Slow Cooperative Folding of a Small Globular Protein HPr

Abstract

The folding of an 85-residue protein, the histidine-containing phosphocarrier protein HPr, has been studied using a variety of techniques including DSC, CD, ANS fluorescence, and NMR spectroscopy. In both kinetic and equilibrium experiments the unfolding of HPr can be adequately described as a two-state process which does not involve the accumulation of intermediates. Thermodynamic characterization of the native and the transition states has been achieved from both equilibrium and kinetic experiments. The heat capacity change from the denatured state to the transition state (3. 2 kJ mol-1 K-1) is half of the heat capacity difference between the native and denatured states (6.3 kJ mol-1 K-1), while the solvent accessibility of the transition state (0.36) indicates that its compactness is closer to that of the native than that of the denatured state. The high value for the change in heat capacity upon unfolding results in the observation of cold denaturation at moderate denaturant concentrations. Refolding from high denaturant concentrations is, however, slow. The rate constant of folding in water, (14.9 s-1), is small compared to that reported for other proteins of similar size under similar conditions. This indicates that very fast refolding is not a universal character of small globular proteins which fold in the absence of detectable intermediates.

Characterization of Single-Tryptophan Mutants of Histidine-Containing Phosphocarrier Protein: Evidence for Local Rearrangements during Folding from High Concentrations of Denaturant

“…This finding is again in good agreement with the dependency of the refolding rates on GdnHCl concentration observed for the WT protein refolded in the presence of ANS (13). This result further supports the assumption that the slow phase involves a local rearrangement of tertiary structure and as such is not very sensitive to changes in the denaturant concentration.…”

“…Kinetic refolding studies of F2W, F29W, and F48W showed a major single phase, independent of the probe used (CD, fluorescence, and fluorescence anisotropy) and similar to that of the wild-type protein. In contrast, F22W showed two phases in the fluorescence experiments corresponding to the two phases previously observed in ANS binding studies of the wild-type protein [Van Nuland et al (1998) Biochemistry 37,[622][623][624][625][626][627][628][629][630][631][632][633][634][635][636][637]. Residue 22 was found from NMR studies to be part of the binding interface on HPr for ANS.…”

“…protein (13). This approach enables spectral information to be obtained by integrating the fluorescence intensities above a variable set point.…”

“…As a consequence of the lack of tryptophan and tyrosine residues, however, the spectroscopic probes available for monitoring the folding of wild-type (WT) HPr are limited. Nevertheless, circular dichroism (CD) in the far-and near-ultraviolet (UV) and fluorescence associated with the binding of 8-anilino-1-naphthalenesulfonic acid (ANS) have been combined with nuclear magnetic resonance (NMR) spectroscopy and differential scanning calorimetry (DSC) to investigate equilibrium and kinetic features of the folding and unfolding process (13). From both types of studies it was found that the unfolding of HPr can be adequately described as a two-state process that does not involve significant accumulation of any intermediates.…”

Characterization of the Folding of a 5 2 -Knotted Protein Using Engineered Single-Tryptophan Variants

“…Previous investigations on the folding mechanisms of a few knotted proteins established that these proteins have complex multistep folding pathways with slow kinetics rather than the simple two-state mechanisms that are commonly seen for small, fast-folding, single-domain proteins (6)(7)(8)(9)(10)(11). Despite experimental and computational work over the last 10 years (12)(13)(14)(15)(16)(17), the folding mechanisms of knotted proteins remain poorly understood compared with those described for small model systems that have been studied in great detail (18)(19)(20)(21)(22)(23).…”