Site-Specific Tryptophan Labels Reveal Local Microsecond-Millisecond Motions of Dihydrofolate Reductase.

Morgan B Vaughn,Qun Li,R Brian Dyer,Chloe Biren,Ashwin Ragupathi

doi:10.3390/molecules25173819

Morgan B Vaughn, Qun Li + Show 3 more

Open Access

https://doi.org/10.3390/molecules25173819

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Abstract

Many enzymes are known to change conformations during their catalytic cycle, but the role of these protein motions is not well understood. Escherichia coli dihydrofolate reductase (DHFR) is a small, flexible enzyme that is often used as a model system for understanding enzyme dynamics. Recently, native tryptophan fluorescence was used as a probe to study micro- to millisecond dynamics of DHFR. Yet, because DHFR has five native tryptophans, the origin of the observed conformational changes could not be assigned to a specific region within the enzyme. Here, we use DHFR mutants, each with a single tryptophan as a probe for temperature jump fluorescence spectroscopy, to further inform our understanding of DHFR dynamics. The equilibrium tryptophan fluorescence of the mutants shows that each tryptophan is in a different environment and that wild-type DHFR fluorescence is not a simple summation of all the individual tryptophan fluorescence signatures due to tryptophan–tryptophan interactions. Additionally, each mutant exhibits a two-phase relaxation profile corresponding to ligand association/dissociation convolved with associated conformational changes and a slow conformational change that is independent of ligand association and dissociation, similar to the wild-type enzyme. However, the relaxation rate of the slow phase depends on the location of the tryptophan within the enzyme, supporting the conclusion that the individual tryptophan fluorescence dynamics do not originate from a single collective motion, but instead report on local motions throughout the enzyme.

Highlights

Enzyme dynamics across a wide range of timescales are important for enzymatic catalysis.Conformational changes and protein motions have been implicated in every step of catalysis from crossing the barrier of the chemistry step on the femtosecond timescale to rotating whole domains on the order of milliseconds [1]
The equilibrium fluorescence confirms that each tryptophan residue is in a unique environment and that there are local quenching interactions
The temperature jump (T-jump) dynamics demonstrate that all of the tryptophan residues are sensitive to ligand binding and dissociation in combination with associated conformational changes

Summary

Introduction

Enzyme dynamics across a wide range of timescales are important for enzymatic catalysis. Conformational changes and protein motions have been implicated in every step of catalysis from crossing the barrier of the chemistry step on the femtosecond timescale to rotating whole domains on the order of milliseconds [1]. Dihydrofolate reductase (DHFR) is a classic model for studying enzyme dynamics and remains an active area of research [2,3,4,5,6,7,8,9]. DHFR is a small flexible enzyme with multiple mobile loops. It is well established that mutations that decrease loop flexibility impair DHFR enzymatic activity [11,12], but important conformational changes are not limited to the flexible loops.

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