Compact dimension of denatured states of staphylococcal nuclease

Abstract

Fluorescence and circular dichroism stopped-flow have been widely used to determine the kinetics of protein folding including folding rates and possible folding pathways. Yet, these measurements are not able to provide spatial information of protein folding/unfolding. Especially, conformations of denatured states cannot be elaborated in detail. In this study, we apply the method of fluorescence energy transfer with a stopped-flow technique to study global structural changes of the staphylococcal nuclease (SNase) mutant K45C, where lysine 45 is replaced by cysteine, during folding and unfolding. By labeling the thiol group of cysteine with TNB (5,5'-dithiobis-2-nitrobenzoic acid) as an energy acceptor and the tryptophan at position 140 as a donor, distance changes between the acceptor and the donor during folding and unfolding are measured from the efficiency of energy transfer. Results indicate that the denatured states of SNase are highly compact regardless of how the denatured states (pH-induced or GdmCl-induced) are induced. The range of distance changes between two probes is between 25.6 and 25.4 A while it is 20.4 A for the native state. Furthermore, the folding process consists of three kinetic phases while the unfolding process is a single phase. These observations agree with our previous sequential model: N(0) left arrow over right arrow D(1) left arrow over right arrow D(2) left arrow over right arrow D(3) (Chen et al., J Mol Biol 1991;220:771-778). The efficiency of protein folding may be attributed to initiating the folding process from these compact denatured structures.

Subdomain-Specific Collapse of Denatured Staphylococcal Nuclease Revealed by Single Molecule Fluorescence Resonance Energy Transfer Measurements

“…With the increase of GdmCl concentration, ⟨ E ⟩ of the non-native state of SNase exhibited a gradual decrease, indicating the expansion of the denatured state. Hence, our results of single molecule measurements showed that the equilibrium denaturation of SNase populated at least variable denatured states, the native state, and the possible intermediate, which agrees with recently reported kinetic results …”

“…The extrapolated value for K28C/H124C was a little higher than the respective ⟨ r 2 ⟩ 1/2 of the native state calculated directly from the experimentally determined ⟨ E ⟩ (37 Å for K28C/H124C and 38 Å for K28C/K97C), differing from that of K28C/K97C which is close to the native value. Also, in the same way, the R g of the non-native state extrapolated to native condition is 24 Å for K28C/K97C and 21 Å for K28C/H124C, which are both larger than 15.6 Å for the folded state reported by SAXS . In the calculation of R g , dye-linkers were considered to be equivalent to an additional 6 amino acid residues, and the Gaussian chain model according to Flory scaling law, R g = [149/( n + 6)] 1/2 (⟨ r 2 ⟩/6) 1/2 , was applied.…”

Local Hydrophobicity in Protein Secondary Structure Formation

“…The dimension of the denatured states of SNase and some of its mutants is compact, no matter by acid or GdmCl denaturation. 22) The structure of WT SNase has been solved by both X-ray crystallography and nuclear magnetic resonance. Here we use the structure file 1EY0 for the analysis.…”