Indian Journal of Biochemistry & Biophysics Vol. 49, February 2012, pp. 7-17 Review Residual ordered structure in denatured proteins and the problem of protein folding Mahmud A Basharov* Institute of Theoretical and Experimental Biophysics, Russian Academy of Sciences, Institutskaya 3, Pushchino, Moscow Region, 142290 Russia Received 14 April 2011; revised 24 November 2011 Structural characteristics of numerous globular proteins in the denatured state have been reviewed using literature data. Recent more precise experiments show that in contrast to the conventional standpoint, proteins under strongly denaturing conditions do not unfold completely and adopt a random coil state, but contain significant residual ordered structure. These results cast doubt on the basis of the conventional approach representing the process of protein folding as a spontaneous transition of a polypeptide chain from the random coil state to the unique globular structure. The denaturation of proteins is explained in terms of the physical properties of proteins such as stability, conformational change, elasticity, irreversible denaturation, etc. The spontaneous renaturation of some denatured proteins most probably is merely the manifestation of the physical properties (e.g., the elasticity) of the proteins per se, caused by the residual structure present in the denatured state. The pieces of the ordered structure might be the centers of the initiation of renaturation, where the restoration of the initial native conformation of denatured proteins begins. Studies on the denaturation of proteins hardly clarify how the proteins fold into the native conformation during the successive residue-by-residue elongation of the polypeptide chain on the ribosome. Keywords: Protein folding, Protein residual structure, Protein denaturation Introduction The problem of protein folding i.e., the elucidation of how a protein molecule acquires the native spatial structure following its synthesis on the ribosome or how the native conformation of a protein is formed on the basis of the primary amino acid sequence of the polypeptide chain, is of current interest in physicochemical biology. The overwhelming majority of studies concerning with the elucidation of the mechanism consider the process of protein folding as a spontaneous transition of the protein polypeptide chain from the random statistical coil state* to the unique native globular structure 1-13 . This conventional approach was put forward in the early 1970s 1 on the basis of the results of in vitro denaturation experiments on small globular proteins 1,14,16-19 and especially on bovine pancreatic RNase-A 1,14,16-18 . These experiments demonstrated that proteins unfold completely and acquire a random statistical coil state during denaturation with no residual ordered structure being present (the random coil hypothesis) and denatured proteins restore their initial native state spontaneously after elimination of denaturing action (the principle of spontaneous self-organization of native conformation). On the basis of these results, the experiments on denaturation and renaturation of native proteins have been widely used for about past four decades as convenient in vitro models to understand the mechanism of the protein folding in vivo. Against the background of the traditional approach representing protein folding as a coil – globule transition, several studies of kinetics of denaturation and structural characteristics of denatured proteins have been carried out in the past two decades by using a new generation of more precise physicochemical and biochemical techniques. These studies have ——————— *Corresponding author: Tel: 7 496 773 9406 Fax: 7 496 733 0553 E-mail: m-basharov@rambler.ru Abbreviations: CD, circular dichroism; Cyt c, cytochrome c; FA, fluorescence analysis; FTIR, Fourier-transform infrared spectroscopy; GuHCl, guanidine hydrochloride; NMR, nuclear magnetic resonance; RNase-A, ribonuclease A; SANS, small angle neutron scattering; SAXS, small-angle X-ray scattering; SNase, staphylococcal nuclease. *A polypeptide chain in the random coil state is believed to have an arbitrary kinetically-allowed unordered conformation with no spatially ordered structural segments; Some other definitions of the random coil are given in the refs 4-7,9-11,14,15 .