Resolving dynamics and function of transient states in single enzyme molecules

Hugo Sanabria,Mykola Dimura,Suren Felekyan,Ralf Kühnemuth,Katherina Hemmen,Claus A M Seidel,Mark R Fleissner,Felix Koberling,Holger Gohlke,Wayne Hubbell,Thomas-Otavio Peulen,Dmitro Rodnin

doi:10.1038/s41467-020-14886-w

Abstract

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.

Highlights

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 our single-molecule FRET (smFRET)-experiments, we monitor the distance between a donor (D) and acceptor (A) attached to specific amino acids of a T4L variant
We present the experimental evidence for the C3 state and its structural properties

Summary

Introduction

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. 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. Enzymes adopt distinct conformational states during catalysis[1,2], where transiently populated (“excited”) states are often of critical importance in the enzymatic cycle. These states are short-lived and “hidden” to many experimental techniques. We apply and extend the fluorescence analysis toolkit[9,10], developed for dynamic structural biology, to interrogate the catalytic cycle of an enzyme[11]. T4L12 consists of two interrelated subdomains, the N-terminal subdomain (NTsD) and the C-

Methods

Results

Conclusion