Protein Dynamics Is Altered by a High Surface Density of Atomic Transfer Radical Polymerization Polymers.

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.

dGMP Binding to Thymidylate Kinase from Plasmodium falciparum Shows Half-Site Binding and Induces Protein Dynamics at the Dimer Interface.

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.

The Role of Conformational Dynamics in the Recognition and Regulation of Ubiquitination.

The ubiquitination pathway is central to many cell signaling and regulatory events. One of the intriguing aspects of the pathway is the combinatorial sophistication of substrate recognition and ubiquitin chain building determinations. The abundant structural and biological data portray several characteristic protein folds among E2 and E3 proteins, and the understanding of the combinatorial complexity that enables interaction with much of the human proteome is a major goal to developing targeted and selective manipulation of the pathway. With the commonality of some folds, there are likely other aspects that can provide differentiation and recognition. These aspects involve allosteric effects and conformational dynamics that can direct recognition and chain building processes. In this review, we will describe the current state of the knowledge for conformational dynamics across a wide timescale, address the limitations of present approaches, and illustrate the potential to make new advances in connecting dynamics with ubiquitination regulation.

nightshift: A Python program for plotting simulated NMR spectra from assigned chemical shifts from the Biological Magnetic Resonance Data Bank.

Nuclear magnetic resonance (NMR) provides site specific information on local environments through chemical shifts. NMR is widely used in the study of proteins, ranging from determination of three-dimensional (3D) structures to characterizing dynamics and binding of small molecules and other proteins or ligands. Assigned chemical shift data for the atoms within proteins is a treasure trove of information that can facilitate a broad range of biochemical and biophysical studies. The Biological Magnetic Resonance Data Bank (BMRB) is a publicly accessible database that contains a large number of assigned chemical shifts; however, translating this wealth of knowledge into a practical application is not straightforward. Herein we present nightshift: a Python command line utility and library for plotting simulated two-dimensional (2D) and 3D NMR spectra from assigned chemical shifts in the BMRB. This tool allows users to simulate routinely collected amide and methyl fingerprint spectra, backbone triple-resonance assignment spectra, and user-defined custom correlations, including ones that do not necessarily correspond to published experiments. This tool enables experienced NMR spectroscopists, those learning the craft, and interested scientists seeking to utilize NMR the ability to preview or examine a wide range of spectra for proteins whose assignments are deposited in the BMRB, irrespective of whether those experiments have been executed or reported. The tool applies equally to folded and intrinsically disordered proteins, limited only by the existence of a BMRB deposition. The features of nightshift are described along with applications that illustrate the ease with which complicated correlation spectra and binding events can be simulated.

Stabilization of Active Site Dynamics Leads to Increased Activity with 3'-Azido-3'-deoxythymidine Monophosphate for F105Y Mutant Human Thymidylate Kinase.

Thymidylate kinases are essential enzymes with roles in DNA synthesis and repair and have been the target of drug development for antimalarials, antifungals, HIV treatment, and cancer therapeutics. Human thymidylate kinase (hTMPK) conversion of the anti-HIV prodrug 3'-azido-3'-deoxythymidine (AZT or zidovudine) monophosphate to diphosphate is the rate-limiting step in the activation of AZT. A point mutant (F105Y) has been previously reported with significantly increased activity for the monophosphate form of the drug [3'-azidothymidine-5'-monophosphate (AZTMP)]. Using solution nuclear magnetic resonance (NMR) techniques, we show that while the wild-type (WT) and F105Y hTMPK adopt the same structure in solution, significant changes in dynamics may explain their different activities toward TMP and AZTMP. 13C spin-relaxation measurements show that there is little change in dynamics on the ps to ns time scale. In contrast, methyl 1H relaxation dispersion shows that AZTMP alters adenosine nucleotide handling in the WT protein but not in the mutant. Additionally, the F105Y mutant has reduced conformational flexibility, leading to an increase in affinity for the product ADP and a slower rate of phosphorylation of TMP. The dynamics at the catalytic center for F105Y bound to AZTMP are tuned to the same frequency as WT bound to TMP, which may explain the mutant's catalytic efficiency toward the prodrug.