Lac Repressor-DNA Interactions assessed by Ultrafast Force-Clamp Spectroscopy

We recently developed an ultrafast force-clamp laser trap technique [Capitanio et al., Nature Methods 9,1013-1019(2012)] that allows probing, under controlled force, both long- and short-lived biomolecular interactions (100μs to tens/hundreds of seconds), as well as sub-nanometer conformational changes occurring upon bond formation. Here, we show the application of our method to the study of lactose repressor (LacI). Our results show two kinetically well-distinct populations of interactions, which clearly represent strong interactions (targeting the two operators located 100nm apart from each other: long events in the figure) and fast scanning of LacI along non-cognate DNA (during target-search: short events in the figure). Our results demonstrate the effectiveness of the method to study the sequence-dependent affinity of DNA-binding proteins along the DNA molecule and the effects of force on a wide range of interaction durations, including μs time scales not accessible to other methods. This improvement in time resolution provides also important means of investigation on the long-puzzled mechanism of target search on DNA and possible protein conformational changes occurring upon target recognition.