Transient conformational states in proteins followed by differential labeling.
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.
D000089:Acetic Anhydrides; D000477:Alkylating Agents; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D004355:Drug Stability; D007461:Iodoacetates; D007700:Kinetics; D010084:Oxidation-Reduction; D010179:Pancreas / Q000201:enzymology; D010196:Pancreatic Elastase / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis; D011487:Protein Conformation; D011489:Protein Denaturation; D013463:Sulfuric Acid Esters; D013552:Swine
D000089:Acetic Anhydrides; D000477:Alkylating Agents; D000596:Amino Acids; D007461:Iodoacetates; D010446:Peptide Fragments; D013463:Sulfuric Acid Esters; D010196:Pancreatic Elastase; C007482:dimethyl sulfate