CombLabel: rational design of optimized sequence-specific combinatorial labeling schemes. Application to backbone assignment of membrane proteins with low stability.

Assignment of backbone resonances is a necessary initial step in every protein NMR investigation. Standard assignment procedure is based on the set of 3D triple-resonance (1H-13C-15N) spectra and requires at least several days of experimental measurements. This limits its application to the proteins with low stability. To speed up the assignment procedure, combinatorial selective labeling (CSL) can be used. In this case, sequence-specific information is extracted from 2D spectra measured for several selectively 13C,15N-labeled samples, produced in accordance with a special CSL scheme. Here we review previous applications of the CSL approach and present novel deterministic 'CombLabel' algorithm, which generates CSL schemes minimizing the number of labeled samples and their price and maximizing assignment information that can be obtained for a given protein sequence. Theoretical calculations revealed that CombLabel software outperformed previously proposed stochastic algorithms. Current implementation of CombLabel robustly calculates CSL schemes containing up to six samples, which is sufficient for moderately sized (up to 200 residues) proteins. As a proof of concept, we calculated CSL scheme for the first voltage-sensing domain of human Nav1.4 channel, a 134 residue four helical transmembrane protein having extremely low stability in micellar solution (half-life ~ 24 h at 45 °C). Application of CSL doubled the extent of backbone resonance assignment, initially obtained by conventional approach. The obtained assignment coverage (~ 50%) is sufficient for ligand screening and mapping of binding interfaces.

[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, MG1655(edd-eda), MG1655(zwf, edd-eda), and MG1655(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.

Comparative study of structure and activity of cytotoxins from venom of the cobras Naja oxiana, Naja kaouthia, and Naja haje.

Cytotoxins are positively charged polypeptides that constitute about 60% of all proteins in cobra venom; they have a wide spectrum of biological activities. By CD spectroscopy, cytotoxins CT1 and CT2 Naja oxiana, CT3 Naja kaouthia, and CT1 and CT2 Naja haje were shown to have similar secondary structure in an aqueous environment, with dominating beta-sheet structure, and to vary in the twisting angle of the beta-sheet and the conformation of disulfide groups. Using dodecylphosphocholine micelles and liposomes, CT1 and CT2 Naja oxiana were shown to incorporate into lipid structures without changes in the secondary structure of the peptides. The binding of CT1 and CT2 Naja oxiana with liposomes was associated with an increase in the beta-sheet twisting and a sign change of the dihedral angle of one disulfide group. The cytotoxins were considerably different in cytotoxicity and cooperativity of the effect on human promyelocytic leukemia cells HL60, mouse myelomonocytic cells WEHI-3, and human erythroleukemic cells K562. The most toxic CT2 Naja oxiana and CT3 Naja kaouthia possessed low cooperativity of interaction (Hill coefficient h = 0.6-0.8), unlike 10-20-fold less toxic CT1 and CT2 Naja haje (h = 1.2-1.7). CT1 Naja oxiana has an intermediate position on the cytotoxicity scale and is characterized by h = 0.5-0.8. The cytotoxins under study induced necrosis of HL60 cells and failed to activate apoptosis. The differences in cytotoxicity are supposed to be related not with features of the secondary structure of the peptides, but with interactions of side chains of variable amino acid residues with lipids and/or membrane proteins.

[ESR study of the interaction of cytotoxin II with model membranes]. / Izuchenie metodom EPR vzaimodeistviia tsitoksina II s model'nymi membranami.

Cytotoxin II from the venom of the Central-Asian cobra Naja oxiana spin-labeled at Lys35 (SLCT II) was studied by ESR spectroscopy in aqueous solution and upon interaction with phospholipid vesicles from egg phosphatidylcholine or its mixture with dimyristoylphosphatidylglycerol (molar ratio 9:1). The distribution of SLCT II between the aqueous and lipid phases depended on the toxin and lipid concentrations and on the solution ionic strength. It was analyzed using the modified Gouy-Chapman equation that takes into account different charges of the cytotoxin in solution and in membrane. The analysis revealed two states of the cytotoxin-lipid complex. The first state corresponds to monomeric SLCT II hydrophobically interacting with the lipid membrane [a binding constant of (8 +/- 3) x 10(3) M-1] and carrying the charge of 4.4 +/- 0.3. On the basis of these parameters and the spatial structure of cytotoxin II in dodecylphosphocholine micelles, we concluded that the cytotoxin is mainly incorporated into the region of polar groups of the lipid bilayer. The second state of SLCT II is realized at high cytotoxin concentrations in the membrane and corresponds to the formation of toxin-lipid complexes that destruct the membrane bilayer structure.