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 (~100 um).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.
Determination of Ultrafast Protein Folding Rates from Loop Formation Dynamics / M. Buscaglia, J. Kubelka, W.A. Eaton, J. Hofrichter. - In: JOURNAL OF MOLECULAR BIOLOGY. - ISSN 0022-2836. - 347:3(2005 Apr 01), pp. 657-664.
|Titolo:||Determination of Ultrafast Protein Folding Rates from Loop Formation Dynamics|
BUSCAGLIA, MARCO (Primo)
|Parole Chiave:||protein folding ; contact rate ; triplet state quenching ; unfolded state ; villin headpiece subdomain|
|Settore Scientifico Disciplinare:||Settore FIS/07 - Fisica Applicata(Beni Culturali, Ambientali, Biol.e Medicin)|
|Data di pubblicazione:||1-apr-2005|
|Digital Object Identifier (DOI):||http://dx.doi.org/10.1016/j.jmb.2005.01.057|
|Appare nelle tipologie:||01 - Articolo su periodico|