Determining biophysical protein stability in lysates by a fast proteolysis assay, FASTpp

PLoS One. 2012;7(10):e46147. doi: 10.1371/journal.pone.0046147. Epub 2012 Oct 3.

Abstract

The biophysical stability is an important parameter for protein activity both in vivo and in vitro. Here we propose a method to analyse thermal melting of protein domains in lysates: Fast parallel proteolysis (FASTpp). Combining unfolding by a temperature gradient in a thermal cycler with simultaneous proteolytic cleavage of the unfolded state, we probed stability of single domains in lysates. We validated FASTpp on proteins from 10 kDa to 240 kDa and monitored stabilisation and coupled folding and binding upon interaction with small-molecule ligands. Within a total reaction time of approximately 1 min, we probed subtle stability differences of point mutations with high sensitivity and in agreement with data obtained by intrinsic protein fluorescence. We anticipate a wide range of applications of FASTpp in biomedicine and protein engineering as it requires only standard laboratory equipment.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Aminoacyltransferases / chemistry
  • Aminoacyltransferases / genetics
  • Aminoacyltransferases / metabolism
  • Bacterial Proteins / chemistry
  • Bacterial Proteins / genetics
  • Bacterial Proteins / metabolism
  • Biophysical Phenomena
  • Chemistry Techniques, Analytical / methods*
  • Cysteine Endopeptidases / chemistry
  • Cysteine Endopeptidases / genetics
  • Cysteine Endopeptidases / metabolism
  • Cytochromes c / chemistry
  • Cytochromes c / metabolism
  • Electrophoresis, Polyacrylamide Gel
  • Heme / chemistry
  • Heme / metabolism
  • Kinetics
  • Maltose / chemistry
  • Maltose / metabolism
  • Maltose-Binding Proteins / chemistry
  • Maltose-Binding Proteins / metabolism
  • Point Mutation
  • Protein Binding
  • Protein Denaturation*
  • Protein Folding
  • Protein Stability
  • Protein Structure, Tertiary*
  • Protein Unfolding
  • Proteins / chemistry*
  • Proteins / genetics
  • Proteins / metabolism
  • Proteolysis
  • Reproducibility of Results
  • Temperature
  • Thermolysin / metabolism

Substances

  • Bacterial Proteins
  • Maltose-Binding Proteins
  • Proteins
  • Heme
  • Maltose
  • Cytochromes c
  • Aminoacyltransferases
  • sortase A
  • Cysteine Endopeptidases
  • Thermolysin

Grant support

SGDR was supported by a Marie-Curie Excellence Grant of the European Union (MEXT-CT-2005-025651), a VIDI career development grant (700.55.421) by the Netherlands Organization for Scientific Research (NWO) and a High Potential Grant of Utrecht University. MMM was supported by the European Research Council (ERC-StG no.242958) and a High Potential Grant of Utrecht University. URLs of funders: NWO: http://www.nwo.nl/; EU: http://europa.eu/index_en.htm; Utrecht University: http://www.uu.nl/; ERC: http://erc.europa.eu/. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.