High-energy intermediates in protein unfolding characterized by thiol labeling under nativelike conditions.
A protein unfolding reaction usually appears to be so dominated by a large free energy barrier that identifying and characterizing high-energy intermediates and, hence, dissecting the unfolding reaction into multiple structural transitions have proven to be a challenge. In particular, it has been difficult to identify any detected high-energy intermediate with the dry (DMG) and wet (WMG) molten globules that have been implicated in the unfolding reactions of at least some proteins. In this study, a native-state thiol labeling methodology was used to identify high-energy intermediates, as well as to delineate the barriers to the disruption of side chain packing interactions and to site-specific solvent exposure in different regions of the small protein, single-chain monellin (MNEI). Labeling studies of four single-cysteine-containing variants of MNEI have identified three high-energy intermediates, populated to very low extents under nativelike conditions. A significant dispersion in the opening rates of the cysteine side chains has allowed multiple steps, leading to the loss of side chain packing, to be resolved temporally. A detailed structural analysis of the positions of the four cysteine residue positions, which are buried to different depths within the protein, has suggested a direct correlation with the structure of a DMG, detected in previous studies. It is observed that side chain packing within the core of the protein is maintained, while that at the surface is disrupted, in the DMG. The core of the protein becomes solvent-exposed only in a WMG populated after the rate-limiting step of unfolding at high denaturant concentrations.
National Centre for Biological Sciences, Tata Institute of Fundamental Research , Bangalore 560065, India.
Pooja
P
Malhotra
Jayant B
JB
Udgaonkar
D003545:Cysteine / Q000493:pharmacokinetics; D004734:Energy Metabolism / Q000502:physiology; D010455:Peptides / Q000737:chemistry / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry* / Q000378:metabolism; D058849:Protein Refolding; D058767:Protein Unfolding; D013438:Sulfhydryl Compounds / Q000737:chemistry* / Q000378:metabolism
D010455:Peptides; D010940:Plant Proteins; D013438:Sulfhydryl Compounds; D003545:Cysteine