Time-resolved fluorescence resonance energy transfer study shows a compact denatured state of the B domain of protein A.
The B domain of protein A (BDPA), a three-helix bundle of 60 residues, folds via a nucleation-condensation mechanism in apparent two-state kinetics. We have applied a time-resolved FRET (tr-FRET) approach to characterize the ensembles of BDPA during chemical denaturation. The distribution of the distance between residues 22 and 55, which are close and separated by helices 2 and 3 in the native state, was determined by global analysis of the time-resolved fluorescence decay curves of the probes. Narrow distributions were observed when the protein was equilibrated in guanidinium chloride (GdmCl) concentrations below 1.5 M (native state, N) and above the transition zone at 2.6-3.0 M GdmCl (denatured state, D). Considerably broader distributions were found around the transition point (2.0 M GdmCl) or much higher GdmCl concentrations (>3.0 M). Comparative global analysis of the tr-FRET data showed a compact denatured state of the protein, characterized by narrow distribution and relatively small mean distance between residues 22 and 55 that was observed at mild denaturing conditions (<3 M GdmCl). This experiment supports the two-state folding mechanism of BDPA and indicates the existence of effective nonlocal, probably hydrophobic, intramolecular interactions that stabilize a pretty uniform ensemble of compact denatured molecules at intermediate denaturing conditions.
Huang
F
F
MRC Center for Protein Engineering, Hills Road, Cambridge CB2 0QH, United Kingdom.
Lerner
E
E
Sato
S
S
Amir
D
D
Haas
E
E
Fersht
A R
AR
D031541:Fluorescence Resonance Energy Transfer / Q000379:methods; D019791:Guanidine / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D011489:Protein Denaturation / Q000235:genetics; D017510:Protein Folding; D017434:Protein Structure, Tertiary / Q000235:genetics; D013205:Staphylococcal Protein A / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D013997:Time Factors
D013205:Staphylococcal Protein A; D019791:Guanidine