Probing the high energy states in proteins by proteolysis.
Unless the native conformation has an unstructured region, proteases cannot effectively digest a protein under native conditions. Digestion must occur from a higher energy form, when at least some part of the protein is exposed to solvent and becomes accessible by proteases. Monitoring the kinetics and denaturant dependence of proteolysis under native conditions yields insight into the mechanism of proteolysis as well as these high-energy conformations. We propose here a generalized approach to exploit proteolysis as a tool to probe high-energy states in proteins. This "native state proteolysis" experiment was carried out on Escherichia coli ribonuclease HI. Mass spectrometry and N-terminal sequencing showed that thermolysin cleaves the peptide bond between Thr92 and Ala93 in an extended loop region of the protein. By comparing the proteolysis rate of the folded protein and a peptidic substrate mimicking the sequence at the cleavage site, the energy required to reach the susceptible state (Delta G(proteolysis)) was determined. From the denaturant dependence of Delta G(proteolysis), we determined that thermolysin digests this protein through a local fluctuation, i.e. localized unfolding with minimal change in solvent assessable surface area. Proteolytic susceptibilities of proteins are discussed based on the finding of this local fluctuation mechanism for proteolysis under native conditions.
Park
Chiwook
C
Department of Molecular and Cell Biology, QB3 Institute, University of California, Berkeley, CA 94720, USA.
Marqusee
Susan
S
D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D007700:Kinetics; D010447:Peptide Hydrolases / Q000378:metabolism*; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D016914:Ribonuclease H / Q000737:chemistry / Q000378:metabolism; D013816:Thermodynamics; D013820:Thermolysin / Q000378:metabolism; D013997:Time Factors; D014508:Urea / Q000378:metabolism
D011506:Proteins; D014508:Urea; D016914:Ribonuclease H; D010447:Peptide Hydrolases; D013820:Thermolysin