Amide Signals Research Articles

Experimental and DFT Investigation on the Influence of Electron Donour/Acceptor on the Hydrogen Bonding Interactions of 1-(1,3-Benzothiazol-2-yl)-3-(R-benzoylthiourea)

The presence of two different chromophores in benzothiazole molecule namely benzothiazole and aromatic rings lead to interesting chemical and biological properties that attract more researches on the compounds. Three new benzothiazolyl-benzoythiourea compounds namely 1-(1,3-benzothiazol-2-yl)-3-(benzoylthiourea) (BBT), 1-(1,3-benzothiazol-2-yl)-3-(4-chlorobenzoylthiourea) (BBT-4Cl) and 1-(1,3-benzothiazol-2-yl)-3-(4-methoxybenzoylthiourea) (BBT-4OCH3) with different electron withdrawing substituents (R) at the para positions on the benzene ring of benzoylthiourea ring have been synthesized from the reaction of R-benzoyl isothiocyanate (R= H, Cl, and OCH3) and 2-aminobenzothiazole. The compounds were characterized by spectroscopic techniques (infrared, 1H proton NMR and UV-Vis). The IR spectra showed the frequency signals of n (C=O), n (C=S), n (N-H) at 1664-1673, 1238-1249 and 3031-3055 cm-1, respectively. The 1H proton NMR spectra showed the presence of N-H amine and amide signals in the region of (12.14-12.35) and (14.17-14.43) ppm, respectively. The proton signals of the two benzothiazole and benzoylthiourea moieties appear at 7.08-8.16 ppm. A theoretical study based on Density Functional Theory (DFT) and Time-Dependent (TD) DFT was conducted to optimize the geometrical structure and investigate the electronic properties of title compounds. The highest occupied molecular orbital (HOMO) was found on the benzothiazole moiety; while, the lowest-unoccupied molecular orbital (LUMO) was located at the benzoylthiourea fragment. The DFT optimized structures possessed an intramolecular hydrogen bonding and the types of para substituents used influenced the properties of hydrogen bonding.

Dimerization and Conformational Exchanges of the Receiver Domain of Response Regulator PhoB from Escherichia coli.

PhoB is a response regulator of PhoR/PhoB two-component system from Escherichia coli that is involved in the environmental phosphate regulation. It has been reported that the N-terminal receiver domain (PhoBN) forms a dimer using the α1-α5 face in the apo state and a dimer using the α4-β5-α5 face in the active state investigated by X-ray crystallography. However, it is not clear whether the conformational switch of the dimer is dependent on phosphorylation. Here, we report the NMR studies of PhoBN in solution in its apo form. We observed that the secondary structural fragments of apo PhoBN characterized by NMR are almost the same as those determined by crystallography, but the NMR spectrum of PhoBN shows inhomogeneous amide signals. Concentration dilution experiments and backbone relaxation parameters showed that PhoBN exists in equilibrium between monomer and dimer states. Using paramagnetic relaxation enhancement experiments, we demonstrated that the dimer of apo PhoBN forms several transient dimer interfaces in solution. This finding suggested that, in addition to the monomer-to-dimer exchange, the inactive conformation of PhoBN has different domain arrangements, which are independent of phosphorylation. It provides an experimental data for the conformational selection mechanism of the phosphorylation of PhoBN.

Total Synthesis of Scytonemide A Employing Weinreb AM Solid-Phase Resin.

The human 20S proteasome inhibitor scytonemide A (1), a macrocyclic imine originally isolated from the cyanobacterium Scytonema hofmanni, was synthesized via a biomimetic solid-phase peptide synthesis (SPPS) approach employing the Weinreb AM resin. Utilizing this approach, cyclization of the protected heptapeptide via formation of the imine bond occurred spontaneously upon cleavage from the resin in the presence of a reducing agent and subsequent aqueous workup. The final deprotection step necessary to produce the natural product was accomplished under slightly basic conditions, facilitating cleavage of the silyl ether group while leaving the macrocycle intact. Purification of the synthetic scytonemide A was accomplished via normal-phase flash column chromatography, potentially facilitating larger scale preparation of the compound necessary for future mechanistic and SAR studies. The structure of the target compound was confirmed by NMR spectroscopy, which also shed light on differences in the spectroscopic data obtained for the synthetic and natural scytonemide A samples for some of the amide and alcohol signals in the 1H NMR spectrum.

Solid state NMR of isotope labelled murine fur: a powerful tool to study atomic level keratin structure and treatment effects

We have prepared mouse fur extensively 13C,15N-labelled in all amino acid types enabling application of 2D solid state NMR techniques which establish covalent and spatial proximities within, and in favorable cases between, residues. 13C double quantum–single quantum correlation and proton driven spin diffusion techniques are particularly useful for resolving certain amino acid types. Unlike 1D experiments on isotopically normal material, the 2D methods allow the chemical shifts of entire spin systems of numerous residue types to be determined, particularly those with one or more distinctively shifted atoms such as Gly, Ser, Thr, Tyr, Phe, Val, Leu, Ile and Pro. Also the partial resolution of the amide signals into two signal envelopes comprising of α-helical, and β-sheet/random coil components, enables resolution of otherwise overlapped α-carbon signals into two distinct cross peak families corresponding to these respective secondary structural regions. The increase in resolution conferred by extensive labelling offers new opportunities to study the chemical fate and structural environments of specific atom and amino acid types under the influence of commercial processes, and therapeutic or cosmetic treatments.Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-016-0056-7) contains supplementary material, which is available to authorized users.

Nuclear Magnetic Resonance Observation of α-Synuclein Membrane Interaction by Monitoring the Acetylation Reactivity of Its Lysine Side Chains.

The interaction between α-synuclein (αS) protein and lipid membranes is key to its role in synaptic vesicle homeostasis and plays a role in initiating fibril formation, which is implicated in Parkinson’s disease. The natural state of αS inside the cell is generally believed to be intrinsically disordered, but chemical cross-linking experiments provided evidence of a tetrameric arrangement, which was reported to be rich in α-helical secondary structure based on circular dichroism (CD). Cross-linking relies on chemical modification of the protein’s Lys Cε amino groups, commonly by glutaraldehyde, or by disuccinimidyl glutarate (DSG), with the latter agent preferred for cellular assays. We used ultra-high-resolution homonuclear decoupled nuclear magnetic resonance experiments to probe the reactivity of the 15 αS Lys residues toward N-succinimidyl acetate, effectively half the DSG cross-linker, which results in acetylation of Lys. The intensities of both side chain and backbone amide signals of acetylated Lys residues provide direct information about the reactivity, showing a difference of a factor of 2.5 between the most reactive (K6) and the least reactive (K102) residue. The presence of phospholipid vesicles decreases reactivity of most Lys residues by up to an order of magnitude at high lipid:protein stoichiometries (500:1), but only weakly at low ratios. The decrease in Lys reactivity is found to be impacted by lipid composition, even for vesicles that yield similar αS CD signatures. Our data provide new insight into the αS–bilayer interaction, including the pivotal state in which the available lipid surface is limited. Protection of Lys Cε amino groups by αS–bilayer interaction will strongly impact quantitative interpretation of DSG cross-linking experiments.

Conformational Dynamics and the Binding of Specific and Nonspecific DNA by the Autoinhibited Transcription Factor Ets-1.

The affinity of the Ets-1 transcription factor for DNA is autoinhibited by an intrinsically disordered serine-rich region (SRR) and a helical inhibitory module (IM) appended to its winged helix-turn-helix ETS domain. Using NMR spectroscopy, we investigated how Ets-1 recognizes specific versus nonspecific DNA, with a focus on the roles of protein dynamics and autoinhibition in these processes. Upon binding either DNA, the two marginally stable N-terminal helices of the IM predominantly unfold, but still sample partially ordered conformations. Also, on the basis of amide chemical shift perturbation mapping, Ets-1 associates with both specific and nonspecific DNA through the same canonical ETS domain interface. These interactions are structurally independent of the SRR, and thus autoinhibition does not impart DNA-binding specificity. However, relative to the pronounced NMR spectroscopic changes in Ets-1 resulting from specific DNA binding, the spectra of the nonspecific DNA complexes showed conformational exchange broadening and lacked several diagnostic amide and indole signals attributable to hydrogen bonding interactions seen in reported X-ray crystallographic structures of this transcription factor with its cognate DNA sequences. Such differences are highlighted by the chemical shift and relaxation properties of several interfacial lysine and arginine side chains. Collectively, these data support a general model in which Ets-1 interacts with nonspecific DNA via dynamic electrostatic interactions, whereas hydrogen bonding drives the formation of well-ordered complexes with specific DNA.

Amide proton signals as pH indicator for in vivo MRS and MRI of the brain—Responses to hypercapnia and hypothermia

Using proton MRS and MRI of mouse brain at 9.4T, this work provides the first in vivo evidence of pH-dependent concurrent changes of three amide signals and related metabolic responses to hypercapnia and hypothermia. During hypercapnia, amide proton MRS signals of glutamine at 6.8–6.9ppm and 7.6ppm as well as of unspecific compounds at 8.1–8.3ppm increase by at least 50% both at 37°C and 22°C. These changes reflect a reduced proton exchange with water. They are strongly correlated with intracellular pH which ranges from 6.75±0.10 to 7.13±0.06 as determined from a shift in creatine phosphokinase equilibrium. In MRI, saturation transfer from aliphatic as well as aromatic and/or amide protons alters slightly during hypercapnia and significantly during hypothermia. The asymmetry in magnetization transfer ratios decreased slightly during hypercapnia and hypothermia. Regardless of pH or temperature, saturation transfer from aliphatic protons between −2 and −4ppm frequency offset to water protons is significantly greater than that from aromatic/amide protons at corresponding offsets between +2 and +4ppm. Irradiation of aliphatic compounds at −3.5ppm frequency offset from water predominantly saturates lipids and water associated with myelin. Taken together, the results indicate that, for the B1 power used in this study, dipolar coupling between aliphatic and water protons rather than proton exchange is the dominant factor in Z-spectra and magnetization transfer ratio asymmetry of the brain in vivo.

Protein residue linking in a single spectrum for magic-angle spinning NMR assignment.

Here we introduce a new pulse sequence for resonance assignment that halves the number of data sets required for sequential linking by directly correlating sequential amide resonances in a single diagonal-free spectrum. The method is demonstrated with both microcrystalline and sedimented deuterated proteins spinning at 60 and 111 kHz, and a fully protonated microcrystalline protein spinning at 111 kHz, with as little as 0.5 mg protein sample. We find that amide signals have a low chance of ambiguous linkage, which is further improved by linking in both forward and backward directions. The spectra obtained are amenable to automated resonance assignment using general-purpose software such as UNIO-MATCH.

Relaxation-compensated CEST-MRI of the human brain at 7 T: Unbiased insight into NOE and amide signal changes in human glioblastoma

Endogenous chemical exchange saturation transfer (CEST) effects of protons resonating near to water protons are always diluted by competing effects such as direct water saturation and semi-solid magnetization transfer (MT). This leads to unwanted T2 and MT signal contributions that contaminate the observed CEST signal. Furthermore, all CEST effects appear to be scaled by the T1 relaxation time of the mediating water pool. As MT, T1 and T2 are also altered in tumor regions, a recently published correction algorithm yielding the apparent exchange-dependent relaxation AREX, is used to evaluate in vivo CEST effects. This study focuses on CEST effects of amides (3.5ppm) and Nuclear-Overhauser-mediated saturation transfer (NOE, −3.5ppm) that can be properly isolated at 7T. These were obtained in 10 glioblastoma patients, and this is the first comprehensive study where AREX is applied in human brain as well as in human glioblastoma. The correction of CEST effects alters the contrast significantly: after correction, the CEST effect of amides does not show significant contrast between contrast enhancing tumor regions and normal tissue, whereas NOE drops significantly in the tumor area. In addition, new features in the AREX contrasts are visible. This suggests that previous CEST approaches might not have shown pure CEST effects, but rather water relaxation shine-through effects. Our insights help to improve understanding of the CEST effect changes in tumors and correlations on a cellular and molecular level.

Long-chain flavodoxin FldB from Escherichia coli.

Flavodoxins are a family of small electron transferases widely distributed in prokaryotes. They utilize a noncovalently bound flavin mononucleotide (FMN) molecule as the redox center, and are able to switch between three different redox states, namely the oxidized (ox) state, the one-electron reduced semiquinone (sq) state, and the twoelectron reduced hydroquinone (hq) state (Knight and Hardy 1967). Since flavodoxins display two redox potentials and can transfer either one or two electrons at a time, they are functionally versatile. Flavodoxins have been identified to play important roles in various biological processes, including photosynthesis, methionine synthesis, biotin synthesis, and the activation of important enzymes such as pyruvate-formate lyase and ribonucleotide reductase (Sancho 2006). In eukaryotes, flavodoxins are integrated into multi-domain proteins and carry out similar redox functions. Flavodoxins can be further classified into the long-chain and short-chain subfamilies, which are distinguished based on the presence or absence of a 20-amino acid extra segment. The flavodoxin-like domains in eukaryotic multidomain proteins are more closely related to the short-chain subfamily. Previous investigations on the extra sequence suggested it is not directly involved in cofactor binding but may play a role in protein partner recognition and interactions, and it was proposed that the short-chain may derive from the long-chain group during evolution (LopezLlano et al. 2004a, b). However, the underlying molecular mechanism is yet unclear and the structural determinants for the distinct biological functions of different flavodoxins remain elusive. The Escherichia coli genome harbors several genes encoding flavodoxin proteins. Among them, the fldA gene encodes the well-characterized flavodoxin 1 protein which belongs to the long-chain subfamily and is essential for bacteria survival (Gaudu and Weiss 2000). The fldB gene encodes another long-chain flavodoxin which shares over 40 % sequence identity with FldA yet is functionally distinct. Insertion mutation of the fldB gene is not lethal to E. coli, and over expression of FldB could not substitute FldA (Gaudu and Weiss 2000). Evidence suggested that the fldB gene is a member of the superoxide response soxRS regulon, and its expression level was induced by paraquat (methyl viologen) (Gaudu and Weiss 2000). However, the exact function of FldB protein remains unclear. We have previously carried out structural and dynamic studies of E. coli flavodoxins including the short-chain MioC and YqcA proteins as well as the long-chain FldA by solution NMR spectroscopy (Hu et al. 2006; Ye et al. 2014). The FldA protein exhibit global conformational exchanges without the presence of FMN cofactor, rendering one third of its backbone amide signals missing in the NMR spectra, and we were unable to determine its solution structures in the apo-form. In contrast, the FldB protein Q. Ye W. Fu Y. Hu (&) C. Jin (&) Beijing Nuclear Magnetic Resonance Center, Peking University, Beijing 100871, China e-mail: yunfei@pku.edu.cn

MRI ParaCEST agents that improve amide based pH measurements by limiting inner sphere water T2exchange

A series of Dy3+ and Tm3+ tetra-substituted DOTAM paraCEST agents incorporating para-substituted anilines has been synthesized and their paraCEST and relaxation properties evaluated. The response of selected agents (aniline and p-methoxyaniline) to changes in pH near the physiological range has also been studied. A crystal structure of Tm3+-p-OMe agent indicates a TSAP geometry with the absence of an inner sphere water molecule (denoted as TSAP'), which is thought to be due to the bulky nature of the amide nitrogen substituent. Two distinct amide signals are observed in the CEST spectrum for this compound, one for each of the isomers that exist in solution: SAP and TSAP with the larger chemical shift centred at −83 ppm compared to bulk water. Due to the non-optimal inner sphere water exchange, these agents produce minimal shortening of T1 and T2 relaxation time constants and therefore decrease relaxation induced signal losses during imaging.

Imaging of Endogenous Exchangeable Proton Signals in the Human Brain Using Frequency Labeled Exchange Transfer Imaging

To image endogenous exchangeable proton signals in the human brain using a recently reported method called frequency labeled exchange transfer (FLEX) MRI. As opposed to labeling exchangeable protons using saturation (i.e., chemical exchange saturation transfer, or CEST), FLEX labels exchangeable protons with their chemical shift evolution. The use of short high-power frequency pulses allows more efficient labeling of rapidly exchanging protons, while time domain acquisition allows removal of contamination from semi-solid magnetization transfer effects. FLEX-based exchangeable proton signals were detected in human brain over the 1-5 ppm frequency range from water. Conventional magnetization transfer contrast and the bulk water signal did not interfere in the FLEX spectrum. The information content of these signals differed from in vivo CEST data in that the average exchange rate of these signals was 350-400 s(-1) , much faster than the amide signal usually detected using direct saturation (∼30 s(-1) ). Similarly, fast exchanging protons could be detected in egg white in the same frequency range where amide and amine protons of mobile proteins and peptides are known to resonate. FLEX MRI in the human brain preferentially detects more rapidly exchanging amide/amine protons compared to traditional CEST experiments, thereby changing the information content of the exchangeable proton spectrum. This has the potential to open up different types of endogenous applications as well as more easy detection of rapidly exchanging protons in diaCEST agents or fast exchanging units such as water molecules in paracest agents without interference of conventional magnetization transfer contrast.

Electron self-exchange of cytochrome c measuredvia13Cdetected protonless NMR

The use of protonless13C′–13C′ EXSY (COCO-EXSY) is proposed here to measure electron self-exchange rates. The experiment is compared to the commonly employed1H and15N EXSY experiments using as a reference system human cytochrome c. In COCO-EXSY, the exchange peaks are stronger than in the other experiments with respect to the self peaks and their intensity is less dependent on the choice of the EXSY mixing time. The use of13C directed detection may be essential for all those cases where T2relaxation is detrimental, as in the case of proteins containing highly paramagnetic metal centers, or rotating slowly in solution, or where the amide signals are difficult to detect due to chemical or conformational exchange. The proposed experiment has a general applicability and can be used to monitor exchange phenomena different from electron self-exchange.

Backbone NMR Assignments of a Prokaryotic Molecular Chaperone, Hsp33 from Escherichia coli

The prokaryotic molecular chaperone Hsp33 achieves its holdase activity upon response to oxidative stress particularly at elevated temperature. Despite many structural studies of Hsp33, which were conducted mainly by X-ray crystallography, the actual structures of the Hsp33 in solution remains controversial. Thus, we have initiated NMR study of the reduced, inactive Hsp33 monomer and backbone NMR assignments were obtained in the present study. Based on a series of triple resonance spectra measured on a triply isotope-[<TEX>$^2H/^{13}C/^{15}N$</TEX>]-labeled protein, sequence-specific assignments of the backbone amide signals observed in the 2D-[<TEX>$^1H/^{15}N$</TEX>]TROSY spectrum could be completed up to more than 96%. However, even considering the small portion of non-assigned resonances due to the lack of sequential connectivity, we confirmed that the total number of observed signals was quite smaller than that expected from the number of amino acid residues in Hsp33. Thus, it is postulated that peculiar dynamic properties would be involved in the solution structure of the inactive Hsp33 monomer. We expect that the present assignment data would eventually provide the most fundamental and important data for the progressing studies on the 3-dimensional structure and molecular dynamics of Hsp33, which are critical for understanding its activation process.

Nuclear Magnetic Resonance Spectral Assignments of α-1,4-Galactosyltransferase LgtC from Neisseria meningitidis: Substrate Binding and Multiple Conformational States

Lipopolysaccharide α-1,4-galactosyltransferase C (LgtC) from Neisseria meningitidis is responsible for a key step in lipooligosaccharide biosynthesis involving the transfer of α-galactose from the sugar donor UDP-galactose to a terminal acceptor lactose. Crystal structures of the complexes of LgtC with Mn(2+) and the sugar donor analogue UDP-2-deoxy-2-fluorogalactose in the absence and presence of the sugar acceptor analogue 4'-deoxylactose provided key insights into the galactosyl-transfer mechanism. Combined with kinetic analyses, the enzymatic mechanism of LgtC appears to involve a "front-side attack" S(N)i-like mechanism with a short-lived oxocarbenium-phosphate ion pair intermediate. As a prerequisite for investigating the required roles of structural dynamics in this catalytic mechanism by nuclear magnetic resonance techniques, the transverse relaxation-optimized amide (15)N heteronuclear single-quantum correlation and methyl (13)C heteronuclear multiple-quantum correlation spectra of LgtC in its apo, substrate analogue, and product complexes were partially assigned. This was accomplished using a suite of complementary spectroscopic approaches, combined with selective isotopic labeling and mutagenesis of all the isoleucine residues in the protein. Only ~70% of the amide signals could be detected, whereas more than the expected number of methyl signals were observed, indicating that LgtC adopts multiple interconverting conformational states. Chemical shift perturbation mapping provided insights into substrate and product binding, including the demonstration that the sugar donor analogue (UDP-2FGal) associates with LgtC only in the presence of a metal ion (Mg(2+)). These spectral assignments provide the foundation for detailed studies of the conformational dynamics of LgtC.

A new method for detecting exchanging amide protons using chemical exchange rotation transfer

In this study, we introduce a new method for amide proton transfer imaging based on chemical exchange rotation transfer. It avoids several artifacts that plague conventional chemical exchange saturation transfer approaches by creating label and reference scans based on varying the irradiation pulse rotation angle (π and 2π radians) instead of the frequency offset (3.5 and -3.5 ppm). Specifically, conventional analysis is sensitive to confounding contributions from magnetic field (B(0)) inhomogeneities and, more problematically, inherently asymmetric macromolecular resonances. In addition, the lipid resonance at -3.5 ppm complicates the interpretation of the reference scan and decreases the resulting contrast. Finally, partial overlap of the amide signal by nearby amines and hydroxyls obscure the results. By avoiding these issues, our new method is a promising approach for imaging endogenous protein and peptide content and mapping pH.

Local and Global Dynamics of the G Protein-Coupled Receptor CXCR1

The local and global dynamics of the chemokine receptor CXCR1 are characterized using a combination of solution NMR and solid-state NMR experiments. In isotropic bicelles (q = 0.1), only 13% of the expected number of backbone amide resonances is observed in (1)H/(15)N HSQC solution NMR spectra of uniformly (15)N-labeled samples; extensive deuteration and the use of TROSY made little difference in the 800 MHz spectra. The limited number of observed amide signals is ascribed to mobile backbone sites and assigned to specific residues in the protein; 19 of the signals are from residues at the N-terminus and 25 from residues at the C-terminus. The solution NMR spectra display no evidence of local backbone motions from residues in the transmembrane helices or interhelical loops of CXCR1. This finding is reinforced by comparisons of solid-state NMR spectra of both magnetically aligned and unoriented bilayers containing either full-length or doubly N- and C-terminal truncated CXCR1 constructs. CXCR1 undergoes rapid rotational diffusion about the normal of liquid crystalline phospholipid bilayers; reductions in the frequency span and a change to axial symmetry are observed for both carbonyl carbon and amide nitrogen chemical shift powder patterns of unoriented samples containing (13)C- and (15)N-labeled CXCR1. In contrast, when the phospholipids are in the gel phase, CXCR1 does not undergo rapid global reorientation on the 10(4) Hz time scale defined by the carbonyl carbon and amide nitrogen chemical shift powder patterns.

Glycosidic intermediates identified in1H MR spectra of intact tumour cells may contribute to the clarification of aspects of glycosylation pathways

The glycosylation process, through the addition of carbohydrates, is a major post-translational modification of proteins and glycolipids. Proteins may be glycosylated in either the secretory pathway leading to N-linked or O-linked glycoproteins or as nucleocytoplasmic glycosylation that targets only single proteins involving a single β-linked N-acetylglucosamine. In both cases, the key precursors are the uridine diphospho-N-acetylhexosamines synthesised by the hexosamine biosynthetic pathway. Furthermore, uridine diphospho-N-acetylglucosamine participates in the biosynthesis of sialic acid. In this work, we propose MRS for the detection of uridine diphospho-N-acetylhexosamines visible in high-resolution MR spectra of intact cells from different human tumours. Signals from the nucleotide and amino sugar moieties, including amide signals observed for the first time in whole cells, are assigned, also taking advantage of spectral changes that follow cell treatment with ammonium chloride. Finally, parallel changes in uridine diphospho-N-acetylhexosamines and glutamine pools, observed after pH changes induced by ammonium chloride in the different tumour cell lines, may provide more details on the glycosylation processes.

Interaction of HIV‐1 Reverse Transcriptase Ribonuclease H with an Acylhydrazone Inhibitor

HIV-1 reverse transcriptase is a bifunctional enzyme, having both DNA polymerase (RNA- and DNA-dependent) and ribonuclease H activities. HIV-1 reverse transcriptase has been an exceptionally important target for antiretroviral therapeutic development, and nearly half of the current clinically used antiretrovirals target reverse transcriptase DNA polymerase. However, no inhibitors of reverse transcriptase ribonuclease H are on the market or in preclinical development. Several drug-like small molecule inhibitors of reverse transcriptase ribonuclease H have been described, but little structural information is available about the interactions between reverse transcriptase ribonuclease H and inhibitors that exhibit antiviral activity. In this report, we describe NMR studies of the interaction of a new ribonuclease H inhibitor, BHMP07, with a catalytically active HIV-1 reverse transcriptase ribonuclease H domain fragment. We carried out solution NMR experiments to identify the interaction interface of BHMP07 with the ribonuclease H domain fragment. Chemical shift changes of backbone amide signals at different BHMP07 concentrations clearly demonstrate that BHMP07 mainly recognizes the substrate handle region in the ribonuclease H fragment. Using ribonuclease H inhibition assays and reverse transcriptase mutants, the binding specificity of BHMP07 was compared with another inhibitor, dihydroxy benzoyl naphthyl hydrazone. Our results provide a structural characterization of the ribonuclease H inhibitor interaction and are likely to be useful for further improvements of the inhibitors.

Characterization of Glycosaminoglycans by15N NMR Spectroscopy and in Vivo Isotopic Labeling

Characterization of glycosaminoglycans (GAGs), including chondroitin sulfate (CS), dermatan sulfate (DS), and heparan sulfate (HS), is important in developing an understanding of cellular function and in assuring quality of preparations destined for biomedical applications. While use of (1)H and (13)C NMR spectroscopy has become common in characterization of these materials, spectra are complex and difficult to interpret when a more heterogeneous GAG type or a mixture of several types is present. Herein a method based on (1)H-(15)N two-dimensional NMR experiments is described. The (15)N- and (1)H-chemical shifts of amide signals from (15)N-containing acetylgalactosamines in CSs are shown to be quite sensitive to the sites of sulfation (4-, 6-, or 4,6-) and easily distinguishable from those of DS. The amide signals from residual (15)N-containing acetylglucosamines in HS are shown to be diagnostic of the presence of these GAG components as well. Most data were collected at natural abundance of (15)N despite its low percentage. However enrichment of the (15)N-content in GAGs using metabolic incorporation from (15)N-glutamine added to cell culture media is also demonstrated and used to distinguish metabolic states in different cell types.