Photo-induced electron transfer between tryptophan and cysteine: pH dependence

Photo-induced electron transfer (PET) between tryptophan (W) and cysteine (C) is used to measure intra-molecular contact formation times in intrinsically disordered proteins (IDPs) and polymers. In this technique, a unique W and C pair are introduced into the protein sequence, at specific positions. Following nanosecond UV laser pulse excitation the W is excited to its triplet state, and the excited state triplet population is monitored in time via transient absorption. Quenching of the W triplet state occurs via electron transfer when C contacts W, due to stochastic collisions occurring in the disordered chain. The measured W triplet relaxation times report on intra-molecular contact formation times. By measuring such relaxation times under varying pH conditions, our group studies how electrostatic interactions affect IDP conformational ensembles and dynamics1-3 (see poster on NTAIL by L. Otteson et al.). The quantitative interpretation of the data requires knowledge of the bi-molecular quenching rate between W and C, freely diffusing in solution, at different pH values.We present an experimental study of the pH-dependence of W-C bimolecular quenching rates in solution, comparing near-physiological pH (7.6) to low pH (4.0), a range that is useful for our IDP experiments. We find that while the natural relaxation rate of the W triplet state (in the absence of quencher) is significantly larger at this pH, the bimolecular quenching rate (in the presence of C) does not vary much. We discuss the reasons for this effect and the implications to studying IDP dynamics.