Quenching of the triplet state of tryptophan by cysteine after nanosecond UV excitation has provided a new tool for measuring the rate of forming a specific intramolecular contact in disordered polypeptides. Here, we use this technique to measure the kinetics of loop formation in two unfolded single domain proteins: CspTm-C, a 67 residues cold-shock protein from Thermotoga maritima, engineered to contain a single tryptophan (W29) and a single cysteine at the C terminus; C-HP-35, a 36 residues variant of the headpiece subdomain of the F-actin-binding protein villin, containing a single tryptophan (W24) and engineered to contain a cysteine residue at the N terminus. We investigate the kinetic of intramolecular loop formation between tryptophan and cysteine for different denaturant concentrations. For both proteins, the measured triplet decay rates in the unfolded state are tenfold faster than the rates observed in the native state. Experiments on the unfolded proteins without the added cysteine residue show that the faster rate results entirely from contact quenching with cysteine. Differently, the triplet decay rate measured in the native state is equal or slower than the decay rate of free tryptophan in solution, since contact formation with cysteine is prevented in the native conformation of both proteins.
|Titolo:||Kinetics of Intramolecular Contact Formation in Unfolded Proteins|
|Autori interni:||BUSCAGLIA, MARCO|
|Data di pubblicazione:||2005|
|Parole Chiave:||protein folding; contact rate; triplet state quenching; unfolded state; villin headpiece subdomain; collapse; cold shock protein|
|Settore Scientifico Disciplinare:||Settore FIS/07 - Fisica Applicata(Beni Culturali, Ambientali, Biol.e Medicin)|
|Citazione:||Kinetics of Intramolecular Contact Formation in Unfolded Proteins / Marco Buscaglia, Benjamin Schuler, Jan Kubelka, Lisa J. Lapidus, William A. Eaton, James Hofrichter. ((Intervento presentato al 2005. convegno 17. Faltertage tenutosi a Lutherstadt Wittenberg, Germania nel 8 - 10 Aprile.|
|Appare nelle tipologie:||14 - Intervento a convegno non pubblicato|