Tyrosyl fluorescence quenching by oxidized dithiothreitol (DTTo) in N-acetyl-L-tyrosine N'-methylamide, and native bovine pancreatic ribonuclease A and its reduced, S-methylated form, in aqueous solution is studied at pH 3.0. From the temperature dependence of the fluorescence quenching, it is demonstrated that the mechanism of the quenching process is probably static (formation of a complex), and not dynamic (collisional), in origin. Although other quenching mechanisms cannot be ruled out, our proposition that the quenching of tyrosyl fluorescence in these molecules is due to the formation of a complex between the tyrosyl moieties and DTTo is consistent with previously reported evidence indicating a strong tendency for aromatics to complex with various disulfide-containing compounds. The strength of binding is approximately the same for these three tyrosine-containing compounds, indicating that the microenvironments of their tyrosyl residues may be similar. With 1 M as the reference standard state, the following average thermodynamic parameters are established for the complexation (at 298 K): delta G0 = -3.32 kcal/mol, delta H0 = -1.1 kcal/mol, and delta S0 = 7.4 eu. The large positive value of delta S0 suggests that hydrophobic interactions may play an important role in the stabilization of such tyrosyl-disulfide complexes; the negative value of delta H0 suggests that polar interactions may also contribute to the formation of these complexes. Some possible implications with regard to protein-folding studies are discussed.

The CEH proton resonance of His-12 of reduced cysteine S-sulfonated bovine pancreatic ribonuclease A exhibits a nonlinear temperature dependence of the chemical shift in its 1H-NMR spectrum at an apparent pH of 3.0. At temperatures below ca. 35TC, the temperature dependence of the chemical shift of the His-12 CEH resonance is opposite in sign to those of His-48, His-105, and His-119. At temperatures above ca. 35TC, the temperature dependence of the chemical shift of the His-12 CEH resonance is similar to those of the other three His C6H resonances. These data indicate the existence of an equilibrium between locally ordered and locally disordered environments of His-12 in the sulfonated protein at temperatures below ca. 35TC. The ordered and disordered conformations interconvert at a rate that is fast relative to the 1H-NMR chemical shift time scale i.e., the locally ordered structure has a lifetime of <<7 msec. These results demonstrate that short-and medium-range interactions can define short-lived local structures under conditions of temperature and solution composition at which the native protein structure is stable. Furthermore, they demonstrate the utility of reduced derivatives of disulfide-containing proteins as model systems for the identification of local structures that may play a role as early-forming chain-folding initiation structures.Locally ordered structures, believed to be determined primarily by short-and medium-range sequence-specific interactions, are thought to play an important role in the initial stages of protein folding (1-8; unpublished data). Under folding conditions (i.e., under conditions of temperature and solution composition at which the native protein structure is. thermodynamically stable), the formation of these local structures is thought (1-8; unpublished data) to represent the first step(s) in the folding mechanism. These chain-folding initiation structures are presumed to limit the conformational space accessible to the protein in the initial stages of folding, thereby directing subsequent folding events. 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 (9-11) 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.The problem of determining the conformations of proteinThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby mark...

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