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 physiochemical, 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.
- Research Organization:
- Universite de Paris, Orsay, France
- OSTI ID:
- 7032454
- Journal Information:
- Biophys. J.; (United States), Journal Name: Biophys. J.; (United States) Vol. 32:1; ISSN BIOJA
- Country of Publication:
- United States
- Language:
- English
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Related Subjects
551000 -- Physiological Systems
59 BASIC BIOLOGICAL SCIENCES
BIOCHEMICAL REACTION KINETICS
CARBON 14 COMPOUNDS
CRYSTAL STRUCTURE
ELASTICITY
ENZYME ACTIVITY
ENZYMES
KINETICS
LABELLED COMPOUNDS
MECHANICAL PROPERTIES
MICROSTRUCTURE
MOLECULAR STRUCTURE
MORPHOLOGICAL CHANGES
PROTEIN DENATURATION
REACTION KINETICS
STRUCTURAL CHEMICAL ANALYSIS
TENSILE PROPERTIES
TIME DEPENDENCE
TRITIUM COMPOUNDS
ULTRASTRUCTURAL CHANGES