Alexander G. Sobol

Three-dimensional structure of ectatomin from Ectatomma tuberculatum ant venom

SummaryTwo-dimensional 1H NMR techniques were used to determine the spatial structure of ectatomin, a toxin from the venom of the ant Ectatomma tuberculatum. Nearly complete proton resonance assignments for two chains of ectatomin (37 and 34 amino acid residues, respectively) were obtained using 2D TOCSY, DQF-COSY and NOESY experiments. The cross-peak volumes in NOESY spectra were used to define the local structure of the protein and generate accurate proton-proton distance constraints employing the MARDIGRAS program. Disulfide bonds were located by analyzing the global fold of ectatomin, calculated with the distance geometry program DIANA. These data, combined with data on the rate of exchange of amide protons with deuterium, were used to obtain a final set of 20 structures by DIANA. These structures were refined by unrestrained energy minimization using the CHARMm program. The resulting rms deviations over 20 structures (excluding the mobile N- and C-termini of each chain) are 0.75 Å for backbone heavy atoms, and 1.25 Å for all heavy atoms. The conformations of the two chains are similar. Each chain consists of two α-helices and a hinge region of four residues; this forms a hairpin structure which is stabilized by disulfide bridges. The hinge regions of the two chains are connected together by a third disulfide bridge. Thus, ectatomin forms a four-α-helical bundle structure.

Spatial Structure of the Dimeric Transmembrane Domain of the Growth Factor Receptor ErbB2 Presumably Corresponding to the Receptor Active State

Proper lateral dimerization of the transmembrane domains of receptor tyrosine kinases is required for biochemical signal transduction across the plasma membrane. The spatial structure of the dimeric transmembrane domain of the growth factor receptor ErbB2 embedded into lipid bicelles was obtained by solution NMR, followed by molecular dynamics relaxation in an explicit lipid bilayer. ErbB2 transmembrane segments associate in a right-handed α-helical bundle through the N-terminal tandem GG4-like motif Thr652-X3-Ser656-X3-Gly660, providing an explanation for the pathogenic power of some oncogenic mutations.

Conformation and mode of membrane interaction in cyclotides

Cyclotides are a family of bioactive plant peptides that are characterized by a circular protein backbone and three conserved tightly packed disulfide bonds. The antimicrobial and hemolytic properties of cyclotides, along with the relative hydrophobicity of the peptides, point to the biological membrane as a target for cyclotides. To assess the membrane‐induced conformation and orientation of cyclotides, the interaction of the Möbius cyclotide, kalata B1, from the African perennial plant Oldenlandia affinis, with dodecylphosphocholine micelles was studied using NMR spectroscopy. Under conditions where the cyclotide formed a well‐defined complex with micelles, the spatial structure of kalata B1 was calculated from NOE and J couplings data, and the model for the peptide–micelle complex was built using 5‐ and 16‐doxylstearate relaxation probes. The binding of divalent cations to the peptide–micelle complex was quantified by Mn2+ titration. The results show that the peptide binds to the micelle surface, with relatively high affinity, via two hydrophobic loops (loop 5, Trp19–Val21; and loop6, Leu27–Val29). The charged residues (Glu3 and Arg24), along with the cation‐binding site (near Glu3) are segregated on the other side of the molecule and in contact with polar head groups of detergent. The spatial structure of kalata B1 is only slightly changed during incorporation into micelles and represents a distorted triple‐stranded β‐sheet cross‐linked by a cystine knot. Detailed structural analysis and comparison with other knottins revealed structural conservation of the two‐disulfide motif in cyclic and acyclic peptides. The results thus obtained provide the first model for interaction of cyclotides with membranes and permit consideration of the cyclotides as membrane‐active cationic antimicrobial peptides.

Lipid-protein nanoscale bilayers: a versatile medium for NMR investigations of membrane proteins and membrane-active peptides.

In the present Communication we demonstrate the possibility to use high-resolution NMR for the investigation of membrane proteins in reconstituted high-density lipoprotein (rHDL) particles. The rHDL particles are nanoscale phospholipid bilayers wrapped around by a dimer of apolipoprotein A-1 (Bayburt, T. H.; Grinkova, Y. V.; Sligar, S. G. Nano Lett. 2002, 2, 853−856). In contrast to the commonly used spherical micelles, the rHDL particles incorporate a lipid bilayer like in biological membranes. These particles still undergo isotropic motion on the NMR time scale, providing the application of high-resolution NMR spectroscopy of the peptides and proteins embedded into their bilayer. As an example, the topology of the membrane-active peptide Antiamoebin-I in the bilayer of the rHDL particles was determined by using the lipid-soluble relaxation probe technique.

Sequence-specific resonance assignment and secondary structure of (1–71) bacterioopsin

SummaryThe conformation of chymotryptic fragment C2 of bacteriorhodopsin (residues 1–71) was studied by 2D1H NMR. The fragment was solubilized in a mixture of chloroform/methanol (1∶1), 0.1 M LiClO4. Most of the resonances in1H NMR spectra of fragment C2 were assigned using phase-sensitive DQF-COSY, TOCSY, and NOESY techniques. To simplify the assignment procedure for overlapping regions of NMR spectra, an analog of fragment C2 with leucines deuterated in β-positions was used. Deuterium exchange rates for amide protons were measured in a series of TOCSY spectra. Two right-handed α-helical regions Pro8-Lys30 and Lys41-Leu62 were identified on the basis of NOE connectivities and deuterium exchange rates. The N-terminal part of the fragment (Ala.2-Gly6) adopts the helical conformation stabilized by 3 hydrogen bonds.

Simultaneous measurement of residual dipolar couplings for proteins in complex using the isotopically discriminated NMR approach

One-bond residual dipolar couplings (RDCs) measured for the amide groups of proteins partially aligned in a magnetic field provide valuable information regarding the relative orientation of protein units. In order for RDCs obtained for individual proteins to be useful in the structure determination of heterodimer complexes, they should be measured for exactly the same alignment of the complex. Here, an isotopically discriminated IDIS-RDC-TROSY NMR experiment is proposed, which enables the measurement of HN RDCs for two proteins simultaneously and independently, but in the same sample, while they are part of the same complex. The signals for both proteins, one of which should be labeled with (15)N and the other with (15)N and (13)C, are observed in different subspectra, thus reducing spectral overlap. The approach uniquely ensures that RDCs measured for both proteins relate to exactly the same alignment tensor, allowing accurate measurement of the relative angle between the two proteins. The method is also applicable for complexes containing three or more protein components. The experiment can speed up and lead to automation of protein-protein docking on the basis of angular restraints.

2D [1H,13C] NMR study of carbon fluxes during glucose utilization by Escherichia coli MG1655

Carbon fluxes through main pathways of glucose utilization in Escherichia coli cells-glycolysis, pentose phosphate pathway (PPP), and Enther-Doudoroff pathway (EDP)—were studied. Their ratios were analyzed in E. coli strains MG1655, MG1655Δ(edd-eda), MG1655Δ(zwf, edd-eda), and MG1655Δ(pgi, edd-eda). It was shown that the carbon flux through glycolysis was the main route of glucose utilization, averaging ca. 80%. Inactivation of EDP did not affect growth parameters. Nevertheless, it altered carbon fluxes through the tricarboxylic acid cycles and energy metabolism in the cell. Inactivation of PPP decreased growth rate to a lesser degree than glycolysis inactivation.

In-situ studies on the micro-structure evolution of A2W2O7 (A = Li, Na, K) during melting by high temperature Raman spectroscopy and density functional theory

In-situ high temperature Raman spectroscopic (HTRS) technique in combination with density functional theory (DFT) analysis has been adopted to investigate the micro-structure of solid and molten A2W2O7 (A=Li, Na, K). The [WO6] octahedra were found to be connected to each other by corner and edge sharing in the crystalline Li2W2O7 and K2W2O7 compounds. In the crystal lattice of Na2W2O7, on the other hand, the [WO4] tetrahedra and [WO6] octahedra were found to coexist and paired by corner sharing. Although the structural diversity has clearly led to distinct Raman spectra of the crystalline A2W2O7 compounds, the spectra of their melts tended to be analogous, showing the typical vibration modes of (W2O7)2- dimer. A mechanism was then proposed to explain the structure evolution occurring during the melting process of A2W2O7. The effect of A+ cation on the Raman bands of (W2O7)2- dimer in molten A2W2O7 has also been investigated. Both the wavenumber and full width at half-height (FWHH) of the characteristic band assigned to the symmetrical stretching vibration mode of WOnb (non-bridging oxygen) in (W2O7)2- were found to decrease in the sequence of Li+, Na+ and K+, indicating the cation effect on the mean bond length and its distribution range of WOnb. In addition, the relative intensity of this band was also influenced by the cation and it was increased in the order of Li2W2O7, Na2W2O7 and K2W2O7, which has been explained by the charge transfer process and confirmed by Mulliken overlap population analysis.