Evidence for a three-state model of protein folding from kinetic analysis of ubiquitin variants with altered core residues

Abstract

To elucidate the kinetic importance of structural intermediates in single-domain proteins, we measured the effect of solution conditions and amino-acid changes at a central core residue of ubiquitin (Val 26) on the kinetics of folding and unfolding. Kinetic analysis in terms of a sequential three-state mechanism provides insight into the contribution of specific interactions within the ubiquitin core to the structural stability of the native and intermediate states. The observations that disruptive mutations and/or addition of denaturants result in an apparent two-state folding process with slower rates is explained by the destabilization of a partially folded intermediate, which is in rapid equilibrium with unfolded states. The model predicts that under sufficiently stabilizing conditions kinetic intermediates may become populated even for proteins showing apparent two-state kinetics.

Molecular dynamics simulations of hydrophobic collapse of ubiquitin

“…Khorasanizadeh et al (1996) report the formation of an on-pathway collapsed intermediate within the 2-3 ms dead time of the experiments, which they describe as a "loosely folded state held together mainly by hydrophobic contacts" (also see Roder & Colbn, 1997). This state affords little protection from exchange of labile amide hydrogens, indicating that it "still lacks stable, persistent hydrogen-bonded structure," but it appears to contain secondary structure (Khorasanizadeh et al, 1993(Khorasanizadeh et al, , 1996. These experimental observations and interpretations are consistent with our "intermediate" (I-C and I-D) states, which have a dynamic and loose hydrophobic core and some secondary structure, although their dynamic nature leads to fluctuations in solvent exposure of the amide hydrogens and only transient formation of hydrogen bonds.…”

“…The folding of ubiquitin has been studied extensively by Roder and co-workers (Briggs & Roder, 1992;Khorasanizadeh et al, 1993Khorasanizadeh et al, , 1996 and reviewed by Baldwin (1 996) and Roder and Col6n (1 997). They conclude that a three state mechanism best describes the kinetics for the folding of ubiquitin.…”

Identification and characterization of protein folding intermediates

“…However, despite the rapidity of their formation, the presence of intermediates can often be inferred indirectly from endpoint analysis. In such analysis, the initial signal (at time zero) and the final signal (at infinite time) of a reaction, determined from extrapolation of one or more exponential functions, are plotted as a function of denaturant concentration [22]. The dependence of the signal at infinite time, the endpoint of the reaction, on denaturant concentration should describe the equilibrium transition, as the signals of the native and denatured states in refolding and unfolding experiments are defined by the endpoint of the reaction kinetics.…”

“…While in a two-state system, the initial signal in refolding experiments defines the signal of the denatured state under refolding conditions (i.e. the refolding amplitude reflects the difference between the native and denatured state at every denaturant concentrations), a significant deviation from the expected signal has been observed in many proteins [22,23]. In these cases, the observed initial signal reflects the endpoint of a faster transition of the unfolded state to a transient intermediate, implying…”

“…that a rapid event is lost in the dead-time of the stopped-flow experiment (burst phase) [22]. The concept of "burst phase" was originally introduced in enzymology by Hartley and Kilby [24], for the reaction of chymotrypsin with excess of p-nitrophenyl acetate, and it is routinely used to detect…”