Jacob Anglister

Resonance Rayleigh scattering of cyanine dyes in solution

Every process of light absorption is inherently associated with a resonance scattering of the incident beam regardless of the nature of the absorbing molecules. Both the real and imaginary parts of the refractive index contribute to the resonance scattering, and marked depolarization is expected to be observed as a rule in the light scattered by complex molecules. The resonance and near‐resonance Rayleigh scattering has been measured for a number of cyanine dyes: pseudocyanine, orthochrome T, 1,1′‐diethyl‐2, 2′‐pyridyl‐quinolyl cyanine, and pinacyanol chloride, in a number of solvents. In no case was the measured scattering intensity weaker than that expected theoretically, and good agreement between theory and experiment was found as a rule at the blue part of the absorption bands. In contrast, the measured scattering intensities exceeded (by up to fourfold) the theoretically expected values at the red parts of the absorption bands. Possible reasons for this discrepancy are discussed.

Alternative Conformations of HIV-1 V3 Loops Mimic β Hairpins in Chemokines, Suggesting a Mechanism for Coreceptor Selectivity

The V3 loop of the HIV-1 envelope glycoprotein gp120 is involved in binding to the CCR5 and CXCR4 coreceptors. The structure of an HIV-1(MN) V3 peptide bound to the Fv of the broadly neutralizing human monoclonal antibody 447-52D was solved by NMR and found to be a beta hairpin. This structure of V3(MN) was found to have conformation and sequence similarities to beta hairpins in CD8 and CCR5 ligands MIP-1alpha, MIP-1beta, and RANTES and differed from the beta hairpin of a V3(IIIB) peptide bound to the strain-specific murine anti-gp120(IIIB) antibody 0.5beta. In contrast to the structure of the bound V3(MN) peptide, the V3(IIIB) peptide resembles a beta hairpin in SDF-1, a CXCR4 ligand. These data suggest that the 447-52D-bound V3(MN) and the 0.5beta-bound V3(IIIB) structures represent alternative V3 conformations responsible for selective interactions with CCR5 and CXCR4, respectively.

The human type I interferon receptor. NMR structure reveals the molecular basis of ligand binding.

The potent antiviral and antiproliferative activities of human type I interferons (IFNs) are mediated by a single receptor comprising two subunits, IFNAR1 and IFNAR2. The structure of the IFNAR2 IFN binding ectodomain (IFNAR2-EC), the first helical cytokine receptor structure determined in solution, reveals the molecular basis for IFN binding. The atypical perpendicular orientation of its two fibronectin domains explains the lack of C domain involvement in ligand binding. A model of the IFNAR2-EC/IFNalpha2 complex based on double mutant cycle-derived constraints uncovers an extensive and predominantly aliphatic hydrophobic patch on the receptor that interacts with a matching hydrophobic surface of IFNalpha2. An adjacent motif of alternating charged side chains guides the two proteins into a tight complex. The binding interface may account for crossreactivity and ligand specificity of the receptor. This molecular description of IFN binding should be invaluable for study and design of IFN-based biomedical agents.

Depolarized rayleigh light scattering in absorption bands measured in lycopene solution

Abstract The depolarized scattering intensity from the polyene pigment lycopene was measured in the visible absorptiion band and was found to be in good agreement with theoretical calculations at the short wavelength part of the band whereas significant deviations were observed at the red edge of this band.

A Monomeric 310-Helix Is Formed in Water by a 13-Residue Peptide Representing the Neutralizing Determinant of HIV-1 on gp41 †,‡

The peptide gp41(659-671) (ELLELDKWASLWN) comprises the entire epitope for one of the three known antibodies capable of neutralizing a broad spectrum of primary HIV-1 isolates and is the only such epitope that is sequential. Here we present the NMR structure of gp41(659-671) in water. This peptide forms a monomeric 3(10)-helix stabilized by i,i+3 side chain-side chain interactions favored by its primary sequence. In this conformation the peptide presents an exposed surface, which is mostly hydrophobic and consists of conserved HIV-1 residues. The presence of the 3(10)-helix is confirmed by its characteristic CD pattern. Studies of the 3(10)-helix have been hampered by the absence of a model peptide adopting this conformation. gp41(659-671) can serve as such a model to investigate the spectral characteristics of the 3(10)-helix, the factors that influence its stability, and the propensity of different amino acids to form a 3(10)-helix. The observation that the 3(10)-helical conformation is highly populated in the peptide gp41(659-671) indicates that the corresponding segment in the cognate protein is an autonomous folding unit. As such, it is very likely that the helical conformation is maintained in gp41 throughout the different tertiary structures of the envelope protein that form during the process of viral fusion. However, the exposure of the gp41(659-671) segment may vary, leading to changes in the reactivity of anti-gp41 antibodies in the different stages of viral fusion. Since gp41(659-671) is an autonomous folding unit, peptide immunogens consisting of the complete gp41(659-671) sequence are likely to induce antibodies highly cross-reactive with HIV-1.

Isotope-Edited Multidimensional NMR of Calcineurin-B in the Presence of the Non-Deuterated Detergent Chaps

SummaryAt the concentration needed for NMR, the calcium-saturated form of calcineurin B dissolved in water shows resonance line widths that indicate aggregation of this protein. Although the line width or aggregation state can be influenced to some degree by temperature, pH, and salt concentrations, in the absence of detergent no conditions could be found where the protein behaved as a monomeric unit. In the presence of a 10- to 20-fold molar excess of the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethyl-ammonio]-1-propanesulfonate (CHAPS), resonance line widths were considerably narrower and were compatible with a protein of ∼25 kDa. The presence of the NMR signals of the non-deuterated CHAPS does not interfere with modern isotope-directed NMR studies as the signals from protons not attached to 15N or 13C are removed by isotope filtering and purge pulses.

Solid–state NMR evidence for an antibody–dependent conformation of the V3 loop of HIV–1 gp120

Solid–state NMR measurements have been carried out on frozen solutions of the complex of a 24–residue peptide derived from the third variable (V3) loop of the HIV–1 envelope glycoprotein gp120 bound to the Fab fragment of an anti–gp120 antibody. The measurements place strong constraints on the conformation of the conserved central GPGR motif of the V3 loop in the antibody–bound state. In combination with earlier crystal structures of V3 peptide–antibody complexes and existing data on the cross–reactivity of the antibodies, the solid–state NMR measurements suggest that the Gly–Pro–Gly–Arg (GPGR) motif adopts an antibody–dependent conformation in the bound state and may be conformationally heterogeneous in unbound, full–length gp120. These measurements are the first application of solid–state NMR methods in a structural study of a peptide–protein complex.

Peptides in the treatment of AIDS.

Fusion of HIV-1 and target cells is mediated by the envelope protein gp41 that undergoes a series of conformational changes during the process of infection. Knowledge of the structural biology of gp41 allows the design of potent peptide inhibitors that prevent the virus from entering lymphocytes and macrophages. The design of such inhibitors is the subject of this review.

Molecular switch for alternative conformations of the HIV-1 V3 region: Implications for phenotype conversion

HIV-1 coreceptor usage plays a critical role in virus tropism and pathogenesis. A switch from CCR5- to CXCR4-using viruses occurs during the course of HIV-1 infection and correlates with subsequent disease progression. A single mutation at position 322 within the V3 loop of the HIV-1 envelope glycoprotein gp120, from a negatively to a positively charged residue, was found to be sufficient to switch an R5 virus to an X4 virus. In this study, the NMR structure of the V3 region of an R5 strain, HIV-1JR-FL, in complex with an HIV-1-neutralizing antibody was determined. Positively charged and negatively charged residues at positions 304 and 322, respectively, oppose each other in the β-hairpin structure, enabling a favorable electrostatic interaction that stabilizes the postulated R5 conformation. Comparison of the R5 conformation with the postulated X4 conformation of the V3 region (positively charged residue at position 322) reveals that electrostatic repulsion between residues 304 and 322 in X4 strains triggers the observed one register shift in the N-terminal strand of the V3 region. We posit that electrostatic interactions at the base of the V3 β-hairpin can modulate the conformation and thereby influence the phenotype switch. In addition, we suggest that interstrand cation-π interactions between positively charged and aromatic residues induce the switch to the X4 conformation as a result of the S306R mutation. The existence of three pairs of identical (or very similar) amino acids in the V3 C-terminal strand facilitates the switch between the R5 and X4 conformations.

NMR Structure of an Anti-Gp120 Antibody Complex with a V3 Peptide Reveals a Surface Important for Co-Receptor Binding

BACKGROUND The protein 0.5beta is a potent strain-specific human immunodeficiency virus type 1 (HIV-1) neutralizing antibody raised against the entire envelope glycoprotein (gp120) of the HIV-1(IIIB) strain. The epitope recognized by 0.5beta is located within the third hypervariable region (V3) of gp120. Recently, several HIV-1 V3 residues involved in co-receptor utilization and selection were identified. RESULTS Virtually complete sidechain assignment of the variable fragment (Fv) of 0.5beta in complex with the V3(IIIB) peptide P1053 (RKSIRIQRGPGRAFVTIG, in single-letter amino acid code) was accomplished and the combining site structure of 0.5beta Fv complexed with P1053 was solved using multidimensional nuclear magnetic resonance (NMR). Five of the six complementarity determining regions (CDRs) of the antibody adopt standard canonical conformations, whereas CDR3 of the heavy chain assumes an unexpected fold. The epitope recognized by 0.5beta encompasses 14 of the 18 P1053 residues. The bound peptide assumes a beta-hairpin conformation with a QRGPGR loop located at the very center of the binding pocket. The Fv and peptide surface areas buried upon binding are 601 A and 743 A(2), respectively, in the 0.5beta Fv-P1053 mean structure. The surface of P1053 interacting with the antibody is more extensive and the V3 peptide orientation in the binding site is significantly different compared with those derived from the crystal structures of a V3 peptide of the HIV-1 MN strain (V3(MN)) complexed to three different anti-peptide antibodies. CONCLUSIONS The surface of P1053 that is in contact with the anti-protein antibody 0.5beta is likely to correspond to a solvent-exposed region in the native gp120 molecule. Some residues of this region of gp120 are involved in co-receptor binding, and in discrimination between different chemokine receptors utilized by the protein. Several highly variable residues in the V3 loop limit the specificity of the 0.5beta antibody, helping the virus to escape from the immune system. The highly conserved GPG sequence might have a role in maintaining the beta-hairpin conformation of the V3 loop despite insertions, deletions and mutations in the flanking regions.