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Balliu, A. & Baltzer, L. (2017). Conjugation of a Dipicolyl Chelate to Polypeptide Conjugates Increases Binding Affinities for Human Serum Albumin and Survival Times in Human Serum. ChemBioChem (Print), 18(14), 1408-1414
Open this publication in new window or tab >>Conjugation of a Dipicolyl Chelate to Polypeptide Conjugates Increases Binding Affinities for Human Serum Albumin and Survival Times in Human Serum
2017 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 18, no 14, p. 1408-1414Article in journal (Refereed) Published
Abstract [en]

The affinity for human serum albumin (HSA) of a series of 2–5 kDa peptides covalently linked to 3,5-bis[[bis(2-pyridylmethyl)amino]methyl]benzoic acid, a dipicolyl chelator with micromolar affinity for Zn2+, was found by surface plasmon resonance to increase in the presence of 1 μm ZnCl2 at physiological pH. The dependence on polypeptide hydrophobicity was found to be minor, thus suggesting that the conjugates bound to the metal-binding site and not to the fatty-acid-binding site. The affinity of the conjugates increased strongly with the positive charge of the polypeptides, thus implicating the negatively charged protein surface surrounding the metal-binding site. The survival times of the peptides in human serum were extended as a consequence of stronger binding to HSA, thus suggesting that Zn2+-chelating agents might provide a general route to increased survival time of peptides in serum in therapeutic and diagnostic applications without significantly increasing their molecular weights.

Keywords
affinity, biosensor, human serum albumin, peptides, zinc-binding site
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-326838 (URN)10.1002/cbic.201700049 (DOI)000405726100010 ()28301711 (PubMedID)
Available from: 2017-07-31 Created: 2017-07-31 Last updated: 2017-11-02Bibliographically approved
Balliu, A. & Baltzer, L. (2017). Exploring Non-obvious Hydrophobic Binding Pockets on Protein Surfaces: Increasing Affinities in Peptide–Protein Interactions. ChemBioChem (Print), 18(14), 1396-1407
Open this publication in new window or tab >>Exploring Non-obvious Hydrophobic Binding Pockets on Protein Surfaces: Increasing Affinities in Peptide–Protein Interactions
2017 (English)In: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 18, no 14, p. 1396-1407Article in journal (Refereed) Published
Abstract [en]

A 42-residue polypeptide conjugated to a small-molecule organic ligand capable of targeting the phosphorylated side chain of Ser15 was shown to bind glycogen phosphorylase a (GPa) with a KD value of 280 nm. The replacement of hydrophobic amino acids by Ala reduced affinities, whereas the incorporation of l-2-aminooctanoic acid (Aoc) increased them. Replacing Nle5, Ile9 and Leu12 by Aoc reduced the KD value from 280 to 27 nm. “Downsizing” the 42-mer to an undecamer gave rise to an affinity for GPa an order of magnitude lower, but the undecamer in which Nle5, Ile9 and Leu12 were replaced by Aoc showed a KD value of 550 nm, comparable with that of the parent 42-mer. The use of Aoc residues offers a convenient route to increased affinity in protein recognition as well as a strategy for the “downsizing” of peptides essentially without loss of affinity. The results show that hydrophobic binding sites can be found on protein surfaces by comparing the affinities of polypeptide conjugates in which Aoc residues replace Nle, Ile, Leu or Phe with those of their unmodified counterparts. Polypeptide conjugates thus provide valuable opportunities for the optimization of peptides and small organic compounds in biotechnology and biomedicine.

Keywords
affinity enhancement, aminooctanoic acid, hydrophobic amino acids, peptide conjugation, peptides
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-326803 (URN)10.1002/cbic.201700048 (DOI)000405726100009 ()28432776 (PubMedID)
Available from: 2017-07-31 Created: 2017-07-31 Last updated: 2017-11-02Bibliographically approved
Yang, J., Gustavsson, A.-L., Haraldsson, M., Karlsson, G., Norberg, T. & Baltzer, L. (2017). High-affinity recognition of the human C-reactive protein independent of phosphocholine. Organic and biomolecular chemistry, 15(21), 4644-4654
Open this publication in new window or tab >>High-affinity recognition of the human C-reactive protein independent of phosphocholine
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2017 (English)In: Organic and biomolecular chemistry, ISSN 1477-0520, E-ISSN 1477-0539, Vol. 15, no 21, p. 4644-4654Article in journal (Refereed) Published
Abstract [en]

A high-affinity polypeptide conjugate 4-C25L22-DQ, has been developed for the molecular recognition of the human C-reactive protein, CRP, a well-known inflammation biomarker. CRP is one of the most frequently quantified targets in diagnostic applications and a target in drug development. With the exception of antibodies, most molecular constructs take advantage of the known affinity for CRP of phosphocholine that depends on Ca2+ for its ability to bind. 4-C25L22-DQ which is unrelated to phosphocholine binds in the absence of Ca2+ with a dissociation constant of 760 nM, an order of magnitude lower than that of phosphocholine, the KD of which is 5 μM. The small organic molecule 2-oxo-1,2-dihydroquinoline-8-carboxylic acid (DQ) was designed based on the structural similarities between three hits from a set of compounds selected from a building block collection and evaluated with regards to affinity for CRP by NMR spectroscopy. 4-C25L22-DQ was shown in a competition experiment to bind CRP three orders of magnitude more strongly than DQ itself, and in a pull-down experiment 4-C25L22-DQ was shown to extract CRP from human serum. The development of a robust and phosphocholine-independent recognition element provides unprecedented opportunities in bioanalytical applications in vivo and in vitro under conditions where the concentration of Ca2+ ions is low, or where Ca2+ binding agents such as EDTA or heparin are needed to prevent blood coagulation. The identification from a compound library of a small organic molecule and its conjugation to a small set of polypeptides, none of which were previously known to bind CRP, illustrates a convenient and general route to selective high-affinity binders for proteins with dissociation constants in the μM to nM range for which no small molecule ligands are known.

National Category
Organic Chemistry
Identifiers
urn:nbn:se:uu:diva-326501 (URN)10.1039/c7ob00684e (DOI)000402742300021 ()28513744 (PubMedID)
Funder
Swedish Research Council, 2014-4057
Available from: 2017-08-14 Created: 2017-08-14 Last updated: 2017-10-05Bibliographically approved
Yang, J., Koruza, K., Fisher, Z., Knecht, W. & Baltzer, L. (2017). Improved molecular recognition of Carbonic Anhydrase IX by polypeptide conjugation to acetazolamide. Bioorganic & Medicinal Chemistry, 25(20), 5838-5848
Open this publication in new window or tab >>Improved molecular recognition of Carbonic Anhydrase IX by polypeptide conjugation to acetazolamide
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2017 (English)In: Bioorganic & Medicinal Chemistry, ISSN 0968-0896, E-ISSN 1464-3391, Vol. 25, no 20, p. 5838-5848Article in journal (Refereed) Published
Keywords
Human Carbonic Anhydrase, Acetazolamide, Recognition, Surface plasmon resonance, Tumor
National Category
Chemical Sciences
Identifiers
urn:nbn:se:uu:diva-330361 (URN)10.1016/j.bmc.2017.09.017 (DOI)000413402100064 ()
Note

The small molecule inhibitor acetazolamide (AZM) was conjugated to a set of designed polypeptides and the resulting conjugates were evaluated for their affinity to Human Carbonic Anhydrase II (HCA II) using surface plasmon resonance. The dissociation constant of the AZM-HCA II complex was 38 nM and that of the AZM conjugated polypeptide (4-C10L17-AZM) to HCA II was found to be 4 nM, an affinity enhancement of a factor of 10 due to polypeptide conjugation. For Human Carbonic Anhydrase IX (HCA IX) the dissociation constant of AZM was 3 nM, whereas that of the 4-C10L17-AZM conjugate was 90 pM, a 33-fold affinity enhancement. This dramatic affinity increase due to polypeptide conjugation was achieved for a small molecule ligand with an already high affinity to the target protein. This supports the concept that enhancements due to polypeptide conjugation are not limited to small molecule ligands that bind proteins in the mM to μM range but may be used also for nM ligands to provide recognition elements with dissociation constants in the pM range. Evaluations of two HCA IX constructs that do not carry the proteoglycan (PG) domain did not show significant affinity differences between AZM and the polypeptide conjugate, providing evidence that the improved binding of 4-C10L17-AZM to HCA IX emanated from interactions between the polypeptide segment and the PG domain found only in one carbonic anhydrase, HCA IX.

Available from: 2017-09-28 Created: 2017-09-28 Last updated: 2018-02-08Bibliographically approved
Giandomenico, V., Modlin, I. M., Pontén, F., Nilsson, M., Landegren, U., Bergquist, J., . . . Öberg, K. (2013). Improving the Diagnosis and Management of Neuroendocrine Tumors: Utilizing New Advances in Biomarker and Molecular Imaging Science. Neuroendocrinology, 98(1), 16-30
Open this publication in new window or tab >>Improving the Diagnosis and Management of Neuroendocrine Tumors: Utilizing New Advances in Biomarker and Molecular Imaging Science
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2013 (English)In: Neuroendocrinology, ISSN 0028-3835, E-ISSN 1423-0194, Vol. 98, no 1, p. 16-30Article in journal (Refereed) Published
Abstract [en]

Neuroendocrine tumors (NET) are malignant solid tumors that arise in hormone-secreting tissue of the diffuse neuroendocrine system or endocrine glands. Although traditionally understood to be a rare disease, the incidence and prevalence of NET have increased greatly in the past 3 decades. However, during this time, progress in diagnosis and outcome of NET has generally been modest. In order to achieve improved outcome in NET, a better understanding of NET biology combined with more reliable serum markers and better techniques to identify tumor localization and small lesions are needed. Although some NET biomarkers exist, sensitive and specific markers that predict tumor growth and behavior are generally lacking. In addition, the integration of new molecular imaging technologies in patient diagnosis and follow-up has the potential to enhance care. To discuss developments and issues required to improve diagnostics and management of NET patients, with specific focus on the latest advances in molecular imaging and biomarker science, 17 global leaders in the fields of NET, molecular imaging and biomarker technology gathered to participate in a 2-day meeting hosted by Prof. Kjell Oberg at the University of Uppsala in Sweden. During this time, findings were presented regarding methods with potential prognostic and treatment applications in NET or other types of cancers. This paper describes the symposium presentations and resulting discussions.

Keywords
Biomarkers, Circulating tumor cells, Molecular imaging, Neuroendocrine tumors
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-206678 (URN)10.1159/000348832 (DOI)000322688400002 ()
Available from: 2013-09-03 Created: 2013-09-02 Last updated: 2017-12-06Bibliographically approved
Rydberg, J., Baltzer, L. & Sarojini, V. (2013). Intrinsically unstructured proteins by designelectrostatic interactions can control binding, folding, and function of a helix-loop-helix heterodimer. Journal of Peptide Science, 19(8), 461-469
Open this publication in new window or tab >>Intrinsically unstructured proteins by designelectrostatic interactions can control binding, folding, and function of a helix-loop-helix heterodimer
2013 (English)In: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 19, no 8, p. 461-469Article in journal (Refereed) Published
Abstract [en]

Intrinsically disordered proteins that exist as unordered monomeric structures in aqueous solution at pH7 but fold into four-helix bundles upon binding to recognized polypeptide targets have been designed. NMR and CD spectra of the monomeric polypeptides show the hallmarks of unordered structures, whereas in the bound state they are highly helical. Analytical ultracentrifugation data shows that the polypeptides bind to their targets to form exclusively heterodimers at neutral pH. To demonstrate the relationship between binding, folding, and function, a catalytic site for ester hydrolysis was introduced into an unordered and largely inactive monomer, but that was structured and catalytically active in the presence of a specific polypeptide target. Electrostatic interactions between surface-exposed residues inhibited the binding and folding of the monomers at pH7. Charge-charge repulsion between ionizable amino acids was thus found to be sufficient to disrupt binding between polypeptide chains despite their inherent propensities for structure formation and may be involved in the folding and function of inherently disordered proteins in biology. 

Keywords
heterodimer, electrostatic interactions, folding, catalysis
National Category
Natural Sciences
Identifiers
urn:nbn:se:uu:diva-204844 (URN)10.1002/psc.2520 (DOI)000321703200001 ()
Available from: 2013-08-13 Created: 2013-08-12 Last updated: 2017-12-06Bibliographically approved
Ramapanicker, R., Sun, X., Viljanen, J. & Baltzer, L. (2013). Powerful binders for the D-dimer by conjugation of the GPRP peptide to polypeptides from a designed set: illustrating a general route to new binders for proteins. Bioconjugate chemistry, 24(1), 17-25
Open this publication in new window or tab >>Powerful binders for the D-dimer by conjugation of the GPRP peptide to polypeptides from a designed set: illustrating a general route to new binders for proteins
2013 (English)In: Bioconjugate chemistry, ISSN 1043-1802, E-ISSN 1520-4812, Vol. 24, no 1, p. 17-25Article in journal (Refereed) Published
Abstract [en]

The synthetic tetrapeptide GPRP based on the amino-terminal GPR sequence of the fibrin α-chain, binds the D-dimer protein with a dissociation constant KD of 25 μM. The D-dimer protein, a well-known biomarker for thrombosis, contains two cross-linked D fragments from the fibrinogen protein formed upon degradation of the fibrin gel, the core component of blood clots. In order to develop a specific high-affinity binder for the D-dimer protein, GPRP was conjugated via an aliphatic spacer to each member of a set of sixteen polypeptides designed for the development of binder molecules for proteins in general. The binders were individually characterised and ranked using surface plasmon resonance (SPR) analysis. The dissociation constant of the complex formed from the D-dimer and 4-D15L8-GPRP labelled with fluorescein was determined by fluorescense titration and found to be 3 nM, an affinity four orders of magnitude higher than that of free GPRP. According to SPR analysis binding was completely inhibited by free GPRP at mM concentrations and the polypeptide conjugate was therefore shown to bind specifically to the binding site of GPRP. Affinities were further enhanced by dimerisation of the polypeptide conjugates via a bifunctional linker resulting in dissociation constants that were further decreased (affinities increased) by factors of 2-4. The results suggest an efficient route to specific binders for proteins based on short peptides with affinites that need only to be modest, thus shortening the time of binder development dramatically.

National Category
Organic Chemistry Other Chemistry Topics
Identifiers
urn:nbn:se:uu:diva-183200 (URN)10.1021/bc300186z (DOI)000313933700003 ()
Available from: 2012-10-23 Created: 2012-10-23 Last updated: 2017-12-07Bibliographically approved
Sun, X., Yang, J., Norberg, T. & Baltzer, L. (2012). A synthetic polypeptide conjugate from a 42-residue polypeptide and salicylhydroxamic acid binds human myeloperoxidase with high affinity. Journal of Peptide Science, 18(12), 731-739
Open this publication in new window or tab >>A synthetic polypeptide conjugate from a 42-residue polypeptide and salicylhydroxamic acid binds human myeloperoxidase with high affinity
2012 (English)In: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 18, no 12, p. 731-739Article in journal (Refereed) Published
Abstract [en]

Myeloperoxidase (MPO) is a 150 kD tetrameric heme protein consisting of two heavy chains and two light chains, which ispresent in neutrophils, white blood cells, at concentrations between 2% and 5% and plays an important role in the innateimmune system. The MPO concentration in serum or plasma has been shown to be linked to the risk for cardiovasculardiseases, and MPO is considered to be a high potential diagnostic biomarker. To develop a molecule that binds MPO,salicylhydroxamic acid (SHA), a substrate analog inhibitor of MPO with a KD=2uM, was conjugated to a designed set of42-residue polypeptide scaffolds via 9- and 11-carbon atom aliphatic spacers to form 20 different protein binder candidates,and their interactions with MPO were evaluated by surface plasmon resonance analysis. The polypeptide conjugate4C37L34C11SHA was found to bind to MPO with an affinity that could be estimated to have a dissociation constant of around400 pM, nearly four orders of magnitude higher than that of SHA. Inhibition of binding to MPO by free SHA was observed incompetition experiments demonstrating that the binding of the polypeptide conjugate is dominated by the interactions ofSHA with the heme cavity. Although still in the future, the discovery of these new synthetic binders for MPO suggests aroute to clinical diagnostic tests in vivo or in vitro, independent of antibodies.

National Category
Organic Chemistry Other Chemistry Topics
Identifiers
urn:nbn:se:uu:diva-183195 (URN)10.1002/psc.2459 (DOI)000311053700003 ()
Available from: 2012-10-23 Created: 2012-10-23 Last updated: 2017-12-07Bibliographically approved
Fromell, K., Forsberg, P., Karlsson, M., Larsson, K., Nikolajeff, F. & Baltzer, L. (2012). Designed protein binders in combination with nanocrystalline diamond for use in high-sensitivity biosensors. Analytical and Bioanalytical Chemistry, 404(6-7), 1643-1651
Open this publication in new window or tab >>Designed protein binders in combination with nanocrystalline diamond for use in high-sensitivity biosensors
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2012 (English)In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 404, no 6-7, p. 1643-1651Article in journal (Refereed) Published
Abstract [en]

A platform for diagnostic applications showing signal-to-noise ratios that by far surpass those of traditional bioanalytical test formats has been developed. It combines the properties of modified nanocrystalline diamond (NCD) surfaces and those of polyethylene oxide and polypropylene oxide based block copolymers for surface passivation and binder conjugation with a new class of synthetic binders for proteins. The NCD surfaces were fluorine-, hydrogen-, or oxygen-terminated prior to further biofunctionalization and the surface composition was characterized by X-ray photoelectron spectroscopy. In a proof of principle demonstration targeting the C-reactive protein, an ELISA carried out using an F-terminated diamond surface showed a signal-to-noise ratio of 3,900 which compares well to the signal-to-noise of 89 obtained in an antibody-based ELISA on a polystyrene microtiter plate, a standard test format used in most life science laboratories today. The increase in signal-to-noise ratio is to a large extent the result of extremely efficient passivation of the diamond surface. The results suggest that significant improvements can be obtained in standardized test formats using new materials in combination with new types of chemical coatings and receptor molecules.

Keywords
Protein binders, Nanocrystalline diamond, CRP, Biosensor, Surface characterization
National Category
Natural Sciences Engineering and Technology
Research subject
Engineering Science with specialization in Microsystems Technology
Identifiers
urn:nbn:se:uu:diva-184455 (URN)10.1007/s00216-012-6245-7 (DOI)000309348400004 ()
Available from: 2012-11-09 Created: 2012-11-07 Last updated: 2017-12-07Bibliographically approved
Ślósarczyk, A. T. & Baltzer, L. (2012). Efficient formation of heterodimers from peptides and proteins using unsymmetrical polyfluorophenyl esters of dicarboxylic acids. Journal of Peptide Science, 18(4), 261-269
Open this publication in new window or tab >>Efficient formation of heterodimers from peptides and proteins using unsymmetrical polyfluorophenyl esters of dicarboxylic acids
2012 (English)In: Journal of Peptide Science, ISSN 1075-2617, E-ISSN 1099-1387, Vol. 18, no 4, p. 261-269Article in journal (Refereed) Published
Abstract [en]

An efficient method for the heteroconjugation of biomolecules carrying free amino groups was reported previously, where mixed polyfluorophenyl diesters of dicarboxylic acids with varied aliphatic chain length were shown to be efficient reagents for the conjugation of a variety of model biomolecules. The concept was based on the differential reactivity of the esters towards amines. The concept has now been further optimized, and a 2,6-difluorophenyl-pentafluorophenyl diester combination has been demonstrated to be the most efficient, both with respect to selectivity and to reaction rate. A pentafluorophenyl ester reacts faster with an amino group and requires a weaker base than a 2,6-difluorophenyl ester that requires a stronger base and longer reaction time. With the use of this combination of esters, we obtained considerably shortened reaction times compared with those reported previously, yet still retaining the desired selectivity in heteroconjugation. The increased reactivity of the bifunctional reagent allowed the construction of sophisticated peptide heteroconjugates from peptides, carbohydrates and proteins, showing a wide scope of applicability in the field of assembling functional bioconjugates.

Keywords
conjugation, bioconjugation, linker, heteroconjugation, polyfluorophenyl esters
National Category
Chemical Sciences
Research subject
Chemistry; Chemistry with specialization in Bioorganic Chemistry
Identifiers
urn:nbn:se:uu:diva-161012 (URN)10.1002/psc.2394 (DOI)000302017900007 ()
Available from: 2011-11-04 Created: 2011-11-04 Last updated: 2017-12-08Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0001-5807-2726

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