1H, 13C and 15N NMR assignments and secondary structure of the paramagnetic form of rat cytochrome b 5

Summary Modern multidimensional double- and triple-resonance NMR methods have been applied to assign the backbone and side-chain ¹³C resonances for both equilibrium conformers of the paramagnetic form of rat liver microsomal cytochrome b ₅. The assignment of backbone ¹³C resonances was used to confirm previous ¹H and ¹⁵N resonance assignments [Guiles, R.D. et al. (1993) Biochemistry, 32, 8329–8340]. On the basis of short- and medium-range NOEs and backbone ¹³C chemical shifts, the solution secondary structure of rat cytochrome b ₅ has been determined. The striking similarity of backbone ¹³C resonances for both equilibrium forms strongly suggests that the secondary structures of the two isomers are virtually identical. It has been found that the ¹³C chemical shifts of both backbone and side-chain atoms are relatively insensitive to paramagnetic effects. The reliability of such methods in anisotropic paramagnetic systems, where large pseudocontact shifts can be observed, is evaluated through calculations of the magnitude of such shifts.Abbreviations DANTE delays alternating with nutation for tailored excitation - DEAE diethylaminoethyl - DQF-COSY 2D double-quantum-filtered correlation spectroscopy - EDTA ethylenediaminetetraacetic acid - HCCH-TOCSY 3D proton-correlated carbon TOCSY experiment - HMQC 2D heteronuclear multiple-quantum correlation spectroscopy - HNCA 3D triple-resonance experiment correlating amide protons, amide nitrogens and alpha carbons - HNCO 3D triple-resonance experiment correlating amide protons, amide nitrogens and carbonyl carbons - HNCOCA 3D triple-resonance experiment correlating amide protons, amide nitrogens and alpha carbons via carbonyl carbons - HOHAHA 2D homonuclear Hartmann-Hahn spectroscopy - HOHAHA-HMQC 3D HOHAHA relayed HMQC - HSQC 2D heteronuclear single-quantum correlation spectroscopy - IPTG isopropyl thiogalactoside - NOESY 2D nuclear Overhauser enhancement spectroscopy - NOESY-HSQC 3D NOESY relayed HSQC - TOCSY 2D total correlation spectroscopy - TPPI time-proportional phase incrementation - TSP trimethyl silyl propionate     

Multidimensional1H and15N NMR investigation of glutamine-binding protein ofEscherichia coli

Summary Specific and uniform¹⁵N labelings along with site-directed mutagenesis of glutamine-binding protein have been utilized to obtain assignments of the His¹⁵⁶, Trp³² and Trp.²²⁰ residues. These assignments have been made not only to further study the importance of these 3 amino acid residues in protein-ligand and protein-protein interactions associated with the active transport ofl-glutamine across the cytoplasmic membrane ofEscherichia coli, but also to serve as the starting points in the sequence-specific backbone assignment. The assignment of H₂ of His¹⁵⁶ refines the earlier, model where this particular proton formas an intermolecular hydrogen bond to the -carbonyl ofl-glutamine, while assignments of both Trp³² and Trp²²⁰ show the variation in local structures which ensure the specificity in ligand binding and protein-protein interaction. Using 3D NOESY-HMQC NMR, amide connectivities can be traced along 8–9 amino acid residues at a time. This paper illustrates the usefulness of combining¹⁵N isotopic labeling and multinuclear, multidimensional NMR techniques for a structural investigation of a protein with a molecular weight of 25 000.     

NMR solution structure of the non-RGD disintegrin obtustatin

The solution structure of obtustatin, a novel non-RGD disintegrin of 41 residues isolated from Vipera lebetina obtusa venom, and a potent and selective inhibitor of the adhesion of integrin alpha(1)beta(1) to collagen IV, has been determined by two-dimensional nuclear magnetic resonance. Almost the whole set of chemical shifts for 1H, 13C and 15N were assigned at natural abundance from 2D homonuclear and heteronuclear 500 MHz, 600 MHz and 800 MHz spectra at pH 3.0 recorded at 298 K and 303 K. Final structural constraints consisted of 302 non-redundant NOE (95 long-range, 60 medium, 91 sequential and 56 intra-residue), four disulfide bond distances, five chi1 dihedral angles and four hydrogen bonds. The 20 conformers with lowest total energy had no NOE violations greater than 0.35A or dihedral angle violations greater than 12 degrees. The average root-mean-square deviation (RMSD) for backbone atoms of all residues among the 20 conformers was 1.1A and 0.6A for the 29 best-defined residues. Obtustatin lacks any secondary structure. Compared to all known disintegrin structures in which the RGD motif is located at the apex of an 11 residue hairpin loop, the active KTS tripeptide of obtustatin is oriented towards a side of its nine residue integrin-binding loop. The C-terminal tail is near to the active loop, and these two structural elements display the largest atomic displacements due to local conformational disorder. Double cross-peaks for W20, Y28 and H27 in the aromatic region of TOCSY spectra, local RMSD values for these residues, and positive cross-peaks in a ROESY spectrum (600 MHz, 100 ms mixing time), suggest that these residues act as a hinge allowing for the overall flexibility of the entire integrin-binding loop. These distinct structural features, along with its different electrostatic surface potential in relation to other known disintegrins, may confer to obtustatin its reported alpha(1)beta(1) integrin inhibitory selectivity.     

Validating the use of database potentials in protein structure determination by NMR

The refinement of protein structures determined by nuclear magnetic resonance spectroscopy against database potentials of mean force allows for the exclusion of unfavourable conformations of the protein backbone during a structure calculation, resulting in protein structures with a marked improvement in Ramachandran statistics. In this communication, we use multiple sets of residual dipolar couplings as quality assessment criteria for several proteins and show that not only do the Ramachandran and structural quality statistics improve, but a significant improvement in the accuracy of structures is achieved upon refinement.     

HIV/AIDS患者抗病毒治疗前HIV-1耐药情况调查及外周血CD8+T细胞CD38表达水平研究

摘要 目的：分析人免疫缺陷病毒/艾滋病（HIV/AIDS）患者抗病毒治疗前HIV-1耐药以及影响因素,探讨HIV/AIDS患者外周血CD₈⁺T细胞CD₃₈表达（CD₈⁺CD₃₈⁺T淋巴细胞百分比）与CD₄⁺T淋巴细胞计数的相关性。方法：选择2016年3月至2019年12月我院接诊的442例HIV/AIDS患者（HIV/AIDS组）和163例同期于我院进行体检的健康志愿者（对照组）,HIV/AIDS组扩增pol基因,进行HIV-1基因耐药分析,检测CD₈⁺CD₃₈⁺T淋巴细胞百分比、CD₄⁺T淋巴细胞计数、CD₈⁺T淋巴细胞计数。分析HIV/AIDS患者HIV-1耐药的影响因素,分析CD₈⁺CD₃₈⁺T淋巴细胞百分比与CD₄⁺T淋巴细胞计数、CD₈⁺T淋巴细胞计数相关性。结果：HIV/AIDS组442例HIV/AIDS患者中376例获得HIV-1 pol基因序列,HIV-1耐药35例,耐药率9.31%（35/376）。单因素分析结果显示耐药组和非耐药组在年龄、文化程度、感染途径、HIV病毒载量方面差异有统计学意义（P＜0.05）。多因素Logistic回归分析结果显示同性性传播、注射吸毒、高HIV病毒载量是HIV/AIDS患者抗病毒治疗前HIV-1耐药的危险因素（P＜0.05）。HIV/AIDS组外周血CD₄⁺T淋巴细胞计数、CD₈⁺T淋巴细胞计数低于对照组（P＜0.05）,CD₈⁺CD₃₈⁺T淋巴细胞百分比高于对照组（P＜0.05）。CD₈⁺CD₃₈⁺T淋巴细胞百分比与CD₄⁺T淋巴细胞计数、CD₈⁺T淋巴细胞计数呈负相关（P＜0.05）。结论：抗病毒治疗前HIV/AIDS患者存在一定HIV-1耐药率,传播途径、HIV-1病毒载量与HIV-1耐药有关。CD₈⁺T细胞表面CD₃₈过表达与HIV/AIDS 患者CD₄⁺T T细胞的过度消耗有关。     

The NMR structure of the domain II of a chloroplastic NifU-like protein OsNifU1A

NifU-like proteins are a highly conserved protein that serves as the scaffold for assembly of Fe-S clusters. Chloroplastic NifU-like proteins have tandem NifU like domains, named domain I and domain II. Although the amino acid sequences of these domains are very similar to each other, the predicted functional region for the Fe-S cluster assembly, the CXXC motif, exists only in domain I. The structure of the domain II of chloroplastic NifU-like protein OsNifU1A has an α-β sandwich structure containing two α helices located on one side of the β-sheet. The electrostatic surface potential of OsNifU1A domain II is predominantly positively charged. Chloroplastic NifU-like proteins are targeted to ferredoxin for transferring the Fe-S cluster. The ferredoxin presents an overall negatively charged surface, which may evoke an electrostatic association with OsNifU1A domain II.     

The structure of F1-ATPase from Saccharomyces cerevisiae inhibited by its regulatory protein IF1

The structure of F₁-ATPase from Saccharomyces cerevisiae inhibited by the yeast IF₁ has been determined at 2.5 Å resolution. The inhibitory region of IF₁ from residues 1 to 36 is entrapped between the C-terminal domains of the α_DP- and β_DP-subunits in one of the three catalytic interfaces of the enzyme. Although the structure of the inhibited complex is similar to that of the bovine-inhibited complex, there are significant differences between the structures of the inhibitors and their detailed interactions with F₁-ATPase. However, the most significant difference is in the nucleotide occupancy of the catalytic β_E-subunits. The nucleotide binding site in β_E-subunit in the yeast complex contains an ADP molecule without an accompanying magnesium ion, whereas it is unoccupied in the bovine complex. Thus, the structure provides further evidence of sequential product release, with the phosphate and the magnesium ion released before the ADP molecule.     

NMR structure of the human doppel protein

The NMR structure of the recombinant human doppel protein, hDpl(24-152), contains a flexibly disordered "tail" comprising residues 24-51, and a globular domain extending from residues 52 to 149 for which a detailed structure was obtained. The globular domain contains four alpha-helices comprising residues 72-80 (alpha1), 101-115 (alpha2(a)), 117-121 (alpha2(b)), and 127-141 (alpha3), and a short two-stranded anti-parallel beta-sheet comprising residues 58-60 (beta1) and 88-90 (beta2). The fold of the hDpl globular domain thus coincides nearly identically with the structure of the murine Dpl protein. There are close similarities with the human prion protein (hPrP) but, similar to the situation with the corresponding murine proteins, hDpl shows marked local differences when compared to hPrP: the beta-sheet is flipped by 180 degrees with respect to the molecular scaffold formed by the four helices, and the beta1-strand is shifted by two residues toward the C terminus. A large solvent-accessible hydrophobic cleft is formed on the protein surface between beta2 and alpha3, which has no counterpart in hPrP. The helix alpha2 of hPrP is replaced by two shorter helices, alpha2(a) and alpha2(b). The helix alpha3 is shortened by more than two turns when compared with alpha3 of hPrP, which is enforced by the positioning of the second disulfide bond in hDpl. The C-terminal peptide segment 144-149 folds back onto the loop connecting beta2 and alpha2. All but four of the 20 conserved residues in the globular domains of hPrP and hDpl appear to have a structural role in maintaining a PrP-type fold. The conservation of R76, E96, N110 and R134 in hDpl, corresponding to R148, E168, N183 and R208 in hPrP suggests that these amino acid residues might have essential roles in the so far unknown functions of PrP and Dpl in healthy organisms.     

Spectral processing methods for the removal of t1 noise and solvent artifacts from NMR spectra

Summary A data processing method is described which reduces the effects of t₁ noise artifacts and improves the presentation of 2D NMR spectral data. A t₁ noise profile is produced by measuring the average noise in each column. This profile is then used to determine weighting coefficients for a sliding weighted smoothing filter that is applied to each row, such that the amount of smoothing each point receives is proportional to both its estimated t₁ noise level and the level of t₁ noise of neighbouring points. Thus, points in the worst t₁ noise bands receive the greatest smoothing, whereas points in low-noise regions remain relatively unaffected. In addition, weighted smoothing allows points in low-noise regions to influence neighbouring points in noisy regions. This method is also effective in reducing the noise artifacts associated with the solvent resonance in spectra of biopolymers in aqueous solution. Although developed primarily to improve the quality of 2D NMR spectra of biopolymers prior to automated analysis, this approach should enhance processing of spectra of a wide range of compounds and can be used whenever noise occurs in discrete bands in one dimension of a multi-dimensional spectrum.     

NMR solution structure of KP-TerB, a tellurite-resistance protein from Klebsiella pneumoniae

Klebsiella pneumoniae (KP), a Gram-negative bacterium, is a common cause of hospital-acquired bacterial infections worldwide. Tellurium (Te) compounds, although relatively rare in the environment, have a long history as antimicrobial and therapeutic agents. In bacteria, tellurite (TeO(3) (-2)) resistance is conferred by the ter (Te(r)) operon (terZABCDEF). Here, on the basis of 2593 restraints derived from NMR analysis, we report the NMR structure of TerB protein (151 amino acids) of KP (KP-TerB), which is mainly composed of seven alpha-helices and a 3(10) helix, with helices II to V apparently forming a four-helix bundle. The ensemble of 20 NMR structures was well-defined, with a RMSD of 0.32 +/- 0.06 A for backbone atoms and 1.11 +/- 0.07 A for heavy atoms, respectively. A unique property of the KP-TerB structure is that the positively and negatively charged clusters are formed by the N-terminal positively and C-terminal negatively charged residues, respectively. To the best of our knowledge, the protein sequence and structures of KP-TerB are unique.     

Cell-based chemical genetic screen identifies damnacanthal as an inhibitor of HIV-1 Vpr induced cell death

Viral protein R (Vpr), one of the human immunodeficiency virus type 1 (HIV-1) accessory proteins, contributes to multiple cytopathic effects, G2 cell cycle arrest and apoptosis. The mechanisms of Vpr have been intensely studied because it is believed that they underlie HIV-1 pathogenesis. We here report a cell-based small molecule screen on Vpr induced cell death in the context of HIV-1 infection. From the screen of 504 bioactive compounds, we identified damnacanthal (Dam), a component of noni [corrected] as an inhibitor of Vpr induced cell death. Our studies illustrate a novel efficient platform for drug discovery and development in anti-HIV therapy which should also be applicable to other viruses.     

Mechanism of HIV-1 viral protein R-induced apoptosis

The paradigm of HIV-1 infection includes the diminution of CD4(+) T cells, loss of immune function, and eventual progression to AIDS. However, the mechanisms that drive host T cell depletion remain elusive. One HIV protein thought to participate in this destructive cascade is the Vpr gene product. Accordingly, we review the biology of the HIV-1 viral protein R (Vpr) an apoptogenic HIV-1 accessory protein that is packaged into the virus particle. In this review we focus specifically on Vpr's ability to induce host cell apoptosis. Recent evidence suggests that Vpr implements a unique mechanism to drive host cell apoptosis, by directly depolarizing the mitochondria membrane potential. Vpr's attack on the mitochondria results in release of cytochrome c resulting in activation of the caspase 9 pathway culminating in the activation of caspase 3 and the downstream events of apoptosis. Vpr may interact with the adenine nucleotide translocator (ANT) to prompt this cascade. The role of Vpr-induced apoptosis in HIV pathogenesis is considered.     

Characterization and molecular basis of the oligomeric structure of HIV-1 nef protein

The Nef protein of human immunodeficiency virus type I (HIV-1) is an important determinant for the onset of AIDS disease. The self-association properties of HIV-1 Nef are analyzed by chemical cross-linking, dynamic light scattering, equilibrium analytical ultracentrifugation, and NMR spectroscopy. The experimental data show that the HIV-1 Nef core domain forms stable homo-dimers and trimers in solution, but not higher oligomers. These Nef homomers are not covalently linked by disulfide bridges, and the equilibrium between these forms is dependent on the Nef concentration. We further provide the molecular basis for the Nef core dimers and trimers obtained by analysis of crystallographic models. Oligomerization of biological polypeptides is a common tool used to trigger events in cellular signaling and endocytosis, both of which are targeted by Nef. The quaternary structure of Nef may be of physiological importance and may help to connect its cellular targets or to increase affinity of the viral molecule for its ligands. The herein described models for Nef dimers and trimers will allow further mutational studies to elucidate their role in vivo. These results provide novel insight into the structural and functional relationships of this important viral protein. Moreover, the oligomer interface may represent a novel target for the design of antiviral agents.     

Characterization of the mechanisms of HIV-1 Vpr(52-96) internalization in cells

Addition of Vpr C-terminus to various cell types provokes cell apoptosis. This property was recently shown useful to develop inhibitors of cell proliferation. In that context, we investigated the cellular uptake of rhodamine- and fluorescein-labeled Vpr(52–96) peptides to understand the mechanism of Vpr C-terminus entry into cells. Dynamic light scattering data indicated that this peptide spontaneously formed polydispersed aggregates in cell culture medium. The fluorescently labeled Vpr(52–96) peptide was efficiently internalized, appearing either as large fluorescent patches in the cytoplasm or in a more diffuse form throughout the cell. Using isothermal titration calorimetry, we demonstrated that Vpr(52–96) can tightly associate with heparin, a glycosaminoglycan analog of heparan sulphate, suggesting a central role of the ubiquitous cell surface-associated heparan sulphate proteoglycans for the internalization of Vpr C-terminus. Fluorescently-labeled transferrin and methyl-β-cyclodextrin showed that the Vpr C-terminus was mediated through clathrin- and caveolae/raft-dependent endocytosis. We found that Vpr C-terminus uptake was partly blocked at 4 °C suggesting the importance of membrane fluidity for Vpr C-terminus entry. In fact, atomic force microscopy and liposome leakage further indicated that the Vpr peptide can destabilize and disrupt model membrane bilayers, suggesting that this mechanism may contribute to the passive entry of the peptide. Finally, using fluorescence lifetime imaging, we found that the Vpr(52–96) peptide was stable in cells for at least 48 h, probably as a consequence of the poor accessibility of the peptide to proteolytic enzymes in aggregates.