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

Abstract

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sequence-specific interactions, are thought to play an important role in the initial stages of protein folding (1-8; unpublished data).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 thermodynamically stable), the formation of these local structures is thought (1-8; unpublished data) to represent the first step(s) in the folding mechanism.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...

Structural studies of a folding intermediate of bovine pancreatic ribonuclease A by continuous recycled flow

“…Experimental results from a combined sample of RNase A and the S-peptide, residues 1-20, demonstrated that resonance D belongs to the C'H of His-12 (see following section). The chemical shift of peak C is very similar to the chemical shift of C'H His-105 in disulfide-reduced and sulfonated RNase A (Swadesh et al, 1984). The C'H His resonances of sulfo-nated RNase A (8S032~-RNase A) were assigned by selective deuteriation procedures.…”

“…In order to examine the formation of structure in the refolding ensemble in greater detail, ID NMR spectra of the nonnative forms of RNase A were taken from 11 to 70 °C. This approach has been used by several investigators to study the formation of compact structures in chemically modified, nonfolding forms of RNase A (Rico et al, 1983(Rico et al, , 1986; Kim & Baldwin, 1984;Swadesh et al, 1984). The term nonnative RNase A refers to conformations found in both the refolding ensemble and the heat-denatured protein.…”

“…These difficulties could be surmounted if it were possible to trap the folding intermediate. Several groups have investigated the folding of RNase A where complete refolding was inhibited by chemical modification Rico et al, 1983Rico et al, , 1986; Kim & Baldwin,1984;Swadesh et al, 1984;Haas et al, 1987). These compounds have been very useful in studying the formation of structures in specific regions of the protein, such as the N-terminal -helix.…”

Implementation of a k/k0 Method to Identify Long-Range Structure in Transition States during Conformational Folding/Unfolding of Proteins

“…The unfolded state ensembles of a number of proteins have been characterized by NMR and other spectroscopic methods. Depending on solution conditions, the unfolded states may show conformational preferences for native-like or non-native residual structure (Swadesh et al, 1984;Dill and Shortle, 1991;Neri et al, 1992;Sosnick and Trewhella, 1992;Shortle, 1993;Logan et al, 1994;Arcus et al, 1995;Buckler et al, 1995;Ittah and Haas, 1995;Dyson and Wright, 1998;Navon et al, 2001;Yao et al, 2001;Schwarzinger et al, 2002). But there are relatively few proteins whose folding transition states have been characterized in detail.…”

Regeneration Studies of an Analog of Ribonuclease A Missing Disulfide Bonds 65−72 and 40−95

“…By monitoring the chemical shifts of the His C 1 H resonances, we identified the local structure in the vicinity of His 12 in reduced S-sulfonated RNase A (Swadesh et al, 1984) and in the disulfide-intact guanidine-denatured protein (Bierzynski & Baldwin, 1982). Predictions, based on hydrophobic interactions in the wild-type protein, have determined that the C-terminal region including residues 105-124 is the primary region of initial structure formation or CFIS (Matheson & Scheraga, 1978).…”