Microsecond folding dynamics of the F13W G29A mutant of the B domain of staphylococcal protein A by laser-induced temperature jump.
The small size (58 residues) and simple structure of the B domain of staphylococcal protein A (BdpA) have led to this domain being a paradigm for theoretical studies of folding. Experimental studies of the folding of BdpA have been limited by the rapidity of its folding kinetics. We report the folding kinetics of a fluorescent mutant of BdpA (G29A F13W), named F13W*, using nanosecond laser-induced temperature jump experiments. Automation of the apparatus has permitted large data sets to be acquired that provide excellent signal-to-noise ratio over a wide range of experimental conditions. By measuring the temperature and denaturant dependence of equilibrium and kinetic data for F13W*, we show that thermodynamic modeling of multidimensional equilibrium and kinetic surfaces is a robust method that allows reliable extrapolation of rate constants to regions of the folding landscape not directly accessible experimentally. The results reveal that F13W* is the fastest-folding protein of its size studied to date, with a maximum folding rate constant at 0 M guanidinium chloride and 45 degrees C of 249,000 s(-1). Assuming the single-exponential kinetics represent barrier-limited folding, these data limit the value for the preexponential factor for folding of this protein to at least approximately 2 x 10(6) s(-1).
Department of Physics and Astronomy and Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, United Kingdom.
George
G
Dimitriadis
Adam
A
Drysdale
Jeffrey K
JK
Myers
Pooja
P
Arora
Sheena E
SE
Radford
Terence G
TG
Oas
D Alastair
DA
Smith
D019943:Amino Acid Substitution; D006358:Hot Temperature; D007834:Lasers; D009154:Mutation; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013057:Spectrum Analysis; D013205:Staphylococcal Protein A / Q000235:genetics / Q000378:metabolism*
D013205:Staphylococcal Protein A