Transient conformational states in proteins followed by differential labeling

Abstract

Refolding of previously denatured and reduced elastase has been followed by titration of chemical reactivities of amino acid side chains to study the topography of the protein in the native state, and the microenvironment variations of protein side chains during the structural transition. Groups accessible to chemical reagents in the denatured form and buried in the "native" form were used as a local conformational probe. Times of labeling, depending on the reagent used, ranged from 100 to 800 ms. The reaction was stopped by isotopic dilution with an excess of unlabeled reagent under denaturing conditions to obtain a chemically homogeneous but heterogeneously labeled material. Peptide fractionation after degradation of the labeled proteins allowed the determination of the amount of radioactive label incorporated by the individual side chains during the refolding. Refolding rates, determined by physicochemical, enzymatic or immunochemical criteria, were compared with the conformational states of protein areas and evaluated by the variation of chemical reactivity at various denaturant concentrations. The importance of the last folding stages is emphasized by the results obtained which indicate that early during the refolding, two domain substructures (H-40 to H-71 and M-180 to H-200)( are stabilized, while the protein remains inactive at the time ranges of the labeling reactions.

Acid and Thermal Denaturation of Barnase Investigated by Molecular Dynamics Simulations

“…Tests of some of the results reported here would be of great interest. Photochemically induced nuclear polarization (Kaptein et al, 1978), nuclear Overhauser effects of water interactions with specific residues (Otting & Wü thrich, 1989), chemical markers (Ghelis, 1980), and NMR of 19 F substituted amino acids (Frieden et al, 1993) are possible approaches. In addition, the early unfolding behavior could be investigated by nanosecond laser photolysis (Jones et al, 1993) and nanosecond temperature jump experiments (M. Grü bele, personal communication).…”

Unfolding-refolding transition of a hinge bending enzyme: horse muscle phosphoglycerate kinase induced by guanidine hydrochloride

“…The existence of a hinge bending motion of the domains implies that each of them behaves like an independent structural unit. In this case, we expect that domains are independent with respect to their folding and stability (Wetlaufer, 1973) as was shown for several proteins, for example, Bence Jones protein (Azuma et al, 1972), serum albumin (Teale & Benjamin 1976a,b, 1977, elastase (Ghelis et al, 1978;Ghelis, 1980), ^-subunit of tryptophan synthase (Zetina & Goldberg 1980a,b) [for reviews, see also Wetlaufer (1981), Ghelis & Yon (1982) and Privalov (1982)]. With the aim to verify such a hypothesis, we studied the unfoldingrefolding of horse muscle PGK induced by guanidine hydrochloride (Gdn-HCl).1 *A detailed study of the transition is reported in this paper.…”

Protein folding: concepts and perspectives

“…Kinetic trapping of intermediates during the refolding of disulphide-bridged proteins has been used to detect intermediate species during the refolding of lysozyme [35] and BPTI [36][37][38]. An elegant method using differential chemical labelling has been developed by Ghélis to detect and characterize transient folding states in any protein [39], and applied successfully to elastase. One approach that is frequently used is the study of protein fragments [40][41][42][43].…”