Distribution of disulfide bonds in the two-disulfide intermediates in the regeneration of bovine pancreatic ribonuclease A: further insights into the folding process.
The distribution of one-disulfide bonds in the two-disulfide intermediates in the oxidative refolding of bovine pancreatic ribonuclease A has been characterized. These two-disulfide intermediates were formed from the fully reduced denatured protein by oxidation with dithiothreitol, then blocked with AEMTS, purified by cation-exchange chromatography, enzymatically digested, and analyzed by reversed-phase high-performance liquid chromatography and mass spectrometry. The relative concentration of each of the 28 possible one-disulfide bonds in the two-disulfide ensemble was determined. Comparison with a statistical mechanical treatment of loop formation shows that the two-disulfide intermediates are probably compact. All 28 disulfide bonds were observed, demonstrating the absence of specific long-range interactions in these intermediates. Thermodynamic arguments suggest that the absence of such specific long-range interactions in the two-disulfide species may elevate the concentration of kinetically important three-disulfide intermediates and thereby increase the folding rate. Bond [65-72] was found to make up approximately 27% of the disulfide bonds of the two-disulfide species, significantly more than all other disulfides, because of stabilization by loop entropy factors and an energetically favorable beta-turn. This turn may be one of several chain-folding initiation sites, accelerating folding by decreasing the dimensionality of the conformational space that has to be searched.
Volles
M J
MJ
Baker Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301, USA.
Xu
X
X
Scheraga
H A
HA
D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism; D004795:Enzyme Stability; D005020:Ethyl Methanesulfonate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D006868:Hydrolysis; D007700:Kinetics; D010084:Oxidation-Reduction; D010449:Peptide Mapping; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000378:metabolism; D013816:Thermodynamics
D004220:Disulfides; C093171:methanethiosulfonate ethylammonium; D005020:Ethyl Methanesulfonate; D012259:Ribonuclease, Pancreatic