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

• Type of Material: Article
• Authors: Morgan Vaughn, Emory University; Chloe Biren, Emory University; Qun Li, Emory University; Ashwin Ragupathi, Emory University; Brian Dyer, Emory University
• Language: English
• Date: 2020-09-01
• Publisher: MDPI
• Publication Version: Final Published Version
• Copyright Statement: © 2020 by the authors.
• License: Creative Commons Attribution 4.0 International
• Final Published Version (URL): http://dx.doi.org/10.3390/molecules25173819
• Title of Journal or Parent Work: MOLECULES
• Volume: 25
• Issue: 17
• Grant/Funding Information: This study was supported in part by the Emory Integrated Genomics Core (EIGC), which is subsidized by the Emory University School of Medicine and is one of the Emory Integrated Core Facilities. Additional support was provided by the Georgia Clinical & Translational Science Alliance of the National Institutes of Health under Award Number UL1TR002378.; This work was supported by National Institutes of Health Grant GM53640 (R.B.D.).
• Keywords: motion; MUTATION; DHFR; Physical Sciences; conformation; enzyme; Biochemistry & Molecular Biology; fluorescence; relaxation; dynamic; spectroscopy; Science & Technology; Life Sciences & Biomedicine; Chemistry, Multidisciplinary; STABILITY; LOOP; Chemistry; HYDRIDE TRANSFER; NETWORK; CONFORMATIONAL DYNAMICS; tryptophan
• Research Categories: Chemistry, Biochemistry; Chemistry, General; Biology, Molecular

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.