Lack of definable nucleation sites in the rate-limiting transition state of barnase under native conditions.
J Mol Biol
; 315(4): 759-70, 2002 Jan 25.
Article
em En
| MEDLINE
| ID: mdl-11812145
ABSTRACT
It has been shown that the burst-phase (submillisecond) intermediate of barnase, if it exists, can be only marginally more stable than the fully unfolded state at pH 6.3 and 25 degrees C. In the study reported here, no stable burst-phase intermediate could be detected, even in the presence of stabilizing salt (0.4 M Na(2)SO(4)). These results suggest that a burst-phase intermediate with even marginal stability does not exist. The absence of such an intermediate in turn suggests the need for re-examination of the rate-limiting transition state (RLTS) under native conditions, which was previously characterized by using a three-state model with a stable intermediate and protein engineering. Surprisingly, mutations throughout the structure of barnase do not significantly affect the folding rate, suggesting a lack of specific favorable interactions among the side-chains in the RLTS. This RLTS is clearly different from that previously characterized under denaturing conditions, indicating that changes take place in the RLTS under native and denaturing conditions. The occurrence of such changes is further supported by the observation that the unfolding rate constants of barnase and its mutants were divergent or convergent as a function of denaturant concentrations. Consistent with changes in the RLTS, a re-analysis of data from native-state hydrogen exchange studies has shown that the logarithm of the unfolding rate constant inflects down under low concentrations of denaturant. Here, we discuss in detail the question of whether changes in the RLTS involve a kinetically silent intermediate that occurs after the initial RLTS.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Ribonucleases
/
Bacillus
/
Dobramento de Proteína
Idioma:
En
Revista:
J Mol Biol
Ano de publicação:
2002
Tipo de documento:
Article
País de afiliação:
Estados Unidos