Folding of ribonuclease A from a partially disordered conformation. Kinetic study under folding conditions

Abstract

Bovine pancreatic ribonuclease A (RNase) was partially disordered with 3.5 M LiClO4 (pH 3.0). The conformation of this partially disordered material was studied by circular dichroism and Raman spectroscopy. Although the partially disordered protein appears to have a lower beta-structure content and disordered tyrosyl side chains, compared to native RNase, it seems to retain some ordered backbone structure that is suggested to be alpha helix. The kinetics of folding of LiClO4-denatured RNase was studied by means of absorption and circular dichroism measurements. For comparison, the kinetics of folding of urea-denatured RNase (which is completely devoid of ordered structure) was examined with the same techniques. Since the kinetics of folding of both denatured species are found to be similar, it appears that the ordered structure present in LiClO4-denatured RNase plays no role in determining the folding pathway. Also, the change in the circular dichroism at 220 nm showed that some of the ordered structure in LiClO4-denatured RNase becomes disordered in the early stages of folding. This implies that all ordered structures in RNase are not equivalent in their influence on the folding pathway; some can play an essential role and some may not.

Local structure involving histidine-12 in reduced S-sulfonated ribonuclease A detected by proton NMR spectroscopy under folding conditions.

“…In view of the body of evidence demonstrating that both the S-peptide (11,24,26,30,31) (i.e., residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] and Cpeptide lactone (25, 28) (i.e., the cyanogen bromide cleavage product corresponding to residues 1-13) fragments of RNase A adopt some amount of a-helical backbone structure in water at temperatures less than ca. 40'C, our finding that, under folding conditions, His-12 of reduced and sulfonated RNase A participates in one or more short-lived local structures (i.e., with a lifetime << 7 msec) suggests that the ahelical backbone conformation is also adopted by these residues (i.e., residues 3-13) in 8SS03-RNase.…”

“…Chemistry: Swadesh et aL sites of the S-protein (residues 21-124) can be fully regenerated from the reduced S-protein in the absence of S-peptide (residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20], the full native enzymatic activity is not regained when S-peptide is added to regenerated S-protein (53,54), suggesting (51) a role for residues [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20] in determining the regenerated conformation(s) of residues 21-124. …”

“…With or without internal rearrangements, these locally ordered structures either grow in size or otherwise merge with other independently initiated structures to form either the native protein structure or metastable intermediates which then fold to the native protein structure. It should be emphasized that chainfolding initiation structures, which may direct subsequent folding processes in the parts of the molecule in which they form, need not be involved in structures that participate in the rate-limiting step in folding to the native structure (9)(10)(11) since chain-folding initiation may take place at more than one site along the polypeptide chain (7, 8; unpublished data) and the structure(s) involved in the rate-limiting step may not include all of these early-forming local structures.…”

“…For bovine pancreatic ribonuclease A (RNase A), several studies demonstrate the presence of partially folded equilibrium structures at temperatures and pH values corresponding to the thermal transition region (15)(16)(17)(18). Spectroscopic studies also indicate the presence of local structure along the polypeptide chain in thermally (19)(20)(21)(22), pH-(23), LiCl04- (10,11), and guanidine-(24) denatured RNase A. Structures important in the folding mechanism under folding conditions, however, may be unstable under these denaturing conditions in which the native protein is destabilized.…”

Protein structure and function, from a colloidal to a molecular view

“…Under some solvent conditions, local ordered structures are stabilized, but they do not necessarily play an essential role in the folding of this protein (55,179). In addition, cis/trans isomerism about peptide bonds preceding proline may play an important role in the folding process, but there is some disagreement as to the stage of folding in which this isomerism takes place (171, 254,338).…”