A thiol labelling competition experiment as a probe for sidechain packing in the kinetic folding intermediate of N-PGK.
Protein folding is directed by the sequence of sidechains along the polypeptide backbone, but despite this the developement of sidechain interactions during folding is not well understood. Here, the thiol-active reagent, dithio-nitrobenzoic acid (DTNB), is used to probe the exposure of the cysteine sidechain thiols in the kinetic folding intermediates of the N-terminal domain of phosphoglycerate kinase (N-PGK) and a number of conservative (I-, L-, or V-to-C) single cysteine variants. Rapid dilution of chemically denatured protein into folding conditions in the presence of DTNB allowed the degree of sidechain protection in any rapidly formed intermediate to be determined through the analysis of the kinetics of labelling. The protection factors derived for the intermediate(s) were generally small (<25), indicating only partial burial of the sidechains. The distribution of protection parallels the previously reported backbone amide protection for the folding intermediate of N-PGK. These observations are consistent with the hypothesis that such intermediates resemble molten globule states; i.e. with native-like backbone hydrogen bonding and overall tertiary structure, but with the sidechains that make up the hydrophobic protein core dynamic and intermittently solvent exposed. The success of the competition technique in characterizing this kinetic intermediate invites application to other model systems.
Cliff
Matthew J
MJ
Department of Molecular Biology and Biotechnology, University of Sheffield, Firth Court, Western Bank, Sheffield, S10 2TN, UK. m.j.cliff@sheffield.ac.uk
Alizadeh
Tooba
T
Jelinska
Clare
C
Craven
C Jeremy
CJ
Staniforth
Rosemary A
RA
Waltho
Jonathan P
JP
D003545:Cysteine; D041961:Deuterium Exchange Measurement; D004228:Dithionitrobenzoic Acid; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D015335:Molecular Probes; D009154:Mutation; D010735:Phosphoglycerate Kinase / Q000737:chemistry* / Q000235:genetics; D011489:Protein Denaturation; D017510:Protein Folding; D020673:Protein Renaturation; D013438:Sulfhydryl Compounds
D015335:Molecular Probes; D013438:Sulfhydryl Compounds; D004228:Dithionitrobenzoic Acid; D010735:Phosphoglycerate Kinase; D003545:Cysteine