Merging microsecond mixing and time-correlated single-photon counting: using time-resolved FRET and time-resolved anisotropy to probe early events in protein folding

摘要

Obtaining geometric snapshots of a protein as it folds will yield insights into how proteins achieve their unique functional native 3-dimensional structure. Time-resolved FRET and time-resolved anisotropy are valuable tools toward obtaining information about site-specific distances and side-chain mobility of the transient structures formed within microseconds of initiating protein folding. To access this timescale we have merged dual-channel TCSPC detection with recently developed laser-micromachining based microsecond turbulent mixer technology to obtain site-specific distance information and rotational correlation times of protein folding intermediates in the 30 microsecond to seconds timescale. Application of this approach shows that chain collapse to globular structures can occur in the several microsecond timescale even for large proteins.