Alternative views of functional protein binding epitopes obtained by combinatorial shotgun scanning mutagenesis

G. Pál, Shun Yin Fong, Anthony A. Kossiakoff, Sachdev S. Sidhu

Research output: Contribution to journalArticle

24 Citations (Scopus)

Abstract

Combinatorial shotgun scanning mutagenesis was used to analyze two large, related protein binding sites to assess the specificity and importance of individual side chain contributions to binding affinity. The strategy allowed for cost-effective generation of a plethora of functional data. The ease of the technology promoted comprehensive investigations, in which the classic alanine-scanning approach was expanded with two additional strategies, serine- and homolog-scanning. Binding of human growth hormone (hGH) to the hGH receptor served as the model system. The entire high affinity receptor-binding sites (site 1) of wild-type hGH (hGHwt) and of an affinity-improved variant (hGHv) were investigated and the results were compared. The contributions that 35 residue positions make to binding were assessed on each hormone molecule by both serine- and homolog-scanning. The hormone molecules were displayed on the surfaces of bacteriophage, and the 35 positions were randomized simultaneously to allow equal starting frequencies of the wild-type residue and either serine or a homologous mutation in separate libraries. Functional selections for binding to the hGH receptor shifted the relative wild-type/mutant frequencies at each position to an extent characteristic of the functional importance of the side chain. Functional epitope maps were created and compared to previous maps obtained by alanine-scanning. Comparisons between the different scans provide insights into the affinity maturation process that produced hGHv. The serine- and homolog-scanning results expand upon and complement the alanine-scanning results and provide additional data on the robustness of the high affinity receptor-binding site of hGH.

Original languageEnglish
Pages (from-to)2405-2413
Number of pages9
JournalProtein Science
Volume14
Issue number9
DOIs
Publication statusPublished - Sep 2005

Fingerprint

Mutagenesis
Firearms
Protein Binding
Serine
Human Growth Hormone
Epitopes
Alanine
Scanning
Binding Sites
Hormones
Bacteriophages
Libraries
Molecules
Technology
Costs and Cost Analysis
Mutation
delta-hGHR

Keywords

  • Functional epitope
  • Human growth hormone
  • Shotgun phage display scanning

ASJC Scopus subject areas

  • Biochemistry

Cite this

Alternative views of functional protein binding epitopes obtained by combinatorial shotgun scanning mutagenesis. / Pál, G.; Fong, Shun Yin; Kossiakoff, Anthony A.; Sidhu, Sachdev S.

In: Protein Science, Vol. 14, No. 9, 09.2005, p. 2405-2413.

Research output: Contribution to journalArticle

Pál, G. ; Fong, Shun Yin ; Kossiakoff, Anthony A. ; Sidhu, Sachdev S. / Alternative views of functional protein binding epitopes obtained by combinatorial shotgun scanning mutagenesis. In: Protein Science. 2005 ; Vol. 14, No. 9. pp. 2405-2413.
@article{0698a9d5907a4a8dacd972511e9b625d,
title = "Alternative views of functional protein binding epitopes obtained by combinatorial shotgun scanning mutagenesis",
abstract = "Combinatorial shotgun scanning mutagenesis was used to analyze two large, related protein binding sites to assess the specificity and importance of individual side chain contributions to binding affinity. The strategy allowed for cost-effective generation of a plethora of functional data. The ease of the technology promoted comprehensive investigations, in which the classic alanine-scanning approach was expanded with two additional strategies, serine- and homolog-scanning. Binding of human growth hormone (hGH) to the hGH receptor served as the model system. The entire high affinity receptor-binding sites (site 1) of wild-type hGH (hGHwt) and of an affinity-improved variant (hGHv) were investigated and the results were compared. The contributions that 35 residue positions make to binding were assessed on each hormone molecule by both serine- and homolog-scanning. The hormone molecules were displayed on the surfaces of bacteriophage, and the 35 positions were randomized simultaneously to allow equal starting frequencies of the wild-type residue and either serine or a homologous mutation in separate libraries. Functional selections for binding to the hGH receptor shifted the relative wild-type/mutant frequencies at each position to an extent characteristic of the functional importance of the side chain. Functional epitope maps were created and compared to previous maps obtained by alanine-scanning. Comparisons between the different scans provide insights into the affinity maturation process that produced hGHv. The serine- and homolog-scanning results expand upon and complement the alanine-scanning results and provide additional data on the robustness of the high affinity receptor-binding site of hGH.",
keywords = "Functional epitope, Human growth hormone, Shotgun phage display scanning",
author = "G. P{\'a}l and Fong, {Shun Yin} and Kossiakoff, {Anthony A.} and Sidhu, {Sachdev S.}",
year = "2005",
month = "9",
doi = "10.1110/ps.051519805",
language = "English",
volume = "14",
pages = "2405--2413",
journal = "Protein Science",
issn = "0961-8368",
publisher = "Cold Spring Harbor Laboratory Press",
number = "9",

}

TY - JOUR

T1 - Alternative views of functional protein binding epitopes obtained by combinatorial shotgun scanning mutagenesis

AU - Pál, G.

AU - Fong, Shun Yin

AU - Kossiakoff, Anthony A.

AU - Sidhu, Sachdev S.

PY - 2005/9

Y1 - 2005/9

N2 - Combinatorial shotgun scanning mutagenesis was used to analyze two large, related protein binding sites to assess the specificity and importance of individual side chain contributions to binding affinity. The strategy allowed for cost-effective generation of a plethora of functional data. The ease of the technology promoted comprehensive investigations, in which the classic alanine-scanning approach was expanded with two additional strategies, serine- and homolog-scanning. Binding of human growth hormone (hGH) to the hGH receptor served as the model system. The entire high affinity receptor-binding sites (site 1) of wild-type hGH (hGHwt) and of an affinity-improved variant (hGHv) were investigated and the results were compared. The contributions that 35 residue positions make to binding were assessed on each hormone molecule by both serine- and homolog-scanning. The hormone molecules were displayed on the surfaces of bacteriophage, and the 35 positions were randomized simultaneously to allow equal starting frequencies of the wild-type residue and either serine or a homologous mutation in separate libraries. Functional selections for binding to the hGH receptor shifted the relative wild-type/mutant frequencies at each position to an extent characteristic of the functional importance of the side chain. Functional epitope maps were created and compared to previous maps obtained by alanine-scanning. Comparisons between the different scans provide insights into the affinity maturation process that produced hGHv. The serine- and homolog-scanning results expand upon and complement the alanine-scanning results and provide additional data on the robustness of the high affinity receptor-binding site of hGH.

AB - Combinatorial shotgun scanning mutagenesis was used to analyze two large, related protein binding sites to assess the specificity and importance of individual side chain contributions to binding affinity. The strategy allowed for cost-effective generation of a plethora of functional data. The ease of the technology promoted comprehensive investigations, in which the classic alanine-scanning approach was expanded with two additional strategies, serine- and homolog-scanning. Binding of human growth hormone (hGH) to the hGH receptor served as the model system. The entire high affinity receptor-binding sites (site 1) of wild-type hGH (hGHwt) and of an affinity-improved variant (hGHv) were investigated and the results were compared. The contributions that 35 residue positions make to binding were assessed on each hormone molecule by both serine- and homolog-scanning. The hormone molecules were displayed on the surfaces of bacteriophage, and the 35 positions were randomized simultaneously to allow equal starting frequencies of the wild-type residue and either serine or a homologous mutation in separate libraries. Functional selections for binding to the hGH receptor shifted the relative wild-type/mutant frequencies at each position to an extent characteristic of the functional importance of the side chain. Functional epitope maps were created and compared to previous maps obtained by alanine-scanning. Comparisons between the different scans provide insights into the affinity maturation process that produced hGHv. The serine- and homolog-scanning results expand upon and complement the alanine-scanning results and provide additional data on the robustness of the high affinity receptor-binding site of hGH.

KW - Functional epitope

KW - Human growth hormone

KW - Shotgun phage display scanning

UR - http://www.scopus.com/inward/record.url?scp=24344495780&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=24344495780&partnerID=8YFLogxK

U2 - 10.1110/ps.051519805

DO - 10.1110/ps.051519805

M3 - Article

C2 - 16131663

AN - SCOPUS:24344495780

VL - 14

SP - 2405

EP - 2413

JO - Protein Science

JF - Protein Science

SN - 0961-8368

IS - 9

ER -