Acyclovir and similar nucleoside analogs form an essential frontline treatment for various herpesvirus infections of the eye. However, these drugs have low ocular retention when delivered topically and need to be administered several times every day. We have previously demonstrated that acyclovir loaded into activated carbon can be used to significantly decrease dosage frequency in a murine model of ocular infection. In this study, we demonstrate that other nucleoside analogs such as ganciclovir, penciclovir and famciclovir have excellent loading and release profile similar to acyclovir. Similarly we also demonstrate that nucleoside analog loaded carbons termed DECON are effective at very low concentrations in treating active viral infection of human corneal epithelial cells. In this study, using a variety of techniques to evaluate corneal dryness, nerve sensitivity, intraocular pressure, corneal thickness, and somatic inflammation, we report that DECON is well tolerated after administration three times daily over the course of four weeks.

Many small molecules have been identified as entry inhibitors of filoviruses. However, a lack of understanding of the mechanism of action for these molecules limits further their development as anti-filoviral agents. Here we provide evidence that toremifene and other small molecule entry inhibitors have at least three distinctive mechanisms of action and lay the groundwork for future development of anti-filoviral agents. The three mechanisms identified here include: (1) direct binding to the internal fusion loop region of Ebola virus glycoprotein (GP); (2) the HR2 domain is likely the main binding site for Marburg virus GP inhibitors and a secondary binding site for some EBOV GP inhibitors; (3) lysosome trapping of GP inhibitors increases drug exposure in the lysosome and further improves the viral inhibition. Importantly, small molecules targeting different domains on GP are synergistic in inhibiting EBOV entry suggesting these two mechanisms of action are distinct. Our findings provide important mechanistic insights into filovirus entry and rational drug design for future antiviral development.

15N labeled amino acids are routinely used to label proteins or nucleic acids for study by NMR. However, NMR studies of 15N labeled amino acids in metabolite studies have not been pursued extensively, presumably due to line broadening present under standard experimental conditions. In this work, we show that lowering the temperature to −5 °C allows facile characterization of 15N-labeled amino acids. Further, we show that this technique can be exploited to measure 15NH3 produced in an enzyme catalyzed reaction and the transport and metabolism of individual amino acids in mammalian cell culture. With respect to 13C-labeled amino acids, 15N-labeled amino acids are less costly and enable direct characterization of nitrogen metabolism in complex biological systems by NMR. In summary, the present work significantly expands the metabolite pools and their reactions for study by NMR.

T-oligo, a guanine-rich oligonucleotide homologous to the 3'-telomeric overhang of telomeres, elicits potent DNA-damage responses in melanoma cells; however, its mechanism of action is largely unknown. Guanine-rich oligonucleotides can form G-quadruplexes (G4), which are stabilized by the hydrogen bonding of guanine residues. In this study, we confirmed the G4-forming capabilities of T-oligo using nondenaturing PAGE, nuclear magnetic resonance, and immunofluorescence. Using an anti-G-quadruplex antibody, we showed that T-oligo can form G4 in the nuclei of melanoma cells. Furthermore, using DNase I in a nuclease degradation assay, G4-T-oligo was found to be more stable than single-stranded T-oligo. G4-T-oligo had decreased antiproliferative effects compared with single-stranded T-oligo. However, G4-T-oligo has similar cellular uptake as single-stranded T-oligo, as shown by FACS analysis. Inhibition of JNK, which causes DNA damage-induced apoptosis, partially reversed the antiproliferative activity of T-oligo. T-oligo also inhibited mRNA expression of human telomerase reverse transcriptase, a catalytic subunit of telomerase that was reversed by JNK inhibition. Furthermore, two shelterin complex proteins TRF2/POT1 were found to be up-regulated and bound by T-oligo, suggesting that T-oligo may mediate dissociation of these proteins from the telomere overhang. These studies show that T-oligo can form a G-quadruplex and that the antitumor effects of T-oligo may be mediated through POT1/TRF2 and via human telomerase reverse transcriptase inhibition through JNK activation.

The effects of Mg2+ on the interaction between ADP, a product of the ATPase reaction, and striated muscle myosin-subfragment 1 (S1) were investigated with both functional and spectroscopic methods. Mg2+ inhibited striated muscle myosin ATPase in the presence of F-actin. Significant effects of Mg2+ were observed in both rate constants of NOE build-up and maximal intensities in WaterLOGSY NMR experiments as F-actin concentration increased. In the absence of F-actin, myosin S1 with Mg2+ bound to a fluorescent ADP analog about five-times tighter than without Mg2+. In the presence of F-actin, the affinity of myosin S1 toward the ADP analog significantly decreased both with and without Mg2+. The equilibrium titration of myosin-S1 into F-actin revealed that in the presence of ADP the apparent dissociation constant (Kd) without Mg2+ was more than five-fold smaller than with Mg2+. Further, we examined effects of F-actin, ADP and Mg2+ binding to myosin on the tertiary structure of myosin-S1 using near UV circular dichroism (CD) spectroscopy. Both in the presence and absence of ADP, there was a Mg2+-dependent difference in the near UV CD spectra of actomyosin. Our results show that Mg2+ affects myosin-ADP and actin-myosin interactions which may be reflected in myosin ATPase activity.

Polysialic acid (polySia) is a unique post-translational modification found on a small set of mammalian glycoproteins. Composed of long chains of α2,8-linked sialic acid, this large, negatively charged polymer attenuates protein and cell adhesion and modulates signaling mediated by its carriers and proteins that interact with these carriers. PolySia is crucial for the proper development of the nervous system and is upregulated during tissue regeneration and in highly invasive cancers. Our laboratory has previously shown that the neural cell adhesion molecule, NCAM, has an acidic surface patch in its first fibronectin type III repeat (FN1) that is critical for the polysialylation of N-glycans on the adjacent immunoglobulin domain (Ig5). We have also identified a polysialyltransferase (polyST) polybasic region (PBR) that may mediate substrate recognition. However, a direct interaction between the NCAM FN1 acidic patch and the polyST PBR has yet to be demonstrated. Here, we have probed this interaction using isothermal titration calorimetry and nuclear magnetic resonance (NMR) spectroscopy. We observe direct and specific binding between FN1 and the PBR peptide that is dependent upon acidic residues in FN1 and basic residues of the PBR. NMR titration experiments verified the role of the FN1 acidic patch in the recognition of the PBR and suggest a conformational change of the Ig5-FN1 linker region following binding of the PBR to the acidic patch. Finally, mutation of residues identified by NMR titration experiments impacts NCAM polysialylation, supporting their mechanistic role in protein-specific polysialylation.

Viral entry into host cells is mediated by membrane proteins in a metastable state that transition to a more stable state upon a stimulus. For example, in the influenza envelope protein hemagglutinin (HA), the low pH in the endosome triggers a transition from the metastable prefusion conformation to the stable fusion conformation. To identify probes that interfere with HA function, here we screened a library of H7 HA peptides for inhibition of H7 HA-mediated entry. We discovered a peptide, PEP87 (WSYNAELLVAMENQHTI), that inhibited H7 and H5 HA-mediated entry. PEP87 corresponds to a highly conserved helical region of the HA2 subunit of HA that self-interacts in the neutral pH conformation. Mutagenesis experiments indicated that PEP87 binds to its native region in the HA trimer. We also found that PEP87 is unstructured in isolation but tends to form a helix as evidenced by CD and NMR studies. Fluorescence, chemical cross-linking, and saturation transfer difference NMR data suggested that PEP87 binds to the neutral pH conformation of HA and disrupts the HA structure without affecting its oligomerization state. Together, this work provides support for a model in which PEP87 disrupts HA function by displacing native interactions of the neutral pH conformation. Moreover, our observations indicate that the HA prefusion structure (and perhaps the metastable states of other viral entry proteins) is more dynamic with transient motions being larger than generally appreciated. These findings also suggest that the ensemble of prefusion structures presents many potential sites for targeting in therapeutic interventions.

The ability of certain oligomeric proanthocyanidins (OPACs) to enhance the biomechanical properties of dentin involves collagen cross-linking of the 1.3-4.5 nm wide space via protein-polyphenol interactions. A systematic interdisciplinary search for the bioactive principles of pine bark has yielded the trimeric PAC, ent-epicatechin-(4β→8)-epicatechin-(2β→O→7,4β→8)-catechin (3), representing the hitherto most potent single chemical entity capable of enhancing dentin stiffness. Building the case from two congeneric PAC dimers, a detailed structural analysis decoded the stereochemistry, spatial arrangement, and chemical properties of three dentin biomodifiers. Quantum-mechanics-driven (1)H iterative full spin analysis (QM-HiFSA) of NMR spectra distinguished previously unrecognized details such as higher order J coupling and provided valuable information about 3D structure. Detection and quantification of H/D-exchange effects by QM-HiFSA identified C-8 and C-6 as (re)active sites, explain preferences in biosynthetic linkage, and suggest their involvement in dentin cross-linking activity. Mapping of these molecular properties underscored the significance of high δ precision in both (1)H and (13)C NMR spectroscopy. Occurring at low- to subppb levels, these newly characterized chemical shift differences in ppb are small but diagnostic measures of dynamic processes inherent to the OPAC pharmacophores and can help augment our understanding of nanometer-scale intermolecular interactions in biomodified dentin macromolecules.

The MYLK gene encodes the multifunctional enzyme, myosin light chain kinase (MLCK), involved in isoform-specific non-muscle and smooth muscle contraction and regulation of vascular permeability during inflammation. Three MYLK SNPs (P21H, S147P, V261A) alter the N-terminal amino acid sequence of the non-muscle isoform of MLCK (nmMLCK) and are highly associated with susceptibility to acute lung injury (ALI) and asthma, especially in individuals of African descent. To understand the functional effects of SNP associations, we examined the N-terminal segments of nmMLCK by 1H-15N heteronuclear single quantum correlation (HSQC) spectroscopy, a 2-D NMR technique, and by in silico molecular modeling. Both NMR analysis and molecular modeling indicated SNP localization to loops that connect the immunoglobulin-like domains of nmMLCK, consistent with minimal structural changes evoked by these SNPs. Molecular modeling analysis identified protein-protein interaction motifs adversely affected by these MYLK SNPs including binding by the scaffold protein 14-3-3, results confirmed by immunoprecipitation and western blot studies. These structure-function studies suggest novel mechanisms for nmMLCK regulation, which may confirm MYLK as a candidate gene in inflammatory lung disease and advance knowledge of the genetic underpinning of lung-related health disparities.

The WaterLOGSY (WL) and saturation transfer difference (STD) NMR experiments have proven to be extremely useful techniques to characterize interactions between small molecules and large biomolecules. In this work we compare the relative sensitivities of WL and STD NMR using 3 experimental systems: ketoprofen (KET)–bovine serum albumin (BSA), tert-butyl hydroquinone (TBHQ)–hemagglutinin (HA), and chloramphenicol (CAM)–ribosome (70S). In all cases we find that WL is more sensitive than STD for a given experimental time with the ratios ranging from 3.2 for KET–BSA to 16 for TBHQ–HA and CAM–70S. We attribute the increased sensitivity of WL to be due to simultaneous saturation of multiple sources of cross correlation, including direct NOEs of 1H of water and exchangeable groups and indirect NOEs of 1H–C groups. We suggest that the outstanding sensitivity of WL make it ideally suited for drug screening efforts targeting very large biomolecules at relatively low concentrations.

NMR has proven to be an invaluable technique for identifying and characterizing ligand interactions with biomolecules. NMR-based detection of ligand binding to protein targets is described. Specifically, the use of the WaterLOGSY NMR experiment to screen mixtures of compounds from a fragment library for binding to influenza H5 hemagglutinin is detailed.

Bdellovibrio bacteriovorus is a δ-proteobacterium that preys upon Salmonella spp., E. coli, and other Gram-negative bacteria. Bdellovibrio can grow axenically (host-independent, HI, rare and mutation-driven) or subsist via a predatory lifecycle (host-dependent, HD, the usual case). Upon contact with prey, B. bacteriovorus enters the host periplasm from where it slowly drains the host cytosol of nutrients for its own replication. At the core of this mechanism is a retractile pilus, whose architecture is regulated by the protein Bd0108 and its interaction with the neighboring gene product Bd0109. Deletion of bd0108 results in negligible pilus formation, whereas an internal deletion (the one that instigates host-independence) causes mis-regulation of pilus length. These mutations, along with a suite of naturally occurring bd0108 mutant strains, act to control the entry to HI growth. To further study the molecular mechanism of predatory regulation, we focused on the apparent lifecycle switch protein Bd0108. Here we characterize the solution structure and dynamics of Bd0108 using nuclear magnetic resonance (NMR) spectroscopy complemented with additional biophysical methods. We then explore the interaction between Bd0108 and Bd0109 in detail utilizing isothermal titration calorimetry (ITC) and NMR spectroscopy. Together our results demonstrate that Bd0108 is an intrinsically disordered protein (IDP) and that the interaction with Bd0109 is of low affinity. Furthermore, we observe that Bd0108 retains an IDP nature while binding Bd0109. From our data we conclude that Bdellovibrio bacteriovorus utilizes an intrinsically disordered protein to regulate its pilus and control predation signaling.

A method was developed to distinguish Vaccinium species based on leaf extracts using nuclear magnetic resonance spectroscopy. Reference spectra were measured on leaf extracts from several species, including lowbush blueberry (Vaccinium angustifolium), oval leaf huckleberry (Vaccinium ovalifolium), and cranberry (Vaccinium macrocarpon). Using principal component analysis, these leaf extracts were resolved in the scores plot. Analysis of variance statistical tests demonstrated that the three groups differ significantly on PC2, establishing that the three species can be distinguished by nuclear magnetic resonance. Soft independent modeling of class analogies models for each species also showed discrimination between species. To demonstrate the robustness of nuclear magnetic resonance spectroscopy for botanical identification, spectra of a sample of lowbush blueberry leaf extract were measured at five different sites, with different field strengths (600 versus 700 MHz), different probe types (cryogenic versus room temperature probes), different sample diameters (1.7 mm versus 5 mm), and different consoles (Avance I versus Avance III). Each laboratory independently demonstrated the linearity of their NMR measurements by acquiring a standard curve for chlorogenic acid (R-2 = 0.9782 to 0.9998). Spectra acquired on different spectrometers at different sites classifed into the expected group for the Vaccinium spp., confirming the utility of the method to distinguish Vaccinium species and demonstrating nuclear magnetic resonance fingerprinting for material validation of a natural health product.

The HIV envelope glycoprotein gp120 plays a critical role in virus entry, and thus, its structure is of extreme interest for the development of novel therapeutics and vaccines. To date, high resolution structural information about gp120 in complex with gp41 has proven intractable. In this study, we characterize the structural properties of gp120 in the presence and absence of gp41 domains by NMR. Using the peptide probe 12p1 (sequence, RINNIPWSEAMM), which was identified previously as an entry inhibitor that binds to gp120, we identify atoms of 12p1 in close contact with gp120 in the monomeric and trimeric states. Interestingly, the binding mode of 12p1 with gp120 is similar for clades B and C. In addition, we show a subtle difference in the binding mode of 12p1 in the presence of gp41 domains, i.e. the trimeric state, which we interpret as small differences in the gp120 structure in the presence of gp41.

Tooth enamel, the hardest material in the human body, is formed within a self-assembled matrix consisting mostly of amelogenin proteins. Here we have determined the complete mouse amelogenin structure under physiological conditions and defined interactions between individual domains. NMR spectroscopy revealed four major amelogenin structural motifs, including an N-terminal assembly of four α-helical segments (S9-V19, T21-P33, Y39-W45, V53-Q56), an elongated random coil region interrupted by two 310 helices (∼P60-Q117), an extended proline-rich PPII-helical region (P118-L165), and a charged hydrophilic C-terminus (L165-D180). HSQC experiments demonstrated ipsilateral interactions between terminal domains of individual amelogenin molecules, i.e. N-terminal interactions with corresponding N-termini and C-terminal interactions with corresponding C-termini, while the central random coil domain did not engage in interactions. Our HSQC spectra of the full-length amelogenin central domain region completely overlapped with spectra of the monomeric Amel-M fragment, suggesting that the central amelogenin coil region did not involve in assembly, even in assembled nanospheres. This finding was confirmed by analytical ultracentrifugation experiments. We conclude that under conditions resembling those found in the developing enamel protein matrix, amelogenin molecules form complex 3D-structures with N-terminal α-helix-like segments and C-terminal PPII-helices, which self-assemble through ipsilateral interactions at the N-terminus of the molecule.

In severe acute respiratory syndrome coronavirus, the envelope heptad repeat 2 (HR2) plays a critical role in viral entry. Moreover, HR2 is both the target for novel antiviral therapies and, as an isolated peptide, presents a potential antiviral therapeutic. The structure of HR2, as determined by NMR spectroscopy in the presence of the co-solvent trifluoroethanol (TFE), is a trimer of parallel helices, whereas the structure of HR2, as determined by X-ray crystallography, is a tetramer of anti-parallel helices. In this work, we added a nitroxide spin label to the N-terminal region of HR2 and used paramagnetic relaxation enhancement to assess the orientation of the HR2 helices under different solution conditions. We find that the relaxation effects are consistent with an orientation corresponding to a trimer of parallel helices in both the presence and absence of TFE. This work suggests that the different orientation and oligomerization states observed by NMR and X-ray are due to the 11 additional residues present at the N-terminus of the NMR construct.

The sections in this article are 1 Introduction 2 Theoretical Considerations 3 Measurement of Dipolar Couplings 4 Alignment Media for Macromolecules 5 Relation between Alignment and Shape 6 Structure Validation 7 Concluding Remarks 8 Biographical Sketch Related Articles

Extracts from the roots and rhizomes of black cohosh (Cimicifuga racemosa) are widely used as dietary supplements to alleviate menopausal symptoms. State-of-the-art quality control measures involve phytochemical fingerprinting of the triterpene glycosides for species identification and chemical standardization by HPLC. In the course of developing materials and methods for standardization procedures, the major C. racemosa triterpene glycoside (1) was isolated and initially thought to be cimicifugoside (2). Detailed HR-LC-MS and 1D and 2D NMR analysis of 1 and 2 unambiguously revealed that 1 is the chlorine-containing derivative of 2, namely, 25-chlorodeoxycimigenol-3-O-beta-d-xyloside. Accordingly, HPLC profiles of black cohosh preparations require revision of the assignments of the chlorinated (1) and nonchlorinated (2) pair. Besides explaining the substantial shift in polarity (DeltatR[RP-18] ca. 20 min), 25-deoxychlorination opens a new pathway of structural diversification in triterpene glycoside chemistry. As chemical conversion of 2 into 1 could be demonstrated, deoxychlorination may be interpreted as artifact formation. Simultaneously, however, it is a potentially significant pathway for the gastric in vivo conversion ("nature's prodrug") of the relatively polar triterpene glycosides into significantly less polar chlorinated derivatives with altered pharmacological properties.