Determination of ultrafast protein folding rates from loop formation dynamics.
Quenching of the triplet state of tryptophan by contact with cysteine can be used to measure the kinetics of loop formation in unfolded proteins. Here we show that cysteine quenching dynamics also provide a novel method for measuring folding rates when the exchange between folded and unfolded states is faster than the unquenched triplet lifetime (approximately 100 micros). We use this technique to investigate folding/unfolding kinetics of the 35 residue headpiece subdomain of the protein villin, which contains a single tryptophan residue and was engineered to contain a cysteine residue at the N terminus. At intermediate concentrations of denaturant the time-course of the triplet decay consists of two relaxations, the rates and amplitudes of which reveal the fast kinetics for folding and unfolding of this protein. The folding rates extracted using a simple kinetic model are close to those reported previously from laser-induced temperature-jump experiments that employ the change in tryptophan fluorescence as a probe. However, the results differ significantly from those reported from dynamic NMR line shape analysis on a variant with methionine at the N terminus, an issue that remains to be resolved. The analysis of the triplet quenching kinetics also shows that the quenching rates in the unfolded state increase with decreasing denaturant concentration, indicating a compaction of the unfolded protein.
Buscaglia
Marco
M
Laboratory of Chemical Physics, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Building 5, Bethesda, MD 20892-0520, USA.
Kubelka
Jan
J
Eaton
William A
WA
Hofrichter
James
J
D000975:Antioxidants / Q000737:chemistry; D003545:Cysteine / Q000737:chemistry; D008958:Models, Molecular; D011487:Protein Conformation; D017510:Protein Folding; D011506:Proteins / Q000737:chemistry; D008063:Thioctic Acid / Q000737:chemistry; D013997:Time Factors; D014364:Tryptophan / Q000737:chemistry
D000975:Antioxidants; D011506:Proteins; D008063:Thioctic Acid; D014364:Tryptophan; D003545:Cysteine