Direct and high resolution characterization of cytochrome c equilibrium folding.
Protein folding has emerged as a central problem in biophysics, and the equilibrium folding mechanism of cytochrome c (cyt c) has served as a model system. Unfortunately, the detailed characterization of both the folding process and of any intermediate that might be populated has been limited by the low structural and/or temporal resolution of the available techniques. Here, we report the use of a recently developed technique to study folding that is based on the site-selective incorporation of carbon-deuterium (C-D) bonds and their characterization by IR spectroscopy. Specifically, we synthesize and characterize the protein with deuterated residues spread throughout four structural motifs: (d3)Leu68 in the 60's helix, (d8)Lys72 and (d8)Lys73 in the 70's helix, (d8)Lys79, (d3)Met80, and (d3)Ala83 in the D-loop, and (d3)Leu94, (d3)Leu98, and (d3)Ala101 in the C-terminal helix. The data reveal correlated behavior of the residues within each structural motif, as well as between the residues of the 60's and C-terminal helices and between residues of the 70's helix and D-loop. Residues of the 70's helix and the D-loop are more stable than those within the 60's and C-terminal helices, although the former are more sensitive to added denaturant. The data also suggest that the hydrophobicity of the heme cofactor plays a central role in folding. These results contrast with those from previous H/D exchange studies and suggest that the low denaturant fluctuations observed in the H/D exchange experiments are not similar to those through which the protein actually unfolds. The inherently fast time scale of IR also allows us to characterize the folding intermediate, long thought to be present, but which has proven difficult to characterize by other techniques.
Sagle
Laura B
LB
Department of Chemistry, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Zimmermann
Jörg
J
Dawson
Philip E
PE
Romesberg
Floyd E
FE
D045304:Cytochromes c / Q000737:chemistry* / Q000378:metabolism; D018625:Microscopy, Atomic Force; D010795:Phthalic Acids / Q000737:chemistry; D011192:Potassium Dichromate / Q000737:chemistry; D017510:Protein Folding; D012996:Solutions / Q000737:chemistry*
D010795:Phthalic Acids; D012996:Solutions; C032279:phthalic acid; D045304:Cytochromes c; D011192:Potassium Dichromate