Nonrandom distribution of the one-disulfide intermediates in the regeneration of ribonuclease A.
The one-disulfide intermediates formed during the oxidative refolding of ribonuclease A (RNase A) have been characterized. This information is important for understanding the folding pathways of RNase A. The one-disulfide intermediates were blocked with 2-aminoethyl methanethiosulfonate, fractionated using ion-exchange chromatography, and digested with trypsin and chymotrypsin. The resulting peptide fragments were fractionated using reversed phase high-performance liquid chromatography, and identified using mass spectrometry. The relative population of each one-disulfide intermediate was determined from its disulfide bond concentration using a postcolumn disulfide detection system. A total of 24 out of 28 possible one-disulfide intermediates were found to be populated (greater than 0.3%) in the one-disulfide mixture. The population of one-disulfide intermediates displays a nonrandom distribution. All four native disulfide pairings have populations greater than those predicted by loop entropy calculations, suggesting the presence of enthalpic contributions stabilizing these species. The one-disulfide intermediate [65, 72], containing the disulfide bond between cysteines 65 and 72, comprises 40% of the entire one-disulfide population. The interactions that stabilize this intermediate may play an important role in the regeneration pathways of RNase A.
Xu
X
X
Baker Laboratory of Chemistry, Cornell University, Ithaca, New York 14853-1301, USA.
Rothwarf
D M
DM
Scheraga
H A
HA
D000818:Animals; D002417:Cattle; D002918:Chymotrypsin; D004220:Disulfides / Q000737:chemistry; D066298:In Vitro Techniques; D007700:Kinetics; D008698:Mesylates; D015394:Molecular Structure; D010084:Oxidation-Reduction; D010446:Peptide Fragments / Q000737:chemistry / Q000302:isolation & purification; D010449:Peptide Mapping; D017510:Protein Folding; D012259:Ribonuclease, Pancreatic / Q000737:chemistry*; D013816:Thermodynamics; D014357:Trypsin
D004220:Disulfides; D008698:Mesylates; D010446:Peptide Fragments; D012259:Ribonuclease, Pancreatic; D002918:Chymotrypsin; D014357:Trypsin