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Exploring Non-obvious Hydrophobic Binding Pockets on Protein Surfaces: Increasing Affinities in Peptide–Protein Interactions
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry. (Baltzer grupp)
Uppsala University, Disciplinary Domain of Science and Technology, Chemistry, Department of Chemistry - BMC, Organic Chemistry.ORCID iD: 0000-0001-5807-2726
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.

Place, publisher, year, edition, pages
2017. Vol. 18, no 14, p. 1396-1407
Keywords [en]
affinity enhancement, aminooctanoic acid, hydrophobic amino acids, peptide conjugation, peptides
National Category
Biochemistry and Molecular Biology
Identifiers
URN: urn:nbn:se:uu:diva-326803DOI: 10.1002/cbic.201700048ISI: 000405726100009PubMedID: 28432776OAI: oai:DiVA.org:uu-326803DiVA, id: diva2:1128984
Available from: 2017-07-31 Created: 2017-07-31 Last updated: 2017-11-02Bibliographically approved
In thesis
1. Exploring molecular interactions between polypeptide conjugates and protein targets: Manipulating affinity by chemical modifications
Open this publication in new window or tab >>Exploring molecular interactions between polypeptide conjugates and protein targets: Manipulating affinity by chemical modifications
2017 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

In this thesis molecular interactions between polypeptide conjugates and protein targets were investigated. Polypeptides were derivatized with small organic molecules, peptides and oligonucleotides. New strategies were developed with the aim to increase affinities for proteins of biological interest.

A 42-residue polypeptide (4-C15L8) conjugated to a small organic molecule 3,5-bis[[bis(2-pyridylmethyl)amino]methyl]benzoic acid (PP1), was shown to bind glycogen phosphorylase a (GPa) in the presence of zinc ions. Under the assumption that hydrophobic interactions dominated the binding energy, the hydrophobic residues of 4-C15L8-PP1 were systematically replaced in order to study their contribution to the affinity enhancement. The replacement of the Nle, Ile and Leu residues by Ala amino acids reduced affinities. The introduction of non-natural L-2-aminooctanoic acid (Aoc) residues into the peptide sequence enhanced the binding affinity for GPa. A decreased KD of 27nM was obtained when Nle5, Ile9 and Leu12 were replaced by Aoc residues, in comparison to the KD value of 280nM obtained for the unmodified 4-C15L8-PP1. It is evident that there are non-obvious hydrophobic binding sites on the surfaces of proteins that could be identified by introducing the more hydrophobic and conformationally flexible Aoc residues. The downsizing of the 42-mer peptide to an 11-mer and the incorporation of three Aoc residues gave rise to a KD of 550 nM, comparable to that of  4-C15L8-PP1 suggesting that bioactive peptides can be downsized by the introduction of Aoc.

Aiming to improve in vivo stability, the affinity for human serum albumin (HSA) of hydrophobic, positively and negatively charged polypeptide-PP1 conjugates was evaluated. Increased hydrophobicity due to the introduction of Aoc residues did not significantly increase the affinity for HSA. No binding was observed in the case of the most negatively charged polypeptides whereas the slightly negatively and positively charged polypeptides conjugated to PP1 bound HSA with affinities that increased with the positive charge. It was found that polypeptide-PP1 conjugates target the zinc binding site of the HSA. Affinity enhancement was obtained due to the incorporation of PP1 and increased by charge to charge interactions between the positively charged amino acids of the polypeptide and the negatively charged residues of HSA, in close proximity to the HSA zinc binding site. The survival times of the peptide-PP1 conjugates in human serum were extended as a result of binding to HSA. Zn2+ ion chelating agents can be incorporated in potential peptide therapeutics with a short plasma half-life, without increasing their molecular weights.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2017. p. 69
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1534
Keywords
polypeptide conjugates, protein targets, molecular recognition, affinity enhancement, conjugation
National Category
Natural Sciences Chemical Sciences Organic Chemistry
Identifiers
urn:nbn:se:uu:diva-327121 (URN)978-91-513-0016-0 (ISBN)
Public defence
2017-10-10, C4:301 BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2017-09-01 Created: 2017-08-03 Last updated: 2017-09-08

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Publisher's full textPubMedhttp://dx.doi.org/10.1002/cbic.201700048

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Balliu, AleksandraBaltzer, Lars

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