title: Probing the folding intermediate of Rd-apocyt b562 by protein engineering and infrared T-jump.
abstract: 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 b(562) , a four-helix bundle protein. Previously, it has been shown that Rd-apocyt b(562) 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 b(562) 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 b(562) . Moreover, it was found that the exponential growth rate of the native state from this intermediate state is roughly (25 microsec)(-1) at 65 degrees C.
authors.affiliation: Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104, USA.
authors.forename: Ting
authors.initials: T
authors.lastname: Wang
authors.forename: Zheng
authors.initials: Z
authors.lastname: Zhou
authors.forename: Michelle R
authors.initials: MR
authors.lastname: Bunagan
authors.forename: Deguo
authors.initials: D
authors.lastname: Du
authors.forename: Yawen
authors.initials: Y
authors.lastname: Bai
authors.forename: Feng
authors.initials: F
authors.lastname: Gai
mesh_terms: D002942:Circular Dichroism; D003573:Cytochrome b Group / Q000737:chemistry* / Q000235:genetics; D009154:Mutation; D015202:Protein Engineering / Q000379:methods*; D017510:Protein Folding; D013696:Temperature
chemical_list: D003573:Cytochrome b Group