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Computational Modeling of the Structure, Function and Dynamics of Biomolecular Systems
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Biochemistry.ORCID iD: 0000-0002-7643-9867
2020 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
Abstract [en]

Proteins are a structurally diverse and functionally versatile class of biomolecules. They perform a variety of life-sustaining biological processes with utmost efficiency. A profound understanding of protein function requires knowledge of its structure. Experimentally determined protein structures can serve as a starting point for computer simulations in order to study their dynamic behavior at a molecular level. In this thesis, computational methods have been used to understand structure-function relationships in two classes of proteins - intrinsically disordered proteins (IDP) and enzymes.

Misfolding and subsequent aggregation of the amyloid beta (Aβ) peptide, an IDP, is associated with the progression of Alzheimer’s disease. Besides enriching our understanding of structural dynamics, computational studies on a medically relevant IDP such as Aβ can potentially guide therapeutic development. In the present work, binding interactions of the monomeric form of this peptide with biologically relevant molecular species such as divalent metal ions (Zn2+, Cu2+, Mn2+) and amphiphilic surfactants were characterized using long timescale molecular dynamics (MD) simulations. Among the metal ions, while Zn2+ and Cu2+ maintained coordination to a well-defined binding site in Aβ, Mn2+-binding was observed to be comparatively weak and transient. Surfactants with charged headgroups displayed strong binding interaction with Aβ. Complemented by biophysical experiments, these studies provided a multifaceted perspective of Aβ interactions with the partner molecules.

Triosephosphate isomerase (TIM), a highly evolved and catalytically proficient enzyme, was studied using empirical valence bond (EVB) calculations to obtain deeper insights into the catalytic reaction mechanism. Multiple structural features of TIM such as the flexible loop and preorganized active site residues were investigated for their role in enzyme catalysis. The effect of substrate binding was also studied using truncated substrates. Finally, using enhanced sampling methods, dynamic behavior of the catalytically important loop 6 was characterized. The importance of structural stability and flexibility on protein function was illustrated by the work presented in this thesis, thus furthering our scientific understanding of proteins at a molecular level.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2020. , p. 72
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1885
Keywords [en]
Molecular Dynamics, Empirical Valence Bond, Enzyme Catalysis, Amyloid Beta, Aβ, Triosephosphate Isomerase, TIM, Computational Biochemistry, Computational Enzymology
National Category
Biochemistry and Molecular Biology Theoretical Chemistry
Research subject
Biochemistry
Identifiers
URN: urn:nbn:se:uu:diva-398169ISBN: 978-91-513-0828-9 (print)OAI: oai:DiVA.org:uu-398169DiVA, id: diva2:1375418
Public defence
2020-02-05, B21, BMC, Husargatan 3, Uppsala, 13:15 (English)
Opponent
Supervisors
Available from: 2020-01-14 Created: 2019-12-04 Last updated: 2020-01-14
List of papers
1. Characterization of Mn(II) ion binding to the amyloid-beta peptide in Alzheimer's disease
Open this publication in new window or tab >>Characterization of Mn(II) ion binding to the amyloid-beta peptide in Alzheimer's disease
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2016 (English)In: Journal of Trace Elements in Medicine and Biology, ISSN 0946-672X, E-ISSN 1878-3252, Vol. 38, p. 183-193Article in journal (Refereed) Published
Abstract [en]

Growing evidence links neurodegenerative diseases to metal exposure. Aberrant metal ion concentrations have been noted in Alzheimer's disease (AD) brains, yet the role of metals in AD pathogenesis remains unresolved. A major factor in AD pathogenesis is considered to be aggregation of and amyloid formation by amyloid-beta (A beta) peptides. Previous studies have shown that A beta displays specific binding to Cu(II) and Zn(II) ions, and such binding has been shown to modulate A beta aggregation. Here, we use nuclear magnetic resonance (NMR) spectroscopy to show that Mn(II) ions also bind to the N-terminal part of the A beta(1-40) peptide, with a weak binding affinity in the milli- to micromolar range. Circular dichroism (CD) spectroscopy, solid state atomic force microscopy (AFM), fluorescence spectroscopy, and molecular modeling suggest that the weak binding of Mn(II) to A beta may not have a large effect on the peptide's aggregation into amyloid fibrils. However, identification of an additional metal ion displaying A beta binding reveals more complex AD metal chemistry than has been previously considered in the literature.

Keywords
Manganese, Neurodegeneration, Metal-protein binding, Spectroscopy, Molecular dynamics
National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-308063 (URN)10.1016/j.jtemb.2016.03.009 (DOI)000385473600023 ()27085215 (PubMedID)
Available from: 2016-11-30 Created: 2016-11-23 Last updated: 2019-12-04Bibliographically approved
2. Amyloid-beta Peptide Interactions with Amphiphilic Surfactants: Electrostatic and Hydrophobic Effects
Open this publication in new window or tab >>Amyloid-beta Peptide Interactions with Amphiphilic Surfactants: Electrostatic and Hydrophobic Effects
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2018 (English)In: ACS Chemical Neuroscience, ISSN 1948-7193, E-ISSN 1948-7193, Vol. 9, no 7, p. 1680-1692Article in journal (Refereed) Published
Abstract [en]

The amphiphilic nature of the amyloid-beta (A beta) peptide associated with Alzheimer's disease facilitates various interactions with biomolecules such as lipids and proteins, with effects on both structure and toxicity of the peptide. Here, we investigate these peptide-amphiphile interactions by experimental and computational studies of A beta(1-40) in the presence of surfactants with varying physicochemical properties. Our findings indicate that electrostatic peptide-surfactant interactions are required for coclustering and structure induction in the peptide and that the strength of the interaction depends on the surfactant net charge. Both aggregation-prone peptide-rich coclusters and stable surfactant-rich coclusters can form. Only A beta(1-40) monomers, but not oligomers, are inserted into surfactant micelles in this surfactant-rich state. Surfactant headgroup charge is suggested to be important as electrostatic peptide-surfactant interactions on the micellar surface seems to be an initiating step toward insertion. Thus, no peptide insertion or change in peptide secondary structure is observed using a nonionic surfactant. The hydrophobic peptide-surfactant interactions instead stabilize the A beta monomer, possibly by preventing self-interaction between the peptide core and C terminus, thereby effectively inhibiting the peptide aggregation process. These findings give increased understanding regarding the molecular driving forces for A beta aggregation and the peptide interaction with amphiphilic biomolecules.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC, 2018
Keywords
Alzheimer's disease, A beta aggregation, surfactant interactions, optical and NMR spectroscopy, mass spectrometry, molecular dynamics simulations
National Category
Neurosciences
Identifiers
urn:nbn:se:uu:diva-386225 (URN)10.1021/acschemneuro.8b00065 (DOI)000439531400017 ()29683649 (PubMedID)
Funder
Swedish Research Council
Available from: 2019-06-19 Created: 2019-06-19 Last updated: 2019-12-04Bibliographically approved
3. Enzyme Architecture: Modeling the Operation of a Hydrophobic Clamp in Catalysis by Triosephosphate Isomerase
Open this publication in new window or tab >>Enzyme Architecture: Modeling the Operation of a Hydrophobic Clamp in Catalysis by Triosephosphate Isomerase
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2017 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 139, no 30, p. 10514-10525Article in journal (Refereed) Published
Abstract [en]

Triosephosphate isomerase (TIM) is a proficient catalyst of the reversible isomerization of dihydroxyacetone phosphate (DHAP) to D-glyceraldehyde phosphate (GAP), via general base catalysis by E165. Historically, this enzyme has been an extremely important model system for understanding the fundamentals of biological catalysis. TIM is activated through an energetically demanding conformational change, which helps position the side chains of two key hydrophobic residues (1170 and L230), over the carboxylate side chain of E165. This is critical both for creating a hydrophobic pocket for the catalytic base and for maintaining correct active site architecture. Truncation of these residues to alanine causes significant falloffs in TIM's catalytic activity, but experiments have failed to provide a full description of the action of this clamp in promoting substrate deprotonation. We perform here detailed empirical valence bond calculations of the TIM-catalyzed deprotonation of DHAP and GAP by both wild type TIM and its 1170A, L230A, and 1170A/L230A mutants, obtaining exceptional quantitative agreement with experiment. Our calculations provide a linear free energy relationship, with slope 0.8, between the activation barriers and Gibbs free energies for these TIM-catalyzed reactions. We conclude that these clamping side chains minimize the Gibbs free energy for substrate deprotonation, and that the effects on reaction driving force are largely expressed at the transition state for proton transfer. Our combined analysis of previous experimental and current computational results allows us to provide an overview of the breakdown of ground-state and transition state effects in enzyme catalysis in unprecedented detail, providing a molecular description of the operation of a hydrophobic clamp in triosephosphate isomerase.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2017
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-334051 (URN)10.1021/jacs.7b05576 (DOI)000407089500046 ()28683550 (PubMedID)
Available from: 2017-11-21 Created: 2017-11-21 Last updated: 2019-12-04Bibliographically approved
4. Uncovering the Role of Key Active-Site Side Chains in Catalysis: An Extended Brønsted Relationship for Substrate Deprotonation Catalyzed by Wild-Type and Variants of Triosephosphate Isomerase
Open this publication in new window or tab >>Uncovering the Role of Key Active-Site Side Chains in Catalysis: An Extended Brønsted Relationship for Substrate Deprotonation Catalyzed by Wild-Type and Variants of Triosephosphate Isomerase
2019 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 141, no 40, p. 16139-16150Article in journal (Refereed) Published
Abstract [en]

We report results of detailed empirical valence bond simulations that model the effect of several amino acid substitutions on the thermodynamic (ΔG°) and kinetic activation (ΔG) barriers to deprotonation of dihydroxyacetone phosphate (DHAP) and d-glyceraldehyde 3-phosphate (GAP) bound to wild-type triosephosphate isomerase (TIM), as well as to the K12G, E97A, E97D, E97Q, K12G/E97A, I170A, L230A, I170A/L230A, and P166A variants of this enzyme. The EVB simulations model the observed effect of the P166A mutation on protein structure. The E97A, E97Q, and E97D mutations of the conserved E97 side chain result in ≤1.0 kcal mol–1 decreases in the activation barrier for substrate deprotonation. The agreement between experimental and computed activation barriers is within ±1 kcal mol–1, with a strong linear correlation between ΔG and Δ for all 11 variants, with slopes β = 0.73 (R2 = 0.994) and β = 0.74 (R2 = 0.995) for the deprotonation of DHAP and GAP, respectively. These Brønsted-type correlations show that the amino acid side chains examined in this study function to reduce the standard-state Gibbs free energy of reaction for deprotonation of the weak α-carbonyl carbon acid substrate to form the enediolate phosphate reaction intermediate. TIM utilizes the cationic side chain of K12 to provide direct electrostatic stabilization of the enolate oxyanion, and the nonpolar side chains of P166, I170, and L230 are utilized for the construction of an active-site cavity that provides optimal stabilization of the enediolate phosphate intermediate relative to the carbon acid substrate.

National Category
Theoretical Chemistry
Identifiers
urn:nbn:se:uu:diva-397177 (URN)10.1021/jacs.9b08713 (DOI)000490358900049 ()31508957 (PubMedID)
Funder
Swedish Research Council, 2015-04298NIH (National Institute of Health), GM03597NIH (National Institute of Health), GM116921Knut and Alice Wallenberg Foundation, 2013.0124Knut and Alice Wallenberg Foundation, 2018.0140Swedish National Infrastructure for Computing (SNIC), 2017/12-11Swedish National Infrastructure for Computing (SNIC), 2018/2-3
Available from: 2019-11-16 Created: 2019-11-16 Last updated: 2019-12-06Bibliographically approved
5. Role of Ligand-Driven Conformational Changes in Enzyme Catalysis: Modeling the Reactivity of the Catalytic Cage of Triosephosphate Isomerase
Open this publication in new window or tab >>Role of Ligand-Driven Conformational Changes in Enzyme Catalysis: Modeling the Reactivity of the Catalytic Cage of Triosephosphate Isomerase
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2018 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 140, no 11, p. 3854-3857Article in journal (Refereed) Published
Abstract [en]

We have previously performed empirical valence bond calculations of the kinetic activation barriers, Delta G(calc) double dagger, for the deprotonation of complexes between TIM and the whole substrate glyceraldehyde-3-phosphate (GAP, Kulkarni et al. J. Am. Chem. Soc. 2017, 139, 10514-10525). We now extend this work to also study the deprotonation of the substrate pieces glycolaldehyde (GA) and GA.HPi [HPi = phosphite dianion]. Our combined calculations provide activation barriers, Delta G(calc)(double dagger) for the TIM-catalyzed deprotonation of GAP (12.9 +/- 0.8 kcal.mol(-1)), of the substrate piece GA (15.0 +/- 2.4 kcal.mol(-1)), and of the pieces GA.HP, (15.5 +/- 3.5 kcal.mol(-1)). The effect of bound dianion on Delta G(calc) double dagger is small (<= 2.6 kcal.mol(-1)), in comparison to the much larger 12.0 and 5.8 kcal.mol(-1) intrinsic phosphodianion and phosphite dianion binding energy utilized to stabilize the transition states for TIM-catalyzed deprotonation of GAP and GA. HP, respectively. This shows that the dianion binding energy is essentially fully expressed at our protein model for the Michaelis complex, where it is utilized to drive an activating change in enzyme conformation. The results represent an example of the synergistic use of results from experiments and calculations to advance our understanding of enzymatic reaction mechanisms.

Place, publisher, year, edition, pages
American Chemical Society (ACS), 2018
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-354362 (URN)10.1021/jacs.8b00251 (DOI)000428356000010 ()29516737 (PubMedID)
Funder
Swedish Research Council, 2015-04928
Available from: 2018-06-25 Created: 2018-06-25 Last updated: 2019-12-04Bibliographically approved
6. Loop Motion in Triosephosphate Isomerase Is Not a Simple Open and Shut Case
Open this publication in new window or tab >>Loop Motion in Triosephosphate Isomerase Is Not a Simple Open and Shut Case
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2018 (English)In: Journal of the American Chemical Society, ISSN 0002-7863, E-ISSN 1520-5126, Vol. 140, no 46, p. 15889-15903Article in journal (Refereed) Published
Abstract [en]

Conformational changes are crucial for the catalytic action of many enzymes. A prototypical and well-studied example is loop opening and closure in triosephosphate isomerase (TIM), which is thought to determine the rate of catalytic turnover in many circumstances. Specifically, TIM loop 6 “grips” the phosphodianion of the substrate and, together with a change in loop 7, sets up the TIM active site for efficient catalysis. Crystal structures of TIM typically show an open or a closed conformation of loop 6, with the tip of the loop moving ∼7 Å between conformations. Many studies have interpreted this motion as a two-state, rigid-body transition. Here, we use extensive molecular dynamics simulations, with both conventional and enhanced sampling techniques, to analyze loop motion in apo and substrate-bound TIM in detail, using five crystal structures of the dimeric TIM from Saccharomyces cerevisiae. We find that loop 6 is highly flexible and samples multiple conformational states. Empirical valence bond simulations of the first reaction step show that slight displacements away from the fully closed-loop conformation can be sufficient to abolish most of the catalytic activity; full closure is required for efficient reaction. The conformational change of the loops in TIM is thus not a simple “open and shut” case and is crucial for its catalytic action. Our detailed analysis of loop motion in a highly efficient enzyme highlights the complexity of loop conformational changes and their role in biological catalysis.

National Category
Biochemistry and Molecular Biology
Research subject
Biochemistry
Identifiers
urn:nbn:se:uu:diva-367313 (URN)10.1021/jacs.8b09378 (DOI)000451496800048 ()30362343 (PubMedID)
Funder
Swedish Research CouncilSwedish National Infrastructure for Computing (SNIC), 2016/1-293Swedish National Infrastructure for Computing (SNIC), 2017/12-11
Available from: 2018-11-29 Created: 2018-11-29 Last updated: 2019-12-04Bibliographically approved

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