Comparison of local and global stability of an analogue of a disulfide-folding intermediate with those of the wild-type protein in bovine pancreatic ribonuclease A: identification of specific regions of stable structure along the oxidative folding pathway.
We have identified specific regions of the polypeptide chain of bovine pancreatic ribonuclease A (RNase A) that are critical for stabilizing the oxidative folding intermediate des-[40-95] (with three native disulfide bonds but lacking the fourth native Cys40-Cys95 disulfide bond) in an ensemble of largely disordered three-disulfide precursors (3S if des-[40-95]). A stable analogue of des-[40-95], viz., [C40A, C95A] RNase A, which contains three out of four native disulfide pairings, was previously found to have a three-dimensional structure very similar to that of the wild-type protein. However, it is determined here from GdnHCl denaturation experiments to have significantly reduced global stability, i.e., = 4.5 kcal /mol at 20 degrees C and pH 4.6. The local stability of [C40A, C95A] RNase A was also examined using site-specific amide (2)H/(1)H exchange measurements at pD 5.0 to determine the individual unfolding free energy of specific residues under both strongly native (12 degrees C) and more destabilizing (20 degrees C) conditions. Comparison of the relative stabilities at specific amide sites of [C40A, C95A] RNase A at both temperatures with the corresponding values for the wild-type protein at 35 degrees C corroborates previous experimental evidence that unidentified intramolecular contacts in the vicinity of the preferentially formed native one-disulfide (C65-C72) loop are crucial for stabilizing early folding intermediates, leading to des-[40-95]. Moreover, values of for residues at or near the third alpha-helix, and in part of the second beta-sheet of [C40A, C95A] RNase A, indicate that these two regions of regular backbone structure contribute to stabilizing the global chain fold of the des-[40-95] disulfide-folding intermediate in the wild-type protein. More significantly, we have identified numerous specific residues in the first alpha-helix and the first beta-sheet of the protein that are stabilized in the final step of the major oxidative regeneration pathway of RNase A (des-[40-95] --> N).
Laity
J H
JH
Baker Laboratory of Chemistry and Chemical Biology, Cornell University, Ithaca, New York 14853-1301, USA.
Montelione
G T
GT
Scheraga
H A
HA
D000577:Amides / Q000737:chemistry; D000818:Animals; D002417:Cattle; D004220:Disulfides / Q000737:chemistry* / Q000378:metabolism*; D004795:Enzyme Stability; D019791:Guanidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007527:Isoenzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D009587:Nitrogen Isotopes; D019906:Nuclear Magnetic Resonance, Biomolecular; D010084:Oxidation-Reduction; D011392:Proline / Q000737:chemistry / Q000378:metabolism; D017510:Protein Folding; D011522:Protons; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D015203:Reproducibility of Results; D012259:Ribonuclease, Pancreatic / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013696:Temperature
D000577:Amides; D004220:Disulfides; D007527:Isoenzymes; D009587:Nitrogen Isotopes; D011522:Protons; D011994:Recombinant Proteins; D011392:Proline; D012259:Ribonuclease, Pancreatic; D019791:Guanidine