We use a hybrid fluorescence spectroscopic toolkit to monitor T4 Lysozyme (T4L) in action by unraveling the kinetic and dynamic interplay of the conformational states. In particular, by combining single-molecule and ensemble multiparameter fluorescence detection, EPR spectroscopy, mutagenesis, and FRET-positioning and screening, and other biochemical and biophysical tools, we characterize three short-lived conformational states over the ns-ms timescale. The use of 33 FRET-derived distance sets, to screen available T4L structures, reveal that T4L in solution mainly adopts the known open and closed states in exchange at 4 µs. A newly found minor state, undisclosed by, at present, more than 500 crystal structures of T4L and sampled at 230 µs, may be actively involved in the product release step in catalysis. The presented fluorescence spectroscopic toolkit will likely accelerate the development of dynamic structural biology by identifying transient conformational states that are highly abundant in biology and critical in enzymatic reactions.
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