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Site-Specific Tryptophan Labels Reveal Local Microsecond

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Site-Specific Tryptophan Labels Reveal Local Microsecond molecules Article Site-Specific Tryptophan Labels Reveal Local Microsecond–Millisecond Motions of Dihydrofolate Reductase Morgan B. Vaughn 1,*, Chloe Biren 2, Qun Li 2, Ashwin Ragupathi 2 and R. Brian Dyer 2,* 1 Division of Natural Sciences, Oglethorpe University, Atlanta, GA 30319, USA 2 Department of Chemistry, Emory University, Atlanta, GA 30322, USA; [email protected] (C.B.); [email protected] (Q.L.); [email protected] (A.R.) * Correspondence: [email protected] (M.B.V.); [email protected] (R.B.D.); Tel.: +1-404-504-1214 (M.B.V.); +1-404-727-6637 (R.B.D.) Academic Editor: Rafik Karaman Received: 24 July 2020; Accepted: 21 August 2020; Published: 22 August 2020 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. Keywords: enzyme; dynamic; conformation; motion; relaxation; spectroscopy; fluorescence; tryptophan; DHFR 1. 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 [2–9]. DHFR is a small flexible enzyme with multiple mobile loops. In particular, the Met20 loop changes conformation throughout the catalytic cycle: it is closed in the substrate-bound complexes and occluded in the product-bound complexes [10]. Enzyme dynamics are paramount for DHFR function. 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. Molecular dynamics simulations and bioinformatics have proposed a network of correlated motions that extends throughout DHFR [13]. Experimental evidence supports Molecules 2020, 25, 3819; doi:10.3390/molecules25173819 www.mdpi.com/journal/molecules Molecules 2020, 25, x FOR PEER REVIEW 2 of 13 Molecules 2020, 25, 3819 2 of 13 network of correlated motions that extends throughout DHFR [13]. Experimental evidence supports thatthat G121, G121, F125, F125, M42, M42, and and I14 I14 are are part part of of the the network network of of correlated correlated amino amino acids, acids, demonstrating demonstrating that that motionsmotions near near and and far far from from the the active active site site are are important important for for catalysis catalysis [ 6[6].]. Recently,Recently, temperature temperature jump jump (T-jump) (T-jump) fluorescence fluorescence spectroscopy spectroscopy has has been been used used to to examine examine the the dynamicsdynamics of of DHFR DHFR on theon microsecond the microsecond to millisecond to mill timescaleisecond [timescale14]. Laser-induced [14]. Laser-induced T-jump spectroscopy T-jump isspectroscopy unique in that is it canunique access in dynamics that it can from access tens of nanosecondsdynamics from to several tens of milliseconds. nanoseconds This to complete several timemilliseconds. regime is difficultThis complete to access time with regime techniques is difficult traditionally to access used with to techniques study enzyme traditionally dynamics used such to asstudy NMR enzyme spectroscopy, dynamics hydrogen such as deuteriumNMR spectroscopy exchange, hydrogen (HDX), stopped-flow, deuterium exchange and molecular (HDX), dynamics stopped- simulationsflow, and molecular [15–18]. Additionally, dynamics simulations because T-jump [15–18]. is aAdditionally, type of relaxation because methodology, T-jump is analyzinga type of T-jumprelaxation transients methodology, provides analyzin informationg T-jump about transients both the forwardprovides and information reverse processes, about both which the forward allows usand to reverse determine processes, the relationship which allows between us to relaxation determine events the relationship based on theirbetween concentration relaxation dependence.events based Previously,on their concentration we measured dependence. the dynamics Previously, of wt-DHFR we withmeasured T-jump the spectroscopy dynamics of usingwt-DHFR tryptophan with T-jump (Trp) fluorescencespectroscopy as ourusing probe tryptophan [14]. DHFR (Trp) has fivefluorescen native tryptophansce as our (W22,probe W30,[14]. W47,DHFR W74, has W133) five located native throughouttryptophans the (W22, enzyme, W30, as W47, shown W74, in Figure W133)1. W22located is located throughout on the the Met20 enzyme, loop; W133as shown is located in Figure at the 1. terminusW22 is located of a beta on sheetthe Met20 near theloop; FG W133 loop; is W30 located is near at the substrate-bindingterminus of a beta site;sheet and near W47 the and FG W74loop; areW30 close is near together the substrate-binding but removed from site; the and active W47 site. and We W74 observed are close relaxation together rates but onremoved a microsecond from the timescaleactive site. strongly We observed coupled torelaxation ligand association rates on /adissociation microsecond and timescale a slower conformationalstrongly coupled change to ligand that wasassociation/dissociation not correlated to ligand and binding. a slower Since conformational wild-type DHFR change (wt-DHFR) that was has not multiple correlated tryptophans, to ligand thebinding. previous Since study wild-type could not DHFR identify (wt-DHFR) the origin has of mult the observediple tryptophans, slow conformational the previous change. study could not identify the origin of the observed slow conformational change. FigureFigure 1. 1.Crystal Crystal structurestructure (Protein(Protein DataData Bank: Bank: 1RX2)1RX2) ofof wild-typewild-type E.E. coli coli dihydrofolate dihydrofolate reductase reductase (DHFR)(DHFR) (magenta) (magenta) with with oxidized oxidized nicotinamide nicotinamide adenine adenine dinucleotide dinucleotide phosphate phosphate (NADP (NADP+)+) cofactor cofactor (blue)(blue) and and folate folate (yellow). (yellow). The The five five native native tryptophan tryptophan (Trp) (Trp) residues residues are are labeled labeled and and shown shown in in green. green. Here,Here, wewe characterizecharacterize thethe equilibriumequilibrium fluorescencefluorescence andand T-jumpT-jump dynamicsdynamics ofof DHFRDHFR mutants,mutants, eacheach with with a a single single tryptophan tryptophan residue residue (denoted (denoted asasmidW midWmutants). mutants). WeWe foundfound thatthat allallof ofthe the midW midW mutantsmutants exhibited exhibited biphasic biphasic relaxation relaxation rates rates similar similar to to wt-DHFR wt-DHFR with with a a concentration-dependent concentration-dependent fast fast relaxationrelaxation rate rate and and a concentration-independenta concentration-independent slow slow relaxation relaxation rate. rate. Notably, Notably, the slowthe slow relaxation relaxation rate ofrate the of midW the midW mutants mutants differs differs based onbased the locationon the loca of thetion Trp of probethe Trp within probe the within enzyme the and enzyme is generally and is fastergenerally than faster the slow than event the slow observed event previously observed inprev wt-DHFR.iously in This wt-DHFR. evidence This supports evidence the supports conclusion the thatconclusion the observed that the relaxation observed rate relaxation in wt-DHFR rate likely in wt-DHFR arises from likely conformational arises from conformational changes that modulate changes thethat Trp–Trp modulate interactions the Trp–Trp and thatinteractions individual and tryptophan that individual residues tryptophan report on local residues motions. report on local motions. 2. Results and Discussion 2.1. Protein Characterization Molecules 2020, 25, x FOR PEER REVIEW 3 of 13 To verify that the Trp to phenylalanine (Phe) mutations did not adversely affect the stability and function Moleculesof midW2020 ,mutant25, 3819 DHFR enzymes, the melting temperature and activity of the enzymes3 of 13 were examined. The
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