The effect of atomic transfer radical polymerization (ATRP) polymers on the structure and dynamics of a 14.5 kDa RNA binding protein, Rho130, was assessed using NMR. A near-homogeneous sample was ...generated by optimizing initiator coupling to maximize the number of modified Lys residues. The reactivity of individual Lys residues was correlated with the average solvent accessible surface area from molecular dynamics (MD) simulations and influenced by local interactions. Larger structural changes were seen with the addition of the initiator alone than with polymer growth. Structural changes were localized to the N-terminal helical domain of the protein and MD simulations suggest stabilization of the terminus of one helix by the addition of the ATRP initiator and an initiator-induced change in interhelical angles. Relaxation dispersion shows that polymer addition, but not attachment of the initiator, causes a reduction in the microsecond–millisecond dynamics of the hydrophobic core.
Nitroxide- and Cu2+-based electron spin resonance (ESR) are combined to provide insight into the conformational states of the functionally important α-helix of the human glutathione S-transferase A1. ...Distance measurements on various spin-labeled dimeric human glutathione S-transferase A1-1 all result in bimodal distance distributions, indicating that the C-terminus exists in two distinct conformations in solution, one of which closely matches that found in the crystal structure of the ligand-bound enzyme. These measurements permit the generation of a model of the unliganded conformation. Room temperature ESR indicates that the second conformation has high mobility, potentially enabling the enzyme’s high degree of substrate promiscuity. This model is then validated using computational modeling and further Cu2+-based ESR distance measurements. Cu2+-based ESR also provides evidence that the secondary structure of the second conformation is of helical nature. Addition of S-hexyl glutathione results in a shift in relative populations, favoring the state that is similar to the previously known structure of the ligand-bound enzyme.
Plasmodium falciparum thymidylate kinase (PfTMK) is an essential enzyme for the growth of the organism because of its critical role in the de novo synthesis of deoxythymidine 5′-diphosphate (TDP), a ...precursor for TTP that is required for DNA replication and repair. The kinetics, thermodynamic parameters, and substrate binding properties of PfTMK for TMP, dGMP, ADP, and ATP were measured and characterized by steady-state kinetics and a combination of isothermal titration calorimetry, tryptophan fluorescence titration, and NMR. Mutational studies were performed to investigate residues that contribute to the unique ability of PfTMK to also utilize dGMP as a substrate. Isothermal titration calorimetry experiments revealed that dGMP binding exhibits a unique half-site binding mechanism. The occlusion of the empty site in the dGMP complex is supported by molecular mechanics calculations. Relaxation dispersion experiments show that the dGMP and enzyme complex is more dynamic at the dimer interface than the TMP complex on the μs–ms time scale. The unique properties of dGMP binding need to be considered in the design of guanosine-based PfTMK-specific inhibitors.
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