Salt‐stabilized globular protein structure in 7 M aqueous urea solution

Abstract

A 7 M aqueous urea solution of the 63-residue Nterminal domain of the 434-repressor at pH 7.5 and 18°C contains a mixture of about 10% native, folded protein and 90% unfolded protein. Interconversion between the two conformations is slow on the NMR chemical shift time scale, so that observation of separate resonances can be used to monitor the equilibrium between folded and unfolded protein when changing the solution conditions. In this paper we describe the influence of various salts or non-ionic compounds on this conformational equilibrium. Solution conditions are described which contain a homogenous preparation of the folded protein in the presence of 6 to 7 M urea, providing a basis for an NMR structure determination in concentrated urea and for studies of the solvation of the folded protein in mixed water/urea/salt environments. basic pancreatic trypsin inhibitor were measured under conditions where this protein is fully folded in the presence of urea, and specific interactions of urea molecules with the protein were detected for 'pockets and grooves on the protein surface' [11]. Similar experiments with unfolded 434-repressor(1-63) in 7 M urea indicated preferential urea solvation of methyl-bearing aliphatic side chains [12]. To pursue these studies on a more systematic level it will be desirable to study urea-protein interactions with folded and unfolded conformations of the same protein. The present work identifies solution conditions where folded and unfolded 434-repressor(1-63) can be studied at the same temperature in the presence of high, 'denaturing' urea concentrations.

Structural Role of a Buried Salt Bridge in the 434 Repressor DNA-binding Domain

“…in the presence of 7 M urea (Dö tsch et al, 1995), and a determination of this ''refolded'' structure is in progress. The mutant protein 434[R10M](1-63) is one out of a series of point mutations studied in our laboratory, and a detailed comparison with the wild-type protein will provide a basis for analyzing experimental observations with other amino acid substitutions in the framework of the three-dimensional structure.…”

“…The preparation of 434(1-63) and 434 [R10M](1-63) followed the general procedure outlined by Dö tsch et al (1995), and will be described in detail elsewhere. For wild-type 434(1-63), several NMR samples with 4 mM concentration of either the uniformly 15 N-labeled or the unlabeled protein were prepared.…”

Protein interactions in enzymatic processes in textiles

“…A comparison with other salts revealed that the sulphate ion is mostly responsible for this effect. Dötsch et al 1995 examined saltstabilization of globular protein structure in very concentrated aqueous urea solutions (6-7 M) and reported 100% stabilization with the addition of 2 M (NH 4 ) 2 SO 4 or 2.5 M NaCl. Carpenter and Crowe, 1988 reported a destabilising influence of "salting-out" salts like ammonium sulphate when applied at concentrations lower than 0.5 M. At higher concentrations, however, all tested salts, even NaCl, which is not regarded a "salting-out" salt, displayed enzyme stabilising properties.…”

Interaction of bovine serum albumin with gemini surfactants

“…The conformation and biological activity of proteins in aqueous media depend on intensive variables, on the presence of salts [1][2][3][4][5], alcohols [6], urea [7], and guanidine hydrochloride [8]. Ionic surfactants were considered too as reactants [9][10][11].…”