Resolving dynamics and function of transient states in single enzyme molecules

TL;DR

This study combines advanced fluorescence and spectroscopic techniques to uncover transient conformational states of T4 Lysozyme, revealing new insights into its dynamic structural behavior and potential catalytic mechanisms.

Contribution

It introduces a hybrid fluorescence toolkit that identifies short-lived conformational states of enzymes, including a novel minor state involved in catalysis.

Findings

T4L mainly adopts open and closed states exchanging at 4 μs.

A minor transient state was discovered at 230 μs, possibly involved in product release.

The toolkit accelerates understanding of enzyme dynamics in structural biology.

Abstract

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.