uu.seUppsala universitets publikasjoner
Endre søk
RefereraExporteraLink to record
Permanent link

Direct link
Referera
Referensformat
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Annet format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annet språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf
Exploring molecular interactions between polypeptide conjugates and protein targets: Manipulating affinity by chemical modifications
Uppsala universitet, Teknisk-naturvetenskapliga vetenskapsområdet, Kemiska sektionen, Institutionen för kemi - BMC, Organisk kemi. (Lars Baltzer)
2017 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Uppsala: Acta Universitatis Upsaliensis, 2017. , s. 69
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Science and Technology, ISSN 1651-6214 ; 1534
Emneord [en]
polypeptide conjugates, protein targets, molecular recognition, affinity enhancement, conjugation
HSV kategori
Identifikatorer
URN: urn:nbn:se:uu:diva-327121ISBN: 978-91-513-0016-0 (tryckt)OAI: oai:DiVA.org:uu-327121DiVA, id: diva2:1129511
Disputas
2017-10-10, C4:301 BMC, Husargatan 3, Uppsala, 09:15 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2017-09-01 Laget: 2017-08-03 Sist oppdatert: 2017-09-08
Delarbeid
1. Exploring Non-obvious Hydrophobic Binding Pockets on Protein Surfaces: Increasing Affinities in Peptide–Protein Interactions
Åpne denne publikasjonen i ny fane eller vindu >>Exploring Non-obvious Hydrophobic Binding Pockets on Protein Surfaces: Increasing Affinities in Peptide–Protein Interactions
2017 (engelsk)Inngår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 18, nr 14, s. 1396-1407Artikkel i tidsskrift (Fagfellevurdert) 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.

Emneord
affinity enhancement, aminooctanoic acid, hydrophobic amino acids, peptide conjugation, peptides
HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-326803 (URN)10.1002/cbic.201700048 (DOI)000405726100009 ()28432776 (PubMedID)
Tilgjengelig fra: 2017-07-31 Laget: 2017-07-31 Sist oppdatert: 2017-11-02bibliografisk kontrollert
2. Conjugation of a Dipicolyl Chelate to Polypeptide Conjugates Increases Binding Affinities for Human Serum Albumin and Survival Times in Human Serum
Åpne denne publikasjonen i ny fane eller vindu >>Conjugation of a Dipicolyl Chelate to Polypeptide Conjugates Increases Binding Affinities for Human Serum Albumin and Survival Times in Human Serum
2017 (engelsk)Inngår i: ChemBioChem (Print), ISSN 1439-4227, E-ISSN 1439-7633, Vol. 18, nr 14, s. 1408-1414Artikkel i tidsskrift (Fagfellevurdert) 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.

Emneord
affinity, biosensor, human serum albumin, peptides, zinc-binding site
HSV kategori
Identifikatorer
urn:nbn:se:uu:diva-326838 (URN)10.1002/cbic.201700049 (DOI)000405726100010 ()28301711 (PubMedID)
Tilgjengelig fra: 2017-07-31 Laget: 2017-07-31 Sist oppdatert: 2017-11-02bibliografisk kontrollert

Open Access i DiVA

fulltext(2343 kB)194 nedlastinger
Filinformasjon
Fil FULLTEXT01.pdfFilstørrelse 2343 kBChecksum SHA-512
4bca1a3bc4edcdbdb282bbab24fa1485ccba9df19f92da4cb9ecb56993b23ea657e927f76ddd4997b06d5d52b492c0eb93b07fa439eb84ca91a7ca7b4970dcdc
Type fulltextMimetype application/pdf
Kjøp publikasjonen >>

Av organisasjonen

Søk utenfor DiVA

GoogleGoogle Scholar
Totalt: 194 nedlastinger
Antall nedlastinger er summen av alle nedlastinger av alle fulltekster. Det kan for eksempel være tidligere versjoner som er ikke lenger tilgjengelige

isbn
urn-nbn

Altmetric

isbn
urn-nbn
Totalt: 1016 treff
RefereraExporteraLink to record
Permanent link

Direct link
Referera
Referensformat
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Annet format
Fler format
Språk
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Annet språk
Fler språk
Utmatningsformat
  • html
  • text
  • asciidoc
  • rtf