  We used a hybrid fluorescence spectroscopic toolkit to monitor T4 Lysozyme
(T4L) in action. By unraveling the kinetic and dynamic interplay of the
conformational states, we sought to elucidate the dynamic structural biology of
T4L. In particular, by combining single-molecule and ensemble multiparameter
fluorescence detection, EPR spectroscopy, mutagenesis, and FRET-positioning and
screening, we characterized three short-lived conformational states within the
conformational landscape of the T4L over the ns-ms timescale. The use of 33
FRET-derived distance sets, to screen known T4L structures, revealed that T4L
in solution mainly adopts the known open and closed states in exchange at 4
$\mu$s. A newly found minor state, undisclosed by at present more than 500
crystal structures of T4L and sampled at 230 $\mu$s, may be actively involved
in the product release step in catalysis. The presented fluorescence
spectroscopic toolkit is anticipated to accelerate the development of dynamic
structural biology.
