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Probing the folding intermediate of Rd-apocyt b₅₆₂ by protein engineering and infrared T-jump

¹ Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA
² Laboratory of Biochemistry, National Cancer Institute, National Institutes of Health, Bethesda, Maryland 20892, USA

(RECEIVED August 17, 2006; FINAL REVISION March 5, 2007; ACCEPTED March 13, 2007)

Small proteins often fold in an apparent two-state manner with the absence of detectable early-folding intermediates. Recently, using native-state hydrogen exchange, intermediates that exist after the rate-limiting transition state have been identified for several proteins. However, little is known about the folding kinetics from these post-transition intermediates to their corresponding native states. Herein, we have used protein engineering and a laser-induced temperature-jump (T-jump) technique to investigate this issue and have applied it to Rd-apocyt b₅₆₂ , a four-helix bundle protein. Previously, it has been shown that Rd-apocyt b₅₆₂ folds via an on-pathway hidden intermediate, which has only the N-terminal helix unfolded. In the present study, a double mutation (V16G/I17A) in the N-terminal helix of Rd-apocyt b₅₆₂ was made to further increase the relative population of this intermediate state at high temperature by selectively destabilizing the native state. In the circular dichroism thermal melting experiment, this mutant showed apparent two-state folding behavior. However, in the T-jump experiment, two kinetic phases were observed. Therefore, these results are in agreement with the idea that a folding intermediate is populated on the folding pathway of Rd-apocyt b₅₆₂ . Moreover, it was found that the exponential growth rate of the native state from this intermediate state is roughly (25 µsec)^–1 at 65°C.

Keywords: T-jump; infrared; Rd-apocyt b₅₆₂ ; intermediate; protein folding

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