Abstract
Several investigators have highlighted a correlation between the basic features of the folding process of a protein and its topology, which dictates the folding pathway. Within this conceptual framework we proposed that different members of the cytochrome c (cyt c) family share the same folding mechanism, involving a consensus partially structured state. Pseudomonas aeruginosa cyt c(551) (Pa cyt c(551)) folds via an apparent two-state mechanism through a high energy intermediate. Here we present kinetic evidence demonstrating that it is possible to switch its folding mechanism from two to three state, stabilizing the high energy intermediate by rational mutagenesis. Characterization of the folding kinetics of one single-site mutant of the Pa cyt c(551) (Phe(7) to Ala) indeed reveals an additional refolding phase and a fast unfolding process which are explained by the accumulation of a partially folded species. Further kinetic analysis highlights the presence of two parallel processes both leading to the native state, suggesting that the above mentioned species is a non obligatory on-pathway intermediate. Determination of the crystallographic structure of F7A shows the presence of an extended internal cavity, which hosts three "bound" water molecules and a H-bond in the N-terminal helix, which is shorter than in the wild type protein. These two features allow us to propose a detailed structural interpretation for the stabilization of the native and especially the intermediate states induced by a single crucial mutation. These results show how protein engineering, x-ray crystallography and state-of-the-art kinetics concur to unveil a folding intermediate and the structural determinants of its stability.
Highlights
Sequential transition states and obligatory high energy intermediates [6], alternative models have been proposed [7]
Pa cyt c551 folds through a complex mechanism involving parallel pathways; a fraction of the molecules reaches the native state within 10 ms following a fast folding track, while the remaining portion folds via a sequential transition state mechanism in which the presence of a high energy intermediate was inferred from kinetic analysis in the ms time range [16]
In agreement with the proposed consensus mechanism, we show in this paper that the high energy intermediate state of Pa cyt c551 can be stabilized by a single critical mutation (Phe7 3 Ala) and becomes kinetically detectable
Summary
Protein Expression and Purification—Pa cyt c551 wt was expressed and purified as described previously [17]. Double mixing kinetic experiments were carried out with a “SX-18” stopped-flow instrument (Applied Photophysics). In these experiments refolding or unfolding were initiated by an asymmetric mixing (1-to-10) of the denatured or native protein with buffer or denaturant, respectively, followed by a symmetric mixing with an unfolding or refolding solution. The program supplied by Applied Photophysics was used to determine the nonlinear least squares fit of the fluorescence kinetic data to monophasic, plus steady-state and biphasic decay equations. Data Analysis—Equilibrium fluorescence and far-UV CD measurements as a function of denaturant concentration were fitted according to a two-state model in which only the native and the fully denatured states are populated, using Equation 1. The free energy of denaturation in the absence of denaturant (⌬G0D-N) was calculated assuming a linear dependence of the free energy of unfolding on denaturant concentration
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