Resolving dynamics and function of transient states in single enzyme molecules.

Nature Communications
Hugo SanabriaClaus A M Seidel

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.

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Citations

Jun 20, 2020·Protein Science : a Publication of the Protein Society·Gloria YangNobuhiko Tokuriki
Jun 15, 2020·The Journal of Chemical Physics·Bastian Schepers, Holger Gohlke
Oct 10, 2020·The Journal of Chemical Physics·Gilad Haran, Hisham Mazal
Oct 22, 2020·Molecules : a Journal of Synthetic Chemistry and Natural Product Chemistry·Reinier CárdenasCarlos Amero
Feb 4, 2021·Physical Chemistry Chemical Physics : PCCP·Joshua L WortBela E Bode
Oct 28, 2020·Nature Communications·Mykola DimuraHolger Gohlke
Nov 27, 2020·Current Opinion in Structural Biology·Huanhuan ChenTai-Yen Chen
Mar 16, 2021·The Journal of Physical Chemistry Letters·Joshua L WortBela E Bode
May 7, 2021·Chemical Society Reviews·Dmytro DziubaYves Mély
May 27, 2021·The Journal of Physical Chemistry. B·Sung Oh WooYongki Choi
Jun 16, 2021·Cytometry. Part a : the Journal of the International Society for Analytical Cytology·Jared HendersonRichard Eric Davis
Jul 19, 2021·Biochemical and Biophysical Research Communications·Francesco TorricellaLucia Banci
Jun 3, 2021·Current Opinion in Structural Biology·Charlotte M MitonNobuhiko Tokuriki
Sep 4, 2021·Bioinformatics·Fabio D SteffenRichard Börner
Sep 11, 2021·The Journal of Physical Chemistry. B·Narendar KolimiJoshua Alper

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Methods Mentioned

BETA
FRET
fluorescence correlation spectroscopy
electron paramagnetic resonance
fluorescence
X-ray
NMR
FCS
reverse phase chromatography
double

Software Mentioned

fastcluster
FPS
DEER
LabVIEW
LongDistances

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