Determining biophysical protein stability in lysates by a fast proteolysis assay, FASTpp.
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.
Cellular Protein Chemistry, Bijvoet Center for Biomolecular Research, Utrecht University, Utrecht, The Netherlands.
David P
DP
Minde
Madelon M
MM
Maurice
Stefan G D
SG
Rüdiger
D019881:Aminoacyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001426:Bacterial Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D055592:Biophysical Phenomena; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D003546:Cysteine Endopeptidases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D045304:Cytochromes c / Q000737:chemistry / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006418:Heme / Q000737:chemistry / Q000378:metabolism; D007700:Kinetics; D008320:Maltose / Q000737:chemistry / Q000378:metabolism; D058619:Maltose-Binding Proteins / Q000737:chemistry / Q000378:metabolism; D017354:Point Mutation; D011485:Protein Binding; D011489:Protein Denaturation; D017510:Protein Folding; D055550:Protein Stability; D017434:Protein Structure, Tertiary; D058767:Protein Unfolding; D011506:Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D059748:Proteolysis; D015203:Reproducibility of Results; D013696:Temperature; D013820:Thermolysin / Q000378:metabolism
D001426:Bacterial Proteins; D058619:Maltose-Binding Proteins; D011506:Proteins; D006418:Heme; D008320:Maltose; D045304:Cytochromes c; D019881:Aminoacyltransferases; C120542:sortase A; D003546:Cysteine Endopeptidases; D013820:Thermolysin