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Measuring TCR-pMHC Binding In Situ using a FRET-based Microscopy Assay
Published on: October 30, 2015
A C-terminal conformational equilibrium in thymidylate synthase observed by electron paramagnetic resonance
C W Carreras1, N Naber, R Cooke
1Department of Pharmaceutical Chemistry, University of California, San Francisco 94143-0448.
Abstract:
A spin-label was attached to the C-terminal side chain of Lactobacillus casei thymidylate synthase (TS, EC2.1.1.45), and EPR spectroscopy was used to study the change in conformational equilibrium that occurs when the enzyme binds nucleotides or the methylenetetrahydrofolate analog CB3717. The C244T/V316C mutant TS has only two cysteines, the active site Cys-198 and an engineered cysteine which replaces valine as the C-terminal residue. dUMP was used to block the active-site cysteine while the C-terminus was reacted with the spin-label 4-maleimido-2,2,6,6- tetramethylpiperidinyl-1-oxy. Exclusive attachment of the label to the C-terminal cysteine was verified by a study of the labeled enzyme's reaction with 5,5'-dithiobis(2-nitrobenzoic acid). EPR spectra of the labeled enzyme and its complexes were composed of two components corresponding to populations of both flexible and more immobilized forms of the C-terminus (tau C = 1 and 9.7 ns, respectively). Ligand binding increased the population of the more immobilized form of the C-terminus with the following series: free enzyme < E.dUMP approximately dTMP approximately E.FdUMP < E.CB3717 < E.dUMP.CB3717. Ligand-induced perturbation of the conformational equilibrium was titratable and indicated approximate Kd values of 3 and 13 microM for formation of the E.dUMP and E.CB3717 binary complexes, respectively, and 7 microM for the binding of CB3717 to the E.dUMP complex. Immobilization of the spin-label correlated well with crystallographic B-factors of the C-terminal residue in corresponding TS crystal structures. These results show that TS has two major conformations which are in equilibrium, and the position of the equilibrium changes in the presence of ligands.
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