Ligand-Dependent Conformational Dynamics of Dihydrofolate Reductase

Abstract

Enzymes are known to change among several conformational states during turnover. The role of such dynamic structural changes in catalysis is not fully understood. The influence of dynamics in catalysis can be inferred, but not proven, by comparison of equilibrium structures of protein variants and protein–ligand complexes. A more direct way to establish connections between protein dynamics and the catalytic cycle is to probe the kinetics of specific protein motions in comparison to progress along the reaction coordinate. We have examined the enzyme model system dihydrofolate reductase (DHFR) from Escherichia coli with tryptophan fluorescence-probed temperature-jump spectroscopy. We aimed to observe the kinetics of the ligand binding and ligand-induced conformational changes of three DHFR complexes to establish the relationship among these catalytic steps. Surprisingly, in all three complexes, the observed kinetics do not match a simple sequential two-step process. Through analysis of the relationship between ligand concentration and observed rate, we conclude that the observed kinetics correspond to the ligand binding step of the reaction and a noncoupled enzyme conformational change. The kinetics of the conformational change vary with the ligand's identity and presence but do not appear to be directly related to progress along the reaction coordinate. These results emphasize the need for kinetic studies of DHFR with highly specific spectroscopic probes to determine which dynamic events are coupled to the catalytic cycle and which are not.

Characterizing the Surface Coverage of Protein–Gold Nanoparticle Bioconjugates

“…A detailed protein expression and purification protocol has been previously described. 45 Briefly, C-terminal hexa-histidine tagged E. coli DHFR was cloned and expressed in BL21(DE3). WT DHFR had only the hexa-histidine tag modification.…”

High-pressure protein crystal structure analysis of Escherichia coli dihydrofolate reductase complexed with folate and NADP+

“…The structural dynamics of ecDHFR have also been investigated using nuclear magnetic resonance (NMR), computational simulations and tryptophan fluorescence probing. In total, more than 90 structures have been deposited in the Protein Data Bank (PDB; Falzone et al, 1994;Kitahara et al, 2000;Schnell et al, 2004;McElheny et al, 2005;Boehr et al, 2006Boehr et al, , 2010Hanoian et al, 2015;Kohen, 2015;Reddish et al, 2016;Abdizadeh et al, 2017;Huang et al, 2017;Oyen et al, 2017).…”

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

“…The concentration dependence of the midW mutant relaxation rates follows the same trend as the wild-type relaxation profile. A fast concentration-dependent relaxation rate and a slow concentration-independent relaxation rate were also observed for wt-DHFR [14]. Assuming a simple two-state model where a ligand binds to an enzyme to form an enzymeligand complex, a linear fit of the fast relaxation rate as a function of the sum of free concentrations ( Figures 6C and S4 B,D,F,H) Assuming a simple two-state model where a ligand binds to an enzyme to form an enzymeligand complex, a linear fit of the fast relaxation rate as a function of the sum of free concentrations ( Figure 6C and Figure S4B,D,F,H) provides additional information.…”

“…Recently, temperature jump (T-jump) fluorescence spectroscopy has been used to examine the dynamics of DHFR on the microsecond to millisecond timescale [ 14 ]. Laser-induced T-jump spectroscopy is unique in that it can access dynamics from tens of nanoseconds to several milliseconds.…”