Mechanism of formation of a productive molten globule form of barstar.
Structural analysis of the initial steps in protein folding is difficult because of the swiftness with which these steps occur. Hence, the link between initial polypeptide chain collapse and formation of secondary and other specific structures remains poorly understood. Here, an equilibrium model has been developed for characterizing the initial steps of folding of the small protein barstar, which lead to the formation of a productive molten globule in the folding pathway. In this model, the high-pH-unfolded form (D form) of barstar, which is shown to be as unstructured as the urea-denatured form, is transformed progressively into a molten globule B form by incremental addition of the salt Na(2)SO(4) at pH 12. At very low concentrations of Na(2)SO(4), the D form collapses into a pre-molten globule (P) form, whose volume exceeds that of the native (N) state by only 20%, and which lacks any specific structure as determined by far- and near-UV circular dichroism. At higher concentrations of Na(2)SO(4), the P form transforms into the molten globule (B) form in a highly noncooperative transition populated by an ensemble of at least two intermediates. The B form is a dry molten globule in which water is excluded from the core, and in which secondary structure develops to 65% and tertiary contacts develop to 40%, relative to that of the native protein. Kinetic refolding experiments carried out at pH 7 and at high Na(2)SO(4) concentrations, in which the rate of folding of the D form to the N state is compared to that of the B form to the N state, indicate conclusively that the B form is a productive intermediate that forms on the direct pathway of folding from the D form to the N state.
Rami
Bhadresh R
BR
National Centre for Biological Sciences, Tata Institute of Fundamental Research, GKVK Campus, Bangalore 560065, India.
Udgaonkar
Jayant B
JB
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics; D002942:Circular Dichroism; D006863:Hydrogen-Ion Concentration; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics; D013431:Sulfates; D013816:Thermodynamics; D014364:Tryptophan / Q000737:chemistry
D001426:Bacterial Proteins; D011994:Recombinant Proteins; D013431:Sulfates; C012036:sodium sulfate; C057438:barstar protein, Bacillus amyloliquefaciens; D014364:Tryptophan