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Emfourin (M4in) from Serratia proteamaculans is a new proteinaceous inhibitor of protealysin-like proteases (PLPs), a subgroup of the well-known and widely represented metallopeptidase M4 family. Although the biological role of PLPs is debatable, data published indicate their involvement in pathogenesis, including bacterial invasion into eukaryotic cells, suppression of immune defense of some animals, and destruction of plant cell walls. Gene colocalization into a bicistronic operon observed for some PLPs and their inhibitors (as in the case of M4in) implies a mutually consistent functioning of both entities. The originality of the amino acid sequence of M4in suggests it belongs to a previously unknown protein family and this encourages structural studies. In this work, we report a near-complete assignment of 1H, 13C, and 15N resonances of recombinant M4in and its structural-dynamic properties derived from the chemical shifts. According the NMR data analysis, the M4in molecule comprises 3–5 helical elements and 4–6 β-strands, at least two of which are apparently antiparallel, ascribing this obviously globular protein to the α + β structural class. Besides, two disordered regions also exist in the central loops between the regular secondary structural elements. The obtained data provide the basis for determining the high-resolution structure as well as functioning mechanism of M4in that can be used for development of new antibacterial therapeutic strategies.

Trichobakin (TBK) is a type-I ribosome-inactivating protein (RIP-I), acting as an extremely potent inhibitor of protein synthesis in the cell-free translation system of rabbit reticulocyte lysate (IC50: 3.5 pM). In this respect, TBK surpasses the well-studied highly homologous RIP-I trichosanthin (IC50: 20-27 pM), therefore creation of recombinant toxins based on it is of great interest. TBK needs to penetrate into cytosol through the cell membrane and specifically bind to α-sarcin/ricin loop of 28S ribosome RNA to perform the function of specific RNA depurination. At the moment, there is no detailed structural-dynamic information in solution about diverse states RIP-I can adopt at different stages on the way to protein synthesis inhibition. In this work, we report a near-complete assignment of 1H, 13C, and 15N TBK (27.3 kDa) resonances and analysis of the secondary structure based on the experimental chemical shifts data. This work will serve as a basis for further investigations of the structure, dynamics and interactions of the TBK with its molecular partners using NMR techniques.

Side chains possess a broader conformational space (compared to the backbone) and are directly affected by intra- and intermolecular interactions, hence their dynamics and the corresponding NMR relaxation data are more sensitive and informative. Nevertheless, transverse relaxation in [Formula: see text] ([Formula: see text] or [Formula: see text]) spin systems is predominantly non-measurable in uniformly [Formula: see text]-labeled proteins due to cross-correlation effects. In the present publication, we propose a number of pulse sequences for accurate and precise measurement of the dipole-dipole transverse cross-correlated relaxation rate [Formula: see text], which, similarly to [Formula: see text] measurements, provides information about the amplitudes of intramolecular dynamics. The suggested approach has allowed us to circumvent a number of obstacles that were limiting earlier applications of [Formula: see text]: (1) impossibility of transmission of the central component of the triplet of [Formula: see text] group to [Formula: see text]-acquisition via INEPT has been solved by transmission of the averaged signal of "inner" and "outer" components of the triplet; (2) direct recording of the entire triplets resulting in substantial overlap of side chain signals has been replaced by recording of individual singlets with the use of [Formula: see text]-modulated approach and constant-time evolution; (3) low sensitivity has been enhanced via proton acquisition which required special attention to a zero-quantum coherence evolution. The proposed method expands the set of "dynamics sensors" covering protein side chains and substantially improves the quality and the level of detail of experimental data describing dynamic processes in proteins and protein complexes.

In the case of soluble proteins, chemical shift mapping is used to identify the intermolecular interfaces when the NOE-based calculations of spatial structure of the molecular assembly are impossible or impracticable. However, the reliability of the membrane protein interface mapping based on chemical shifts or other relevant parameters was never assessed. In the present work, we investigate the predictive power of various NMR parameters that can be used for mapping of helix-helix interfaces in dimeric TM domains. These parameters are studied on a dataset containing three structures of helical dimers obtained for two different proteins in various membrane mimetics. We conclude that the amide chemical shifts have very little predictive value, while the methyl chemical shifts could be used to predict interfaces, though with great care. We suggest an approach based on conversion of the carbon NMR relaxation parameters of methyl groups into parameters of motion, and one of such values, the characteristic time of methyl rotation, appears to be a reliable sensor of interhelix contacts in transmembrane domains. The carbon NMR relaxation parameters of methyl groups can be measured accurately and with high sensitivity and resolution, making the proposed parameter a useful tool for investigation of protein-protein interfaces even in large membrane proteins. An approach to build the models of transmembrane dimers based on perturbations of methyl parameters and TMDOCK software is suggested.

Mature transforming growth factor beta1 (TGF-β1) is a homodimeric protein with a single disulfide bridge between Cys77 on the respective monomers. The synthetic DNA sequence encoding the mature human TGF-β1/C77S (further termed TGF-β1m) was cloned into plasmid pET-32a downstream to the gene of fusion partner thioredoxin (Trx) immediately after the DNA sequence encoding enteropeptidase recognition site. High-level expression (~1.5 g l(-1)) of Trx/TGF-β1m fusion was achieved in Escherichia coli BL21(DE3) strain mainly in insoluble form. The fusion was solubilized and refolded in glutathione redox system in the presence of zwitterionic detergent CHAPS. After refolding, Trx/TGF-β1m fusion was cleaved by enteropeptidase, and the carrier protein of TGF-β1m was separated from thioredoxin on Ni-NTA agarose. Separation of monomeric molecules from the noncovalently bounded oligomers was done using cation-exchange chromatography. The structure of purified TGF-β1m was confirmed by circular dichroism analysis. The developed technology allowed purifying biologically active tag-free monomeric TGF-β1m from bacteria with a yield of about 2.8 mg from 100 ml cell culture. The low-cost and easy purification steps allow considering that our proposed preparation of recombinant monomeric TGF-β1 could be employed for in vitro and in vivo experiments as well as for therapeutic intervention.

Novel disulfide-containing polypeptide toxin was discovered in the venom of the Tibellus oblongus spider. We report on isolation, spatial structure determination and electrophysiological characterization of this 41-residue toxin, called ω-Tbo-IT1. It has an insect-toxic effect with LD50 19 μg/g in experiments on house fly Musca domestica larvae and with LD50 20 μg/g on juvenile Gromphadorhina portentosa cockroaches. Electrophysiological experiments revealed a reversible inhibition of evoked excitatory postsynaptic currents in blow fly Calliphora vicina neuromuscular junctions, while parameters of spontaneous ones were not affected. The inhibition was concentration dependent, with IC50 value 40 ± 10 nM and Hill coefficient 3.4 ± 0.3. The toxin did not affect frog neuromuscular junctions or glutamatergic and GABAergic transmission in rat brains. Ca2+ currents in Calliphora vicina muscle were not inhibited, whereas in Periplaneta americana cockroach neurons at least one type of voltage gated Ca2+ current was inhibited by ω-Tbo-IT1. Thus, the toxin apparently acts as an inhibitor of presynaptic insect Ca2+ channels. Spatial structure analysis of the recombinant ω-Tbo-IT1 by NMR spectroscopy in aqueous solution revealed that the toxin comprises the conventional ICK fold containing an extended β-hairpin loop and short β-hairpin loop which are capable of making “scissors-like mutual motions”.

Neurotoxin II fromNaja oxiana venom is a short-chain snake curaremimetic neurotoxin containing four disulfide bonds. We obtained13C-15N-labeled neurotoxin II to study its internal dynamics and surface properties with atomic resolution. The recombinant protein has the native spatial structure and is biologically active. The nearly complete assignment of1H,13C and15N resonances for neurotoxin II was obtained by heteronuclear triple-resonance nuclear magnetic resonance spectroscopy. Analysis of the secondary chemical shifts of the1Hα,13Cα,13Cβ and13CO nuclei reveal their strong correlation with the protein secondary structure.

The mechanism of the reaction of high temperature solid state catalytic isotope exchange (HSCIE) of hydrogen in peptides with spillover-tritium at 140-180 degrees C was analyzed. This reaction was used for preparing [(3)H]enkephalins such as [(3)H]DALG with specific activity of 138 Ci/mmol and [(3)H]LENK with specific activity of 120 Ci/mmol at 180 degrees C. The analogues of [(3)H]ACTG(4-10) with specific activity of 80 Ci/mmol, [(3)H]zervamicin IIB with specific activity of 70 Ci/mmol and [(3)H]conotoxin G1 with specific activity 35 Ci/mmol were produced. The obtained preparations completely retained their biological activity. [(3)H]Peptide analysis using (3)H NMR spectroscopy on a Varian UNITY-600 spectrometer at 640 MHz was carried out. The reaction ability of amino fragments in HSCIE was shown to depend both of their structures and on the availability and the mobility of the peptide chain. The reaction of HSCIE with the beta-galactosidase from Termoanaerobacter ethanolicus was studied. The selected HSCIE conditions allow to prepare [(3)H] beta-galactosidase with specific activity of 1440 Ci/mmol and completely retained its the enzymatic activity.

Tritium-labeled α-conotoxin G1 with a molar radioactivity of 35 Ci/mmol and full biological activity (according to the binding to nicotinic acetylcholine receptor) was obtained by the high-temperature solid-state catalytic isotope exchange (HSCIE). The tritium distribution in the molecule of α-conotoxin G1 was revealed by3H NMR spectroscopy. Tritium was found in all amino acid residues except for the Asn4-Pro5-Ala6 fragment. The data on the comparative reactivity of C-H bonds, theab initio quantum-chemical calculation of the hydrogen exchange reaction, and the information on the spatial structures of α-conotoxin G1 in solution and in crystal state allowed us to establish that the reactivity of H atoms may be increased by their interaction with the electron donor O and N atoms at the transition state of the HSCIE reaction. A decrease in the rate of the HSCIE reaction could be caused by both a poor spatial accessibility of C-H bonds and a limited mobility of the peptide fragment containing these bonds.