Conformational diversity defines substrate specificity of thymidylate/uridylate kinase from Candida albicans.

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.

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.

Double Histidine Based EPR Measurements at Physiological Temperatures Permit Site-Specific Elucidation of Hidden Dynamics in Enzymes.

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 ß-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.

On-chip colloidal quantum dot devices with a CMOS compatible architecture for near-infrared light sensing.

Solution-processed semiconductors that exhibit tunable light absorption and can be directly integrated into state-of-the-art silicon technologies are attractive for near-infrared (NIR) light detection in applications of medical imaging, night vision cameras, hyperspectral sensing, etc. Colloidal quantum dot (CQD) is regarded as a promising candidate for its solution-processability and superior optoelectronic properties. Here we propose an on-chip CQD photodetector, photodiode-oxide-semiconductor field-effect transistor, for NIR light sensing. This CMOS compatible device architecture utilizes silicon as a channel for carrier transport and PbS CQD as the light absorbing material controlling the channel conductivity. While the light with a wavelength longer than about 1100 nm cannot excite a photocurrent in commercial silicon-based photodetectors due to the absorption cutoff of silicon, the proposed photodetector can have responses owing to the usage of a PbS CQD photodiode. Simulations showed that the photodiode could provide photovoltage to the semiconductor, forming an inversion layer at the oxide-semiconductor interface, and the electron density at the interface is significantly enhanced. As a result, currents could flow through this layer with ease between the source and drain electrodes. For a proof-of-concept demonstration, we experimentally connected a CQD photodiode with a commercial silicon transistor and proved that the current from the transistor could be increased by photovoltage provided by the photodiode under NIR light illumination. The device shows a responsivity of 5.9A/W at the wavelength of 1250 nm.

Interaction of α-Thymidine Inhibitors with Thymidylate Kinase from Plasmodium falciparum.

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.

The Structure of Thymidylate Kinase from Candida albicans Reveals a Unique Structural Element.

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.

Metal Ion Binding at the Catalytic Site Induces Widely Distributed Changes in a Sequence Specific Protein-DNA Complex.

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 ß-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.

Divide and conquer is always best: sensitivity of methyl correlation experiments.

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, Cß, 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, Cß, 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.

Specific labeling of threonine methyl groups for NMR studies of protein-nucleic acid complexes.

Specific (13)C labeling of Thr methyl groups has been accomplished via the growth of a standard laboratory strain of Escherichia coli on [2-(13)C]glycerol in the presence of deuterated isoketovalerate, Ile, and Ala. Diversion of the label from the Thr biosynthetic pathway is suppressed by including Lys, Met, and Ile in the growth medium. This method complements the repertoire of methyl labeling schemes for NMR structural and dynamic studies of proteins and is particularly useful for the study of nucleic acid binding proteins because of the high propensity of Thr residues at protein-DNA and -RNA interfaces.

Organophosphate detoxification by membrane-engineered red blood cells.

Biotherapeutics have achieved global economic success due to their high specificity towards their drug targets, providing exceptional safety and efficiency. The ongoing shift away from small molecule drugs towards biotherapeutics heightens the need to further improve the pharmacokinetics of these biological drugs. Three pervasive obstacles that limit the therapeutic capacity of biotherapeutics are proteolytic degradation, circulating half-life, and the development of anti-drug antibodies. These challenges can culminate in limited efficiency and consequently warrant the need for higher drug doses and more frequent administration. We have explored the coupling of biotherapeutics to long-lived and biocompatible red blood cells (RBCs) to address limited pharmacokinetics. Butyrylcholinesterase (BChE), for example, provides prophylactic protection against organophosphate nerve agents (OPNAs), yet the short circulation life of the drug requires extraordinary doses. Herein, we report the rapid and tunable chemical engineering of BChE to RBC membranes to create a cell-based delivery system that retains the enzyme activity and enhances stability. In a three-step process that first pre-modifies BChE with a cell-reactive polymer chain, primes the cells for engineering, and then grafts the conjugates to the cells, we attached over 2 million BChE molecules to the surface of each RBC without diminishing the bioscavenging capacity of the enzyme. Critically, this membrane-engineering approach was cell-tolerated with minimal hemolysis observed. These results provide strong evidence for the ability of engineered RBCs to serve as an enhanced biotherapeutic delivery vehicle. STATEMENT OF SIGNIFICANCE: Organophosphate nerve agents (OPNAs) are one of the most lethal forms of chemical warfare. After exposure to OPNAs, a patient is given life-saving therapeutics, such as atropine and oxime. However, these drugs are limited, and the patient can still suffer from irreparable injuries. Given the toxicity of OPNAs, access to a prophylactic is vital. We have created an enhanced delivery system for prophylactic butyrylcholinesterase (BChE) by engineering this biotherapeutic to the red blood cell (RBC) surface. In three simple steps that first pre-modifies BChE with a cell-reactive polymer, primes the cells for engineering, and then grafts the conjugates to the cells, we attached over 2 million BChE molecules to a single RBC while retaining the enzyme's activity and enhancing its stability.

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.