Sequential Folding of Globular Protein Initiated by Fast Loop Closure

Our understanding of the mechanism of ultrafast folding of globular protein depends on our ability to monitor transient sub-domain structural elements in a protein molecule, in situ, during the fast folding transition Ultrafast Förster 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, 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. Thus, the kinetics of change of the acceptor emission intensity reports both folding events simultaneously. The method was applied in a study of the folding mechanism of the B domain of staphylococcal protein A (a three helix buldle). We found that the folding pathway start in loop L2 closure and the helix H3 formation which are very fast, followed by a slower helix H2 formation which is prior to the closure of loop L1. These results show the power of the “Transfer-Quench” method for unraveling hidden folding pathway elements in a protein that was considered as a two-state folder. The results support the hypothesis that early formation of loops are essential in the early phase of the folding pathway.