A streamlined one-day protocol is described to produce isotopically methyl-labeled protein with high levels of deuterium for NMR studies. Using this protocol, the D2O and 2H-glucose content of the media and protonation level of ILV labeling precursors (ketobutyrate and ketovalerate) were varied. The relaxation rate of the multiple-quantum (MQ) state that is present during the HMQC-TROSY pulse sequence was measured for different labeling schemes and this rate was used to predict upper limits of molecular weights for various labeling schemes. The use of deuterated solvents (D2O) or deuterated glucose is not required to obtain 1H–13C correlated NMR spectra of a 50 kDa homodimeric protein that are suitable for assignment by mutagenesis. High quality spectra of 100–150 kDa proteins, suitable for most applications, can be obtained without the use of deuterated glucose. The proton on the β-position of ketovalerate appears to undergo partial exchange with deuterium under the growth conditions used in this study.

The catalytic activity of human glutathione S-transferase A1-1 (hGSTA1-1), a homodimeric detoxification enzyme, is dependent on the conformational dynamics of a key C-terminal helix α9 in each monomer. However, the structural details of how the two monomers interact upon binding of substrates is not well understood and the structure of the ligand-free state of the hGSTA1-1 homodimer has not been resolved. Here, we used a combination of electron paramagnetic resonance (EPR) distance measurements and weighted ensemble (WE) simulations to characterize the conformational ensemble of the ligand-free state at the atomic level. EPR measurements reveal a broad distance distribution between a pair of Cu(II) labels in the ligand-free state that gradually shifts and narrows as a function of increasing ligand concentration. These shifts suggest changes in the relative positioning of the two α9 helices upon ligand binding. WE simulations generated unbiased pathways for the seconds-timescale transition between alternate states of the enzyme, leading to the generation of atomically detailed structures of the ligand-free state. Notably, the simulations provide direct observations of negative cooperativity between the monomers of hGSTA1-1, which involve the mutually exclusive docking of α9 in each monomer as a lid over the active site. We identify key interactions between residues that lead to this negative cooperativity. Negative cooperativity may be essential for interaction of hGSTA1-1 with a wide variety of toxic substrates and their subsequent neutralization. More broadly, this work demonstrates the power of integrating EPR distances with WE rare-events sampling strategy to gain mechanistic information on protein function at the atomic level.

Protease-protease interactions lie at the heart of the biological cascades that provide rapid molecular responses to networks, bacterial infection, and virus trafficking have all evolved to be activated and sustained by protease-protease interactions. Biomimetic strategies designed to target drugs to specific locations have generated proprotein drugs that can be activated by proteolytic cleavage to release native protein. We have previously demonstrated that the modification of enzymes with a customdesigned comb-shaped polymer nanoarmor can shield the enzyme surface and eliminate almost all protein-protein interactions. We now describe the synthesis and characterization of protease-sensitive comb-shaped nanoarmor cages using poly(ethylene glycol) [Sundy, J. S. et al. Arthritis Rheum. 2008, 58(9), 2882-2891]methacrylate macromonomers where the PEG tines of the comb are connected to the backbone of the growing polymer chain by peptide linkers. Protease-induced cleavage of the tines of the comb releases a polymer-modified protein that can once again participate in protein-protein interactions. Atom transfer radical polymerization (ATRP) was used to copolymerize the macromonomer and carboxybetaine methacrylate from initiator-labeled chymotrypsin and trypsin enzymes, yielding proprotease conjugates that retained activity toward small peptide substrates but prevented activity against proteins. Native proteases triggered the release of the PEG side chains from the polymer backbone within 20 min, thereby increasing the activity of the conjugate toward larger protein substrates by 100%. Biomimetic cascade initiation of nanoarmored protease-sensitive protein-polymer conjugates may open the door to a new class of responsive targeted therapies.

Thymidylate kinase (TMK) from Candida albicans (CaTMK) contains a unique 15 residue insert, the CaLoop, that is not found on other TMKs. CaTMK is proficient at phosphorylating deoxyuridine monophosphate (dUMP), showing a rate 6-fold higher than TMP. It has been shown that deletion of the CaLoop reduces the activity towards dUMP by 19-fold, but has only a modest 4-fold decrease in activity towards TMP. The molecular dynamics calculations presented here show that the increased activity towards dUMP is due to an increase in flexibility and correlated motions of the protein that allows the enzyme-dUMP complex to more readily approach a catalytically competent state. Deletion of the CaLoop allows the dUMP-enzyme complex to adopt catalytically non-functional conformations. In contrast, TMP stabilizes the deletion such that it remains in a functional conformation that is similar to the conformation of the original enzyme.

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.

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.

Protein dynamics is at the heart of all cellular processes. Here, we utilize the dHis-CuII NTA label to obtain site-specific information on dynamics for both an α-helix and β-sheet site of GB1, the immunoglobulin binding domain of protein G. Spectral features found in our CW-EPR measurements were consistent with the overall rigid nature of GB1 and with predictions from molecular dynamics simulations. Using this information, we show the potential of this approach to elucidate the role of dynamics in substrate binding of a functionally necessary α-helix in human glutathione transferase A1-1 (hGSTA1-1). We observe two dynamical modes for the helix. The addition of the inhibitor GS-Met and GS-Hex resulted in hGSTA1-1 to favor the more rigid active state conformation, while the faster mode potentially aids the search for substrates. Together the results illustrate the remarkable potential of the dHis-based labelling approach to measure site-specific dynamics using room temperature lineshape analysis.

Plasmodium falciparum thymidylate kinase (PfTMK) is a critical enzyme in the de novo biosynthesis pathway of pyrimidine nucleotides. N-(5'-Deoxy-α-thymidin-5'-yl)- N'-[4-(2-chlorobenzyloxy)phenyl]urea was developed as an inhibitor of PfTMK and has been reported as an effective inhibitor of P. falciparum growth with an EC50 of 28 nM [Cui, H., et al. (2012) J. Med. Chem. 55, 10948-10957]. Using this compound as a scaffold, a number of derivatives were developed and, along with the original compound, were characterized in terms of their enzyme inhibition ( Ki) and binding affinity ( KD). Furthermore, the binding site of the synthesized compounds was investigated by a combination of mutagenesis and docking simulations. Although the reported compound is indicated to be highly effective in its inhibition of parasite growth, we observed significantly lower binding affinity and weaker inhibition of PfTMK than expected from the reported EC50. This suggests that significant structural optimization will be required for the use of this scaffold as an effective PfTMK inhibitor and that the inhibition of parasite growth is due to an off-target effect.

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.

Site-directed spin labeling using two strategically placed natural histidine residues allows for the rigid attachment of paramagnetic Cu2+. This double histidine (dHis) motif enables extremely precise, narrow distance distributions resolved by Cu2+-based pulsed ESR. Furthermore, the distance measurements are easily relatable to the protein backbone-structure. The Cu2+ ion has, till now, been introduced as a complex with the chelating agent iminodiacetic acid (IDA) to prevent unspecific binding. Recently, this method was found to have two limiting concerns that include poor selectivity towards α-helices and incomplete Cu2+-IDA complexation. Herein, we introduce an alternative method of dHis-Cu2+ loading using the nitrilotriacetic acid (NTA)-Cu2+ complex. We find that the Cu2+-NTA complex shows a four-fold increase in selectivity toward α-helical dHis sites. Furthermore, we show that 100% Cu2+-NTA complexation is achievable, enabling precise dHis loading and resulting in no free Cu2+ in solution. We analyze the optimum dHis loading conditions using both continuous wave and pulsed ESR. We implement these findings to show increased sensitivity of the Double Electron-Electron Resonance (DEER) experiment in two different protein systems. The DEER signal is increased within the immunoglobulin binding domain of protein G (called GB1). We measure distances between a dHis site on an α-helix and dHis site either on a mid-strand or a non-hydrogen bonded edge-strand β-sheet. Finally, the DEER signal is increased twofold within two α-helix dHis sites in the enzymatic dimer glutathione S-transferase exemplifying the enhanced α-helical selectivity of Cu2+-NTA.

The structure of thymidylate kinase from Candida albicans, determined by X-ray crystallography, is reported to a resolution of 2.45 Å with a final Rfree of 0.223. Thymidylate kinase from C. albicans possesses a unique 15-residue loop that is not seen in thymidylate kinases from other genera. The structure reported here reveals that the conformation of this loop is constrained by both intra- and intersubunit hydrogen bonding, and a number of key residues in this loop are conserved among different Candida species that are medically important. The substrate specificity of the enzyme was determined using a novel nuclear magnetic resonance-based assay as well as a traditional coupled assay. The enzyme is active against 3'-azido-3'-deoxythymidine monophosphate and moderately active with dGMP. The distinct functional and structural differences between the C. albicans enzyme and the human enzyme suggest that thymidylate kinase is an appropriate target for the development of new antifungal agents.

Single-molecule fluorescence techniques were used to characterize the binding of products and inhibitors to human glutathione S-transferase A1-1 (hGSTA1-1). The identification of at least two different bound states for the wild-type enzyme suggests that there are at least two conformations of the protein, consistent with the model that ligand binding promotes closure of the carboxy-terminal helix over the active site. Ligand induced changes in ensemble fluorescence energy transfer support this proposed structural change. The more predominant state in the ensemble of single molecules shows a significantly faster off-rate, suggesting that the carboxy-terminal helix is delocalized in this state, permitting faster exit of the bound ligand. A point mutation (I219A), which is known to interfere with the association of the carboxy-terminal helix with the enzyme, shows increased rates of interconversion between the open and closed state. Kinematic traces of fluorescence from single molecules show that a single molecule readily samples a number of different conformations, each with a characteristic off-rate.

Thymidylate kinases (TMKs) play a central role in the production of nucleotide precursors that are required for the replication of DNA. Consequently, this enzyme is a potential drug target for the discovery of anti-bacterial, anti-fungal, and anti-parasitic drugs. In addition, TMKs are also involved in the activation of prodrugs. In particular, the anti-HIV drug AZT is activated by human TMK (huTMK) and the low efficiency of huTMK towards AZT is a significant problem in the use of AZT in the treatment of HIV. Finally, nucleotide precursors are required in large amounts by cancerous cells, thus the inhibition of huTMK by chemotherapeutic agents may enhanced the arsenal of drugs that are used to treat cancer. Although there has been some effort to develop inhibitors of TMKs, these efforts have been hampered by the difficulty in performing high throughput screening using compound libraries. In addition, the characterization of TMK-drug complexes has been limited to X-ray diffraction studies which provide static information about the enzyme-drug complex. There have been no attempts to apply high-resolution multi-nuclear NMR techniques to determine the fundamental dynamic properties of these enzymes and how the structure and dynamics of the enzyme are altered by the binding of substrates or inhibitors. As a preliminary step in characterizing these enzymes by NMR we have over-expressed TMKs from yeast, human, and two pathogens - Plasmodium falciparum and Candida albicans. Expression of these TMKs was optimized by the design of synthetic genes for expression in bacteria. In the case of the human enzyme, we are able to routinely produce 250 mg of the enzyme/L of culture. Preliminary NMR spectra of the yeast, human, and plasmodium enzyme show that the protein is a homo-dimer in solution, as anticipated from X-ray studies. The amide and methyl spectra are well resolved, indicating that resonance assignment by traditional TROSY based methods will be feasible for both the amides and the methyl resonances. In particular the high sensitivity and dispersion of the methyl spectra will facilitate characterization of the dynamic properties of these enzymes by carbon and deuterium relaxation. Ligand induced changes in the dynamics and structure of huTMK in solution will be characterized using NMR methods. These studies will provide additional insights into the inability to huTMK to effectively activate AZT. The entropic component of the thermodynamics of substrate binding to TMK from the parasite that causes malaria will also be characterized by determining dynamic changes by NMR methods. The development of NMR methods to study these enzymes also provides a method for high throughput screening of compound libraries by detecting chemical shift changes in the NMR spectral of the enzyme due to binding of a potential lead compound.

Metal ion cofactors can alter the energetics and specificity of sequence specific protein-DNA interactions, but it is unknown if the underlying effects on structure and dynamics are local or dispersed throughout the protein-DNA complex. This work uses EcoRV endonuclease as a model, and catalytically inactive lanthanide ions, which replace the Mg2+ cofactor. Nuclear magnetic resonance (NMR) titrations indicate that four Lu3+ or two La3+ cations bind, and two new crystal structures confirm that Lu3+ binding is confined to the active sites. NMR spectra show that the metal-free EcoRV complex with cognate (GATATC) DNA is structurally distinct from the nonspecific complex, and that metal ion binding sites are not assembled in the nonspecific complex. NMR chemical shift perturbations were determined for 1H-15N amide resonances, for 1H-13C Ile-δ-CH3 resonances, and for stereospecifically assigned Leu-δ-CH3 and Val-γ-CH3 resonances. Many chemical shifts throughout the cognate complex are unperturbed, so metal binding does not induce major conformational changes. However, some large perturbations of amide and side chain methyl resonances occur as far as 34 Å from the metal ions. Concerted changes in specific residues imply that local effects of metal binding are propagated via a β-sheet and an α-helix. Both amide and methyl resonance perturbations indicate changes in the interface between subunits of the EcoRV homodimer. Bound metal ions also affect amide hydrogen exchange rates for distant residues, including a distant subdomain that contacts DNA phosphates and promotes DNA bending, showing that metal ions in the active sites, which relieve electrostatic repulsion between protein and DNA, cause changes in slow dynamics throughout the complex.

Thymidylate kinases (TMKs) play a central role in the production of nucleotide precursors that are required for the replication of DNA. Consequently, this enzyme is a potential drug target for the discovery of anti-bacterial, anti-fungal, and anti-parasitic drugs. In addition, TMKs are also involved in the activation of prodrugs. In particular, the anti-HIV drug AZT is activated by human TMK (huTMK) and the low efficiency of huTMK towards AZT is a significant problem in the use of AZT in the treatment of HIV. Finally, nucleotide precursors are required in large amounts by cancerous cells, thus the inhibition of huTMK by chemotherapeutic agents may enhanced the arsenal of drugs that are used to treat cancer. Although there has been some effort to develop inhibitors of TMKs, these efforts have been hampered by the difficulty in performing high throughput screening using compound libraries. In addition, the characterization of TMK-drug complexes has been limited to X-ray diffraction studies which provide static information about the enzyme-drug complex. There have been no attempts to apply high-resolution multi-nuclear NMR techniques to determine the fundamental dynamic properties of these enzymes and how the structure and dynamics of the enzyme are altered by the binding of substrates or inhibitors. As a preliminary step in characterizing these enzymes by NMR we have over-expressed TMKs from yeast, human, and two pathogens - Plasmodium falciparum and Candida albicans. Expression of these TMKs was optimized by the design of synthetic genes for expression in bacteria. In the case of the human enzyme, we are able to routinely produce 250 mg of the enzyme/L of culture. Preliminary NMR spectra of the yeast, human, and plasmodium enzyme show that the protein is a homo-dimer in solution, as anticipated from X-ray studies. The amide and methyl spectra are well resolved, indicating that resonance assignment by traditional TROSY based methods will be feasible for both the amides and the methyl resonances. In particular the high sensitivity and dispersion of the methyl spectra will facilitate characterization of the dynamic properties of these enzymes by carbon and deuterium relaxation. Ligand induced changes in the dynamics and structure of huTMK in solution will be characterized using NMR methods. These studies will provide additional insights into the inability to huTMK to effectively activate AZT. The entropic component of the thermodynamics of substrate binding to TMK from the parasite that causes malaria will also be characterized by determining dynamic changes by NMR methods. The development of NMR methods to study these enzymes also provides a method for high throughput screening of compound libraries by detecting chemical shift changes in the NMR spectral of the enzyme due to binding of a potential lead compound.

Adenosine Deaminases that Act on RNA (ADARs) are a small family of enzymes found in metazoans which edit pre-mRNAs by modifying the base adenosine to inosine. This editing results in translational mutations as inosine is interpreted as guanosine by translational machinery. Two editing events occur in humans on the Ionotropic Glutamate Receptor (GluR) pre-mRNA and are known to modify its ion permeability and resensitization kinetics. ADARs promiscuously edit perfectly complementary dsRNAs, however, in the context of complex secondary and tertiary structure, ADARs gain significant specificity for their substrates. Toward understanding the elements of ADARs which drive specificity in the context of a wild type substrate, we sought to generate a co-crystal structure of human ADAR2 with RNA and determine the structure of a flexible loop of the catalytic domain to ascertain its function. Crystals of GluR-B RNA and an ADAR2 truncation PP-R2D have been produced in small scale screens, and work to produce larger crystals suitable for x-ray diffraction is ongoing. Several mutants of the ADAR2 catalytic domain (CD), S458G and R455A, have been solved. Both of these mutant structures give some insight into the previously undefined electron density of an unstructured loop located near the catalytic site. These structures remain incomplete, yet provide a better descriptor of the dynamicity of the loop. To understand the kinetic role of the loop we have replaced it with glycine and found that editing is strongly inhibited. Current work focuses on further mutations to the loop to determine its kinetic role. The results of these studies have shown that large complexes of ADAR2 and substrate mimics can be crystallized, which is fortuitous for further structure determination, and that the unstructured loop of the catalytic domain remains highly dynamic despite mutations to decrease mobility.

The HMCM [CG]CBCA experiment (Tugarinov and Kay in J Am Chem Soc 125:13868–13878, 2003) correlates methyl carbon and proton shifts to Cγ, Cβ, and Cα resonances for the purpose of resonance assignments. The relative sensitivity of the HMCM[CG]CBCA sequence experiment is compared to a divide-and-conquer approach to assess whether it is best to collect all of the methyl correlations at once, or to perform separate experiments for each correlation. A straightforward analysis shows that the divide-and-conquer approach is intrinsically more sensitive, and should always be used to obtain methyl-Cγ, Cβ, and Cα correlations. The improvement in signal-to-noise associated with separate experiments is illustrated by the detection of methyl-aliphatic correlations in a 65 kDa protein-DNA complex.

Novel fluorescent tools such as green fluorescent protein analogues and fluorogen activating proteins (FAPs) are useful in biological imaging for tracking protein dynamics in real time with a low fluorescence background. FAPs are single-chain variable fragments (scFvs) selected from a yeast surface display library that produce fluorescence upon binding a specific dye or fluorogen that is normally not fluorescent when present in solution. FAPs generally consist of human immunoglobulin variable heavy (VH) and variable light (VL) domains covalently attached via a glycine- and serine-rich linker. Previously, we determined that the yeast surface clone, VH-VL M8, could bind and activate the fluorogen dimethylindole red (DIR) but that the fluorogen activation properties were localized to the M8VL domain. We report here that both nuclear magnetic resonance and X-ray diffraction methods indicate the M8VL forms noncovalent, antiparallel homodimers that are the fluorogen activating species. The M8VL homodimers activate DIR by restriction of internal rotation of the bound dye. These structural results, together with directed evolution experiments with both VH-VL M8 and M8VL, led us to rationally design tandem, covalent homodimers of M8VL domains joined by a flexible linker that have a high affinity for DIR and good quantum yields.