Loop-sequence features and stability determinants in antibody variable domains by high-throughput experiments

Structure. 2014 Jan 7;22(1):9-21. doi: 10.1016/j.str.2013.10.005. Epub 2013 Nov 21.

Abstract

Protein loops are frequently considered as critical determinants in protein structure and function. Recent advances in high-throughput methods for DNA sequencing and thermal stability measurement have enabled effective exploration of sequence-structure-function relationships in local protein regions. Using these data-intensive technologies, we investigated the sequence-structure-function relationships of six complementarity-determining regions (CDRs) and ten non-CDR loops in the variable domains of a model vascular endothelial growth factor (VEGF)-binding single-chain antibody variable fragment (scFv) whose sequence had been optimized via a consensus-sequence approach. The results show that only a handful of residues involving long-range tertiary interactions distant from the antigen-binding site are strongly coupled with antigen binding. This implies that the loops are passive regions in protein folding; the essential sequences of these regions are dictated by conserved tertiary interactions and the consensus local loop-sequence features contribute little to protein stability and function.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Amino Acid Sequence
  • Complementarity Determining Regions / chemistry*
  • Complementarity Determining Regions / immunology
  • High-Throughput Screening Assays
  • Humans
  • Hydrogen Bonding
  • Models, Molecular
  • Molecular Sequence Data
  • Peptide Library
  • Protein Binding
  • Protein Folding
  • Protein Stability
  • Protein Structure, Secondary
  • Protein Structure, Tertiary
  • Single-Chain Antibodies / chemistry*
  • Single-Chain Antibodies / immunology
  • Staphylococcal Protein A / chemistry
  • Staphylococcal Protein A / immunology
  • Structure-Activity Relationship
  • Thermodynamics
  • Vascular Endothelial Growth Factor A / chemistry*
  • Vascular Endothelial Growth Factor A / immunology

Substances

  • Complementarity Determining Regions
  • Peptide Library
  • Single-Chain Antibodies
  • Staphylococcal Protein A
  • VEGFA protein, human
  • Vascular Endothelial Growth Factor A