant, clearly favoring adenosine over inosine and guanosine. Thus, the EVB simulations show that this particular mutation affects the electrostatic preorganization of the active site, which in turn can explain the substrate specificity.

  • 42.
    Isaksen, Geir Villy
    et al.
    Univ Tromso, Dept Chem, Ctr Theoret & Computat Chem, NO-9037 Tromso, Norway..
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Computational Biology and Bioinformatics.
    Brandsdal, Björn Olav
    Univ Tromso, Dept Chem, Ctr Theoret & Computat Chem, NO-9037 Tromso, Norway..
    Thermodynamics of the Purine Nucleoside Phosphorylase Reaction Revealed by Computer Simulations2017In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 56, no 1, p. 306-312Article in journal (Refereed)
    Abstract [en]

    Enzymes are able to catalyze chemical reactions by reducing the activation free energy, yielding significant increases in the reaction rates. This can thermodynamically be accomplished by either reducing the activation enthalpy or increasing the activation entropy. The effect of remote mutations on the thermodynamic activation parameters of human purine nucleoside phosphorylase is examined using extensive molecular dynamics and free energy simulations. More than 2700 independent reaction free energy profiles for six different temperatures have been calculated to obtain high-precision computational Arrhenius plots. On the basis of these, the activation enthalpies and entropies were computed from linear regression of the plots with Delta G(double dagger) as a function of 1/T, and the obtained thermodynamic activation parameters are in very good agreement with those from experiments. The Arrhenius plots immediately show that the 6-oxopurines (INO and GUO) have identical slopes, whereas the 6-aminopurine (ADO) has a significantly different slope, indicating that the substrate specificity is related to the difference in thermodynamic activation parameters. Furthermore, the calculations show that the human PNP specificity for 6-oxopurines over 6-aminopurines originates from significant differences in electrostatic preorganization. The effect of the remote double mutation, K22E and H104R (E:R), has also been examined, as it alters human PNP toward the bovine PNP. These residues are situated on the protein surface, 28-35 angstrom from the active site, and the mutation alters the enthalpy entropy balance with little effect on the catalytic rates. It is thus quite remarkable that the empirical valence bond method can reproduce the enthalpies and entropies induced by these long-range mutations.

  • 43.
    Janfalk Carlsson, Åsa
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Bauer, Paul
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Ma, Huan
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Widersten, Mikael
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.
    Obtaining optical purity for product diols in enzyme-catalyzed epoxide hydrolysis: contributions from changes in both enantio- and regioselectivity2012In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 51, no 38, p. 7627-7637Article in journal (Refereed)
    Abstract [en]

    Enzyme variants of the plant epoxide hydrolase StEH1 displaying improved stereoselectivities in the catalyzed hydrolysis of (2,3-epoxypropyl)benzene were generated by directed evolution. The evolution was driven by iterative saturation mutagenesis in combination with enzyme activity screenings where product chirality was the decisive selection criterion. Analysis of the underlying causes of the increased diol product ratios revealed two major contributing factors: increased enantioselectivity for the corresponding epoxide enantiomer(s) and, in some cases, a concomitant change in regioselectivity in the catalyzed epoxide ring-opening half-reaction. Thus, variant enzymes that catalyzed the hydrolysis of racemic (2,3-epoxypropyl)benzene into the R-diol product in an enantioconvergent manner were isolated.

  • 44.
    Jemth, Per
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Gianni, Stefano
    PDZ domains: folding and binding2007In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 30, p. 8701-8708Article in journal (Refereed)
    Abstract [en]

    The PDZ domain is one of the most common protein-protein interaction domains in humans, and it is found in all kingdoms of life. We will review recent progress in the understanding of biophysical aspects of PDZ domains with emphasis on the folding and binding reactions. Finally, we discuss an intriguing correlation between stability and binding of peptide for PDZ2 from PTP-BL.

  • 45.
    Johansson, Carolina
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Boukharta, Lars
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Eriksson, Jens
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Cell Biology.
    Åqvist, Johan
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology.
    Sundström, Lars
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Medical Biochemistry and Microbiology.
    Mutagenesis and homology modelling of the Tn21 integron integrase IntI12009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 8, p. 1743-1753Article in journal (Refereed)
    Abstract [en]

    Horizontal DNA transfer between bacteria is widespread and a major cause of antibiotic resistance. For logistic reasons, single or combined genes are shuttled between vectors such as plasmids and   bacterial chromosomes. Special elements termed integrons operate in such shuttling and are therefore vital for horizontal gene transfer. Shorter elements carrying genes, cassettes, are integrated in the integrons, or excised from them, by virtue of a recombination site, attC, positioned in the 3' end of each unit. It is a remarkable and   possibly restricting elementary feature of attC that it must be single-stranded while the partner target site, attI, may be double-stranded. The integron integrases belong to the tyrosine recombinase family, and this work reports mutations of the integrase IntI1 from transposon Tn21, chosen within a well-conserved region characteristic of the integron integrases. The mutated proteins were  tested for binding to a bottom strand of an attC substrate, by using an electrophoresis mobility shift assay. To aid in interpreting the   results, a homology model was constructed on the basis of the crystal structure of integron integrase VchIntIA from Vibrio cholerae bound to its cognate attC substrate VCRbs. The local stability and hydrogen bonding network of key domains of the modeled structure were further examined using molecular dynamics simulations. The homology model allowed us to interpret the roles of several amino acid residues, four of which were clearly binding assay responsive upon mutagenesis. Notably, we also observed features indicating that IntI1 may be more prone to base-specific contacts with VCRbs than VchIntIA.

  • 46. Johnson, W. W.
    et al.
    Ueng, Y.-F.
    Widersten, Mikael
    Mannervik, Bengt
    Hayes, John D.
    Sherratt, P. J.
    Ketterer, Brian
    Guengerich, F. P.
    Conjugation of highly reactive aflatoxin B1 exo-8,9-epoxide catalyzed by rat and human glutathione transferases: Estimation of kinetic parameters1997In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 36, p. 3056-3060Article in journal (Refereed)
  • 47. Karkehabadi, Saeid
    et al.
    Satagopan, Sriram
    Taylor, Thomas C
    Spreitzer, Robert J
    Andersson, Inger
    Department of Molecular Biology, Swedish University of Agricultural Sciences.
    Structural analysis of altered large-subunit loop-6/carboxy-terminus interactions that influence catalytic efficiency and CO2/O2 specificity of ribulose-1,5-bisphosphate carboxylase/oxygenase.2007In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 46, no 39, p. 11080-11089Article in journal (Refereed)
    Abstract [en]

    The loop between alpha-helix 6 and beta-strand 6 in the alpha/beta-barrel of ribulose-1,5-bisphosphate carboxylase/oxygenase plays a key role in discriminating between CO2 and O2. Genetic screening in Chlamydomonas reinhardtii previously identified a loop-6 V331A substitution that decreases carboxylation and CO2/O2 specificity. Revertant selection identified T342I and G344S substitutions that restore photosynthetic growth by increasing carboxylation and specificity of the V331A enzyme. In numerous X-ray crystal structures, loop 6 is closed or open depending on the activation state of the enzyme and the presence or absence of ligands. The carboxy terminus folds over loop 6 in the closed state. To study the molecular basis for catalysis, directed mutagenesis and chloroplast transformation were used to create T342I and G344S substitutions alone. X-ray crystal structures were then solved for the V331A, V331A/T342I, T342I, and V331A/G344S enzymes, as well as for a D473E enzyme created to assess the role of the carboxy terminus in loop-6 closure. V331A disturbs a hydrophobic pocket, abolishing several van der Waals interactions. These changes are complemented by T342I and G344S, both of which alone cause decreases in CO2/O2 specificity. In the V331A/T342I revertant enzyme, Arg339 main-chain atoms are displaced. In V331A/G344S, alpha-helix 6 is shifted. D473E causes disorder of the carboxy terminus, but loop 6 remains closed. Interactions between a transition-state analogue and several residues are altered in the mutant enzymes. However, active-site Lys334 at the apex of loop 6 has a normal conformation. A variety of subtle interactions must be responsible for catalytic efficiency and CO2/O2 specificity.

  • 48.
    Knight, Stefan D.
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Biology, Department of Cell and Molecular Biology, Structure and Molecular Biology.
    Presto, Jenny
    Linse, Sara
    Johansson, Jan
    The BRICHOS Domain, Amyloid Fibril Formation, and Their Relationship2013In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 52, no 43, p. 7523-7531Article in journal (Refereed)
    Abstract [en]

    Amyloid diseases are defined by tissue deposition of insoluble, fibrillar beta-sheet polymers of specific proteins, but it appears that toxic oligomeric species rather than the fibrils are the main cause of tissue degeneration. Many proteins can form amyloid-like fibrils in vitro, but only similar to 30 proteins have been found to cause mammalian amyloid disease, suggesting that physiological mechanisms that protect against amyloid formation exist. The transmembrane region of lung surfactant protein C precursor (proSP-C) forms amyloid-like fibrils in vitro, and SP-C amyloid has been found in lung tissue from patients with interstitial lung disease (ILD). ProSP-C contains a BRICHOS domain, in which many ILD-associated mutations are localized, and the BRICHOS domain can prevent SP-C from forming amyloid-like fibrils. Recent data suggest that recombinant BRICHOS domains from proSP-C and Bri2 (associated with familial dementia and amyloid formation) interact with peptides with a strong propensity to form beta-sheet structures, including amyloid beta-peptide associated with Alzheimer's disease. Such interactions efficiently delay formation of fibrils and oligomers. The BRICHOS domain is defined at the sequence level and is found in similar to 10 distantly related proprotein families. These have widely different or unknown functions, but several of the proteins are associated with human disease. Structural modeling of various BRICHOS domains, based on the X-ray structure of the proSP-C BRICHOS domain, identifies a conserved region that is structurally complementary to the beta-sheet- and/or amyloid-prone regions in the BRICHOS domain-containing proproteins. These observations make the BRICHOS domain the first example of a chaperone-like domain with specificity for beta-prone regions.

  • 49. Koutsodontis, George
    et al.
    Moustakas, Aristidis
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Medicinska och farmaceutiska vetenskapsområdet, centrumbildningar mm, Ludwig Institute for Cancer Research.
    Kardassis, Dimitris
    The role of Sp1 family members, the proximal GC-rich motifs, and the upstream enhancer region in the regulation of the human cell cycle inhibitor p21WAF-1/Cip1 gene promoter2002In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 41, no 42, p. 12771-12784Article in journal (Refereed)
    Abstract [en]

    In the present study we establish that specific members of the Sp1 family of transcription factors (Sp1 and Sp3) bind to all six GC-rich motifs (elements 1-6) present in the proximal promoter of the human cell cycle inhibitor p21(WAF-1/Cip1) gene. Competition analysis showed that Sp1 and Sp3 bound with high affinity to elements 1, 3, 4, and 5/6 and with lower affinity to element 2. Transfection experiments in the Sp1-deficient Drosophila SL2 cells established that Sp1 and Sp3 but not Sp2 are potent transactivators of the p21 promoter. Transactivation by Sp1 was compromised either by deletion of element 1 (-119/-114) or by using a truncated Sp1 form lacking the C-terminal regulatory domain D. Point mutagenesis of the -2325/+8 p21 promoter, targeting individual elements 1-6, showed that mutations in element 3 (-82/-77) caused a dramatic reduction (90%) in p21 promoter activity whereas mutations in other elements had a less severe effect. The mutations in element 3 abolished p21 promoter induction by upstream enhancer elements in HepG2 cells. Sp1, but not Sp3, mediated the transactivation of the p21 promoter by the TGFbeta signaling mediator Smad3 and Smad4 proteins whereas none of the individual mutations in elements 1-6 affected the transactivation of the p21 promoter by Smad proteins in HepG2 cells. Our results suggest that functional interactions between Sp1 family members bound to specific elements of the proximal promoter and factors bound to distal enhancer elements govern the hepatic activity of the human p21 promoter under basal or inducible conditions.

  • 50.
    Kurtovic, Sanela
    et al.
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Mannervik, Bengt
    Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Biochemistry and Organic Chemistry.
    Identification of Emerging Quasi-Species in Directed Enzyme Evolution2009In: Biochemistry, ISSN 0006-2960, E-ISSN 1520-4995, Vol. 48, no 40, p. 9330-9339Article in journal (Refereed)
    Abstract [en]

    The bases of enzyme evolution are structural changes in protein scaffolds combined with recognition and propagation of novel variants with valuable functional properties. Structural diversification may be accomplished by a variety of methods, including random mutations, homologous recombinations, and insertions and deletions of coding DNA sequences. The functional consequences of mutations are manifested at the protein level and are dependent on a substrate matrix, when catalytic properties are requested. Libraries of variant enzymes showing promiscuous activities can be interrogated with a set of alternative substrates. We demonstrate using a library of glutathione transferases (GSTs) that the functional properties are not uniformly distributed in substrate-activity space but form clusters, or quasi-species. Multivariate analysis facilitates the identification of such quasi-species, which can be regarded as the proper developing units in molecular evolution.

123 1 - 50 of 106
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