T. V. Ovchinnikova

Lipid-protein nanodiscs: Possible application in high-resolution NMR investigations of membrane proteins and membrane-active peptides

High-resolution NMR is shown to be applicable for investigation of membrane proteins and membrane-active peptides embedded into lipid-protein nanodiscs (LPNs). 15N-Labeled K+-channel from Streptomyces lividans (KcsA) and the antibiotic antiamoebin I from Emericellopsis minima (Aam-I) were embedded in LPNs of different lipid composition. Formation of stable complexes undergoing isotropic motion in solution was confirmed by size-exclusion chromatography and 31P-NMR spectroscopy. The 2D 1H-15N-correlation spectra were recorded for KcsA in the complex with LPN containing DMPC and for Aam-I in LPNs based on DOPG, DLPC, DMPC, and POPC. The spectra recorded were compared with those in detergent-containing micelles and small bicelles commonly used in high-resolution NMR spectroscopy of membrane proteins. The spectra recorded in LPN environments demonstrated similar signal dispersion but significantly increased 1HN line width. The spectra of Aam-I embedded in LPNs containing phosphatidylcholine showed significant selective line broadening, thus suggesting exchange process(es) between several membrane-bound states of the peptide. 15N relaxation rates were measured to obtain the effective rotational correlation time of the Aam-I molecule. The obtained value (∼40 nsec at 45°C) is indicative of additional peptide motions within the Aam-I/LPN complex.

Spatial structure of zervamicin IIB bound to DPC micelles: implications for voltage-gating.

Zervamicin IIB is a 16-amino acid peptaibol that forms voltage-dependent ion channels with multilevel conductance states in planar lipid bilayers and vesicular systems. The spatial structure of zervamicin IIB bound to dodecylphosphocholine micelles was studied by nuclear magnetic resonance spectroscopy. The set of 20 structures obtained has a bent helical conformation with a mean backbone root mean square deviation value of approximately 0.2 A and resembles the structure in isotropic solvents (Balashova et al., 2000. NMR structure of the channel-former zervamicin IIB in isotropic solvents. FEBS Lett 466:333-336). The N-terminus represents an alpha-helix, whereas the C-terminal part has a mixed 3(10)/alpha(R) hydrogen-bond pattern. In the anisotropic micelle environment, the bending angle on Hyp10 (23 degrees) is smaller than that (47 degrees) in isotropic solvents. In the NOESY (Nuclear Overhauser Effect Spectroscopy) spectra, the characteristic attenuation of the peptide signals by 5- and 16-doxylstearate relaxation probes indicates a peripheral mode of the peptaibol binding to the micelle with the N-terminus immersed slightly deeper into micelle interior. Analysis of the surface hydrophobicity reveals that the zervamicin IIB helix is amphiphilic and well suited to formation of a tetrameric transmembrane bundle, according to the barrel-stave mechanism. The results are discussed in a context of voltage-driven peptaibol insertion into membrane.

NMR structure of the channel‐former zervamicin IIB in isotropic solvents

Spatial structure of the membrane channel‐forming hexadecapeptide, zervamicin IIB, was studied by NMR spectroscopy in mixed solvents of different polarity ranging from CDCl3/CD3OH (9:1, v/v) to CD3OH/H2O (1:1, v/v). The results show that in all solvents used the peptide has a very similar structure that is a bent amphiphilic helix with a mean backbone root mean square deviation (rmsd) value of ca. 0.3 Å. Side chains of Trp1, Ile2, Gln3, Ile5 and Thr6 are mobile. The results are discussed in relation to the validity of the obtained structure to serve as a building block of zervamicin IIB ion channels.

Domain structure and ATP‐induced conformational changes in Escherichia coli protease Lon revealed by limited proteolysis and autolysis

Escherichia coli protease Lon (La) is an adenosine triphosphate (ATP)‐regulated homo‐oligomeric proteolytic complex responsible for the recognition and selective degradation of abnormal and unstable proteins. Each subunit of the protease Lon appears to consist of three functional domains: the C‐terminal proteolytic containing a serine active site, the central displaying the ATPase activity, and the N‐terminal with still obscure function. We have used limited proteolysis to probe the domain structure and nucleotide‐induced conformational changes in the enzyme. Limited proteolysis of the native protease Lon generated a low number of stable fragments roughly corresponding to its functional domains. Conformational changes in the wild‐type enzyme and its mutant forms in the presence or absence of adenine and guanine nucleotides were investigated by limited proteolysis. The nucleotide character was shown to play a key role for susceptibility of the protease Lon to limited proteolysis, in particular, for resistance of the ATPase functional domain. ATP and adenosine diphosphate displayed a protective effect of the ATPase domain of the enzyme. We suggest that these nucleotides induce conformational changes of the enzyme, transforming the ATPase domain from the most vulnerable part of the molecule into a spatially inaccessible one. Both limited proteolysis and autolysis demonstrate that the most stable part of the protease Lon molecule is its N‐terminal region. Obvious resistance of the protease Lon C‐terminus to proteolysis indicates that this region of the enzyme molecule including its substrate‐binding and proteolytic domains has a well folded structure.

Purification and primary structure of novel lipid transfer proteins from germinated lentil (Lens culinaris) seeds

A subfamily of eight novel lipid transfer proteins designated as Lc-LTP1-8 was found in the lentil Lens culinaris. Lc-LTP2, Lc-LTP4, Lc-LTP7, and Lc-LTP8 were purified from germinated lentil seeds, and their molecular masses (9268.7, 9282.7, 9121.5, 9135.5 daltons) and complete amino acid sequences were determined. The purified proteins consist of 92–93 amino acid residues, have four disulfide bonds, and inhibit growth of Agrobacterium tumefaciens. Total RNA was isolated from germinated lentil seeds, RT-PCR and cloning were performed, and the cDNAs of six LTPs were sequenced. Precursor 116–118-residue proteins with 24–25-residue signal peptides were found, and two of them are purified proteins Lc-LTP2 and Lc-LTP4.

Backbone dynamics of the channel-forming antibiotic zervamicin IIB studied by 15N NMR relaxation

The backbone dynamics of the channel‐forming peptide antibiotic zervamicin IIB (Zrv‐IIB) in methanol were studied by 15N nuclear magnetic resonance relaxation measurements at 11.7, 14.1 and 18.8 T magnetic fields. The anisotropic overall rotation of the peptide was characterized based on 15N relaxation data and by hydrodynamic calculations. ‘Model‐free’ analysis of the relaxation data showed that the peptide is fairly rigid on a sub‐nanosecond time‐scale. The residues from the polar side of Zrv‐IIB helix are involved in micro–millisecond time‐scale conformational exchange. The conformational exchange observed might indicate intramolecular processes or specific intermolecular interactions of potential relevance to Zrv‐IIB ion channel formation.

Influenza A virus M1 protein structure probed by in situ limited proteolysis with bromelain.

Influenza A virus matrix M1 protein is membrane associated and plays a crucial role in virus assembly and budding. The N-terminal two thirds of M1 protein was resolved by X-ray crystallography. The overall 3D structure as well as arrangement of the molecule in relation to the viral membrane remains obscure. Now a proteolytic digestion of virions with bromelain was used as an instrument for the in situ assessment of the M1 protein structure. The lipid bilayer around the subviral particles lacking glycoprotein spikes was partially disrupted as was shown by transmission electron microscopy. A phenomenon of M1 protein fragmentation inside the subviral particles was revealed by SDS-PAGE analysis followed by in-gel trypsin hydrolysis and MALDI-TOF mass spectrometry analysis of the additional bands. Putative bromelain-digestion sites appeared to be located at the surface of the M1 protein globule and could be used as landmarks for 3D molecular modeling.

Lactoferrin from Canine Neutrophils: Isolation and Physicochemical and Antimicrobial Properties

Lactoferrin has been isolated from canine leukocytes for the first time. Lactoferrin was identified by N-terminal amino acid sequence and by capability to capture ferric cations resulting in a complex with absorbance maximum at 460–470 nm. It is demonstrated that canine lactoferrin resembles the human homolog in some physicochemical properties, i.e. molecular weight, carbohydrate presence, and conditions of protein—iron complex dissociation. Bactericidal activity of dog lactoferrin was demonstrated on the gram-negative bacterium Escherichia coli and gram-positive bacterium Listeria monocytogenes. Bactericidal activity of canine lactoferrin is similar to that of human lactoferrin.

Antimicrobial peptides of invertebrates. Part 1. structure, biosynthesis, and evolution

Antimicrobial peptides and proteins (AMPs) are among the most important components of the immune system of multicellular organisms. The role of AMPs is of particular importance for invertebrates which constitute the vast majority of species diversity of the living world, because these animals lack acquired immunity. The AMPs of animal origin are ribosomally-synthesized molecules that have, as a rule, a positive net charge and amphiphilic properties. They can act against bacteria, yeast and filamentous fungi, protozoa, and enveloped viruses. AMPs can also play a role of mediators of the immune system. Search and investigation for protective factors of the invertebrate host defense provide a better understanding of mechanisms of the innate immunity of humans and other mammals and give a key to a development of new medicines. The first part of this review focuses on special features of a structure, biosynthesis, regulation of gene expression, and molecular evolution of AMPs of invertebrates.

Interaction between tubulin and Na+, K+-ATPase in brain stem neurons

Functionally active Na2+,K2+-ATPase isozymes containing three types of the catalytic subunits (α1, α2, and α3) were obtained from calf brain by two methods: selective removal of contaminating proteins according to Jorgensen (1974) and selective solubilization of the enzyme with subsequent reformation of the membrane structure according to Esmann (1988). All preparations were characterized with respect to ouabain-inhibition constants. The presence of the cytoskeleton protein tubulin (β3 isoform) in the high-molecular-weight complex of Na2+,K2+-ATPase α3β1 isozyme from brain stem axolemma and the junction between Na2+,K2+-ATPase α3 subunit and tubulin β3 subunit are shown for the first time.