NMR Dynamics Studies of Rho130-Polymer Conjugates and Human Thymidylate Kinase

2020-01-08T18:06:08Z (GMT) by Ian Fucci
Nuclear magnetic resonance (NMR) studies on two systems: the RNA binding domain of the E. coli transcriptional termination factor Rho (Rho130), and human thymidylate kinase (hTMPK) will be discussed. Protein-polymer
conjugates produced by atom transfer radical polymerization (ATRP) are an emergent tool in biomedical sciences with uses ranging from pharmaceuticals to biomaterials. Rho130 was used as a model system to explore the structural
and dynamical implications of protein modi fication by polymers grown from the protein surface. Formation of protein-polymer conjugates is a twostep process consisting of: (1) the attachment of an ATRP initiator to the protein and (2) growth of polymers from the protein-attached initiator. By
tracking chemical shift perturbations (CSPs) of 15N- and 13CH3 ILV methyllabeled Rho130 it was found that the covalent attachment of initiators, and not
subsequent polymerization, is more perturbing to protein structure. Initiator attachment results in additional resonance lines in the 15N HSQC corresponding to the amide bonds between the initiator and accessible primary amines
on the protein that were used to map degree of modifi cation, which was confirmed by mass spectrometry. Methyl 1H relaxation dispersion experiments revealed essentially no changes to the s-ms dynamics of Rho130-conjugates.
These fi ndings have implications for the quality control of clinically relevant protein-polymer conjugates. TMPKs catalyze the phosphorylation of TMP using ATP and Mg2+ to form TDP which is subsequently phosphorylated to form the DNA building block TTP. The activation of 3'-azido-3'-deoxythymidine (AZT), an anti-retroviral prodrug, is rate-limited by the conversion of AZT monophosphate (AZTMP)
to the diphosphate by hTMPK; a reaction that occurs at a rate 70-fold slower than TMP conversion. A previously reported point mutant of hTMPK (F105Y) confers a 20-fold increase in activity toward AZTMP while reducing activity with the natural substrate (TMP) by 4-fold. The interaction of wild-type (WT) and F105Y hTMPK with both TMP and AZTMP was investigated using solution state NMR experiments, which showed that all complexes adopt the
same average structure in solution and have few diff erences in dynamics on the ps-ns time scale. Methyl 1H relaxation dispersion experiments indicate that notable changes to the s-ms time scale may lead to di fferences in catalytic rate.
It was found that AZTMP causes signfii cant perturbations to WT hTMPK dynamics in both the ATP- and TMP-binding sites. This active site instability is rescued by the F105Y mutant, which reduces conformational exibility in
these regions leading to the decrease in activity with TMP. Interestingly, the frequency of the motions in the active site is most similar between F105Y hTMPK bound to AZTMP and WT bound to TMP, suggesting the increased AZTMP activity of the F105Y mutant may stem from similar active site tuning.