Resolution of the Time Sequence of Fast Folding Transition by the “Transfer-Quench” Method

Abstract

The rates and efficiency of the folding transition of globular protein might be enhanced by early formation of few sub-domain structures. Ultrafast Forster resonance energy transfer (FRET) based methods are ideal for characterization of the transient ensembles of refolding molecules. However, each site specific labeling modification might affect rates of folding of near neighbor structural elements and thus limit the ability to resolve fine differences in rates of folding of these elements. Therefore, it is highly desirable to be able to study the rates of folding of two or more neighboring sub-domain structures using a single mutant in order to facilitate resolution of the order and interdependence of such steps. Here we report the development of the “Transfer-Quench” method for measuring the rate of formation of two structural elements using a single triple-labeled mutant. This method is based on FRET combined with fluorescence quenching. We placed the donor and acceptor at the loop ends’ and a quencher at an α-helical element involved in the node forming the loop. The folding of the triple labeled mutant is monitored at the acceptor emission. The formation of non-local contact (loop closure) increases the time dependent acceptor emission while the closure of the labeled helix turn reduces this emission. The method was applied in a study of the folding mechanism of the B domain of staphylococcal protein A. Only natural amino acids were used as probes and thus possible structural perturbations were minimized (Tyr and Trp residues as donor and acceptor at the ends of a long loop between helices I and II, and Cys residue as a quencher for the acceptor). We found that the closure of the loop is formed with the same rate constant as the nucleation of helix II in line with the nucleation-condensation model.