Regeneration of RNase A from the reduced protein: models of regeneration pathways.

Abstract

Two models ofprotein-folding pathways are proposed on the basis ofequilibrium and Idnetic data in the literature.One is a growth-type model-.e., nucleation of the native-like structure occurs in the folding process, in the rate-limiting step(s), and subsequent folding around the nucleation sites proceeds smoothly to form the native disulfide bonds and conformation. The other is a rearrangement-type model-i.e., proper On the basis of experimental and theoretical studies, three different models have been proposed forprotein-folding pathways.In the first, the dominant near-neighbor interactions lead to the formation of nucleation sites among nearby residues in the amino-acid sequence in a rate-limiting step(s) in the initial stages of folding, this is followed by a rapid growth phase in which other parts of the chain fold around the nucleation sites '(1-19

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.…”

Respice, Adspice, and Prospice

“…An experimental effort was initiated with a postdoc, Yasuo Konishi, to elucidate the relatively slow oxidative folding pathways of RNase A from the reduced protein to the native structure by using a redox mixture of oxidized and reduced glutathione, GSSG and GSH, respectively (37,38,113). With a wide range of GSSG and GSH concentrations, 7,191 theoretically possible intermediates, in addition to the fully reduced and native forms, exhibiting a combination of intramolecular disulfide bonds, intermolecular disulfide bonds between a cysteine residue of the protein and GSH, and free sulfhydryl groups could appear.…”

Toward an understanding of the folding of ribonuclease A.

“…Several years ago, a mechanism was proposed for the regeneration of ribonuclease A (RNase A; EC 3.1.27.5) from the reduced form by a mixture of oxidized and reduced glutathione (GSSG and GSH, respectively) (1)(2)(3)(4) Others may occur after the rate-limiting step(s) in the regeneration pathways and, therefore, would not participate in the rate-limiting step(s). Nevertheless, a large number of intermediates can still be involved in determining the regeneration pathway(s).…”