[Comparative study of structural stabylity of potato virus X coat protein molecules in solution and in the virus particles].

With help of several optical methods and differential scanning calorimetry we studied the structure and stability of molecules of coat protein (CP) of filamentous of potato virus X (PVX) in free state and in the virions. According to the results of all these methods, at room temperature (25 degrees C) free PVX CP subunits possess some fixed tertiary structure but this structure is highly unstable and is completely disrupted at temperatures as low as 35 degrees C. The free PVX CP tertiary structure was also disrupted by very low sodium dodecylsulfate and cetyltrimetylammonium bromide concentrations: 3 to 5 moleculs of the surfactants per the CP molecule were sufficient to induce its total disruption. At the same time, these treatments did not result in any changes in the PVX CP secondary structure. Incorporation of the CP subunits into the PVX virions resulted in a strong increase in their stability to effects of increased temperatures and surfactants. This combination of highly labile tertiary structure and rather stable secondary structure of free PVX CP subunits may represent a structural basis for recently observed capacity of the PVX CP moleculs to assume two different functional states in the virion.

[Modified model of potato virus X coat protein structure].

We propose the modified model of the structure of coat protein (CP) subunits in filamentous virions of potato virus X (PVX). The model is similar to the one proposed by us in 2001 for the CP of another helical plant virus (potato virus A) belonging to other (potyvirus) group. In this model the PVX CP molecule consist of two main domains--a bundle of four alpha-helices located close to the virion long axis and a so-called RNP-fold (or abCd-fold) located near the virion surface. Basing on this model we suggest possible mechanism of described by J.G. Atabekov and colleagues structural transition ("remodeling") of the PVX virions resulting from their interaction with virus-specific TGB-1 protein.

Low sodium dodecyl sulfate concentrations inhibit tobacco mosaic virus coat protein amorphous aggregation and change the protein stability.

Effects of low SDS concentrations on amorphous aggregation of tobacco mosaic virus (TMV) coat protein (CP) at 52 degrees C and on the protein structure were studied. It was found that SDS completely inhibits the TMV CP (11.5 microM) unordered aggregation at the detergent/CP molar ratio of 15 : 1 (0.005% SDS). As judged by fluorescence spectroscopy, these SDS concentrations did not prevent heating-induced disordering of the large-distance part of the TMV CP subunit, including the so-called "hydrophobic girdle". At somewhat higher SDS/protein ratio (40 : 1) the detergent completely disrupted the TMV CP hydrophobic girdle structure even at room temperature. At the same time, these low SDS concentrations (15 : 1, 40 : 1) strongly stabilized the structure of the small-distance part of the TMV CP molecule (the four alpha-helix bundle) against thermal disordering as judged by the far-UV (200-250 nm) CD spectra. Possible mechanisms of TMV CP heating-induced unordered aggregation initiation are discussed.

Adaptive algorithm of automated annotation.

MOTIVATION: It is common knowledge that the avalanche of data arriving from the sequencing projects cannot be annotated either experimentally or manually by experts. The need for a reliable and convenient tool for automated sequence annotation is broadly recognized. RESULTS: Here, we describe the Adaptive Algorithm of Automated Annotation (A(4)) based on a statistical approach to this problem. The mathematical model relates a set of homologous sequences and descriptions of their functional properties, and calculates the probabilities of transferring a sequence description onto its homologue. The proposed model is adaptive, its parameters (distribution characteristics, transference probabilities, thresholds, etc.) are dynamic, i.e. are generated individually for the sequences and various functional properties (words of the description). The proposed technique significantly outperforms the widely used test for frequency threshold, which is a special case of our model realized for the simplest set of parameters. The prediction technique has been realized as a computer program and tested on a random sequence sampling from SWISS-PROT. AVAILABILITY: The automated annotation program based on the proposed algorithm is available through the Web browser at http://www.genebee.msu.su/services/annot/basic.html.

Macroscopic aggregation of tobacco mosaic virus coat protein.

The relationship between processes of thermal denaturation and heat-induced aggregation of tobacco mosaic virus (TMV) coat protein (CP) was studied. Judging from differential scanning calorimetry "melting" curves, TMV CP in the form of a trimer-pentamer mixture ("4S-protein") has very low thermal stability, with a transition temperature at about 40 degrees C. Thermally denatured TMV CP displayed high propensity for large (macroscopic) aggregate formation. TMV CP macroscopic aggregation was strongly dependent on the protein concentration and solution ionic strength. By varying phosphate buffer molarity, it was possible to merge or to separate the denaturation and aggregation processes. Using far-UV CD spectroscopy, it was found that on thermal denaturation TMV CP subunits are converted into an intermediate that retains about half of its initial alpha-helix content and possesses high heat stability. We suppose that this stable thermal denaturation intermediate is directly responsible for the formation of TMV CP macroscopic aggregates.

Changes in the thermal unfolding of p-phenylenedimaleimide-modified myosin subfragment 1 induced by its 'weak' binding to F-actin.

Differential scanning calorimetry (DSC) was used to analyze the thermal unfolding of myosin subfragment 1 (S1) with the SH1 (Cys-707) and SH2 (Cys-697) groups cross-linked by N,N'-p-phenylenedimaleimide (pPDM-S1). It has been shown that F-actin affects the thermal unfolding of pPDM-S1 only at very low ionic strength, when some part of pPDM-S1 binds weakly to F-actin, but not at higher ionic strength (200 mM KCl). The weak binding of pPDM-S1 to F-actin shifted the thermal transition of pPDM-S1 by about 5 degrees C to a higher temperature. This actin-induced increase in thermal stability of pPDM-S1 was similar to that observed with 'strong' binding of unmodified S1 to F-actin. Our results show that actin-induced structural changes revealed by DSC in the myosin head occur not only upon strong binding but also on weak binding of the head to F-actin, thus suggesting that these changes may occur before the power-stroke and play an important role in the motor function of the head.

Appearance of "beta-like" circular dichroism spectra on protein aggregation that is not accompanied by transition to beta-structure.

CD spectra in the 200 to 250 nm spectral region for small ordered aggregates (trimers-pentamers) of tobacco mosaic virus (TMV) coat protein (CP) and for long virus-like helical aggregates of TMV CP were compared. It was found that small (4S) TMV CP aggregates have a CD spectrum typical of a protein with high alpha-helix content, which agrees well with results of X-ray diffraction studies. But in the long helical aggregates (and in the TMV virions) TMV CP gives "beta-like" CD spectra similar to those of many other aggregated proteins. From X-ray diffraction data, it is well known that TMV CP subunits do not change their secondary or tertiary structure on assembly into virions or the helical repolymerized protein. Thus, the change in the shape of 200 to 250 nm CD spectra cannot be employed as the sole criterion of the conversion of a protein to beta-structure in the course of aggregation.

Activity of penicillin acylase from E. coli in the reversed-micelle system AOT--H2O--octane.

The behavior of a penicillin acylase from E. coli was studied in the reversed-micelle system AOT--H2O--octane. Kinetic studies of the enzymatic hydrolysis of the m-carboxy-p-nitroanilide of phenylacetic acid, titration of the penicillin acylase active site with an irreversible specific inhibitor (phenylmethylsulfonyl fluoride), sedimentation analysis at different hydration degrees, and chemical modification showed that the enzyme loses no more than 20% of its initial activity during 3-4 h in the reversed-micelle systems of different hydration degrees and retains its catalytically active structure.

Thermally induced chain exchange of smooth muscle tropomyosin dimers studied by differential scanning calorimetry.

The thermal unfolding of duck gizzard tropomyosin dimers, alphabeta, alphaalpha, and betabeta, and of a 1:1 mixture of alphaalpha and betabeta homodimers was studied by differential scanning calorimetry (DSC). Both alphaalpha and betabeta homodimers demonstrated a broad thermal transition with maxima at 37.4 degrees C and 44.6 degrees C, respectively. However, a sharp cooperative thermal transition at 41.5 degrees C characteristic for alphabeta heterodimer appeared on the thermogram of the mixture of homodimers. The appearance of this transition was prevented by disulfide cross-linking of polypeptide chains in the homodimers. Thus, DSC studies clearly demonstrate formation of tropomyosin heterodimers during heating of the mixture of homodimers and in agreement with earlier published reports indicate thermally induced chain exchange between tropomyosin dimers.

A comparative differential scanning calorimetric study of tobacco mosaic virus and of its coat protein ts mutant.

The differential scanning calorimetry (DSC) 'melting curves' for virions and coat proteins (CP) of wild-type tobacco mosaic virus (strain U1) and for its CP ts mutant ts21-66 were measured. Strain U1 and ts21-66 mutant (two amino acid substitutions in CP: 121 --> T and D66 --> G) differ in the type of symptoms they induce on some host plants. It was observed that CP subunits of both U1 and ts21-66 at pH 8.0, in the form of small (3-4S) aggregates, possess much lower thermal stability than in the virions. Assembly into the virus particles resulted in a DSC melting temperature increase from 41 to 72 degrees C for U1 and from 38 to 72 degrees C for ts21-66 CP. In the RNA-free helical virus-like protein assemblies U1 and ts21-66 CP subunits had a thermal stability intermediate between those in 3-4S aggregates and in the virions. ts21-66 helical protein displayed a somewhat lower thermal stability than U1.

Interaction of myosin subfragment 1 with F-actin studied by differential scanning calorimetry.

The thermal unfolding of the myosin subfragment 1 (S1) and of filamentous actin (F-actin) in their strong complex obtained in the presence of ADP was studied by differential scanning calorimetry (DSC). It is shown that in the acto-S1 complexes S1 and F-actin melt separately, and thermal transitions of each protein can be easily followed. Interaction of S1 with F-actin significantly increases S1 thermal stability and also affects the thermal stability of F-actin. Although S1 unfolds at much lower temperature than F-actin, the molecules of S1 remain bound to F-actin even after full denaturation. Under these conditions S1 may induce cross-linking between actin filaments. It is concluded that DSC studies on the acto-S1 complexes offer a new and promising approach to investigate the structural changes which occur in the myosin head and in F-actin due to their interaction.

[GeneBee-NET: An Internet based server for biopolymer structure analysis]. / GeneBee-NET: Baziruiushchiisia v seti Internet server po analizu struktur biopolimerov.

A network server providing biopolymer structure databank retrieval as well as some other biocomputing procedures for Internet users is described. Its basic procedures consist in looking for sequence and 3D homologies (similarities). Found homologies are used for constructing multiple alignment, for predicting RNA and protein secondary structures as well as for constructing phylogenetic trees. Alongside traditional methods of sequence homology retrieval, a "matrix-free" (correlation) method is proposed. A similar procedure is used to locate protein 3D similarities. For novel procedures algorithm ideas and their possible applications are discussed. The service ideology is based on the interaction of server and client programs. The client program (GeneBee for IBM PC) can be used to form queries to the server as well as to manipulate a treatment result. In the absence of the client program the interaction with the server can be in the text mode. The E-mail and WWW addresses for the server are as follows: SERVE/INDY.GENEBEE.MSU.SU and WWW.GENEBEE.MSU.SU.

[The effect of the laser microirradiation of the cell center on neutrophil motility]. / Vliianie lazernogo mikrooblucheniia kletochnogo tsentra na podvizhnost' neitrofilov.

The cell center of human neutrophils spread on polylysine-coated coverslips was irradiated with an argon laser microbeam. After the cells were pretreated with acridine orange, the irradiation of the cell center in a dose of over 0.1 J completely and irreversibly suppressed the motility of neutrophils (both random migration and chemotaxis), even though the cells retained their polarization. The same dose, applied to the cell nucleus and the forward and backward edges of the cytoplasm, resulted in little, if any, effect on cell motility, and did not inhibit their movement toward the target. Electron microscopy of the cells with the irradiated center showed the microtubules to persist for no less than 30 minutes; no visible destruction was caused in the cell center structure. Consequently, the cell center directly controls (not through polymerization of microtubules) the motility of neutrophils.

[The effect of high hydrostatic pressure on the cell center and microtubules in tissue culture cells]. / Vliianie vysokogo gidrostaticheskogo davleniia na kletochnyi tsentr i mikrotrubochki v kletkakh kul'tury tkani.

A network of cytoplasmic microtubules in PE cells disassembles at 37 degrees C under 1000 atm pr. in 12 to 14 hours; under 2000 atm pr., the disassembly time is not more than 2 hours. The reconstitution process sets in 20 minutes after pressure dropping to proceed diffusely throughout the cytoplasm. Microtubules attached to the cell center reappear in 45 minutes. The dynamics of microtubular disassembly and reconstitution indicates a complete inactivation of the cell center as a microtubule-organizing center.

High-pressure enzyme kinetics. Lactate dehydrogenase in an optical cell that allows a reaction to be started under high pressure.

A newly designed optical cell allows an enzyme reaction to be started under high pressure and makes it possible to begin measurement of the reaction rate after a 'dead time' no longer than 1-2 s. This device was used to study the kinetics of lactate dehydrogenase reaction at 1 kbar. At this pressure lactate dehydrogenase from rabbit muscle exhibited a rapid deactivation in the presence of NADH if pyruvate was absent. After addition of pyruvate the reaction was initiated and proceeded at a constant rate, i.e., without loss of enzyme activity. It is suggested that pyruvate markedly increases the association constant of this tetrameric enzyme.

[Homogeneous Cl. perfringens alpha-anatoxin: its physicochemical and immunogenic properties]. / Gomogennyi al'fa-anatoksin C1. perfringens: ego fiziko-khimicheskie i immunogennye svoistva.

The authors present a method of obtaining relatively homogeneous preparations of alpha-toxoid of Cl. perfringens, type A, including the primary conception of the alpha-toxin proteins, their chromatography on DEAE-cellulose, fractionation with (NH4)2SO4, detoxication, with the subsequent gel-filtration through sephadex and isoelectric focussing. Sedimentation coefficient of the preparation proved to be 3.8 S, isoelectric point-4.83 +/- 0.07. In studying the immunogenic properties of alpha-toxoid in experiments on guinea pigs and rabbits their high immunogenicity, exceeding that of the industrial toxoid 8- and 6-fold, respectively, was established. Homogeneous preparations of alpha-toxoid provided intense anti-microbial immunity. Interlinear differences in the levels of the immune response of inbred mice, highly-reactive (BALB/c) and low-reactive (C57BL/6) to alpha-toxoid, reached 20-fold; in combination with the high immunogenicity of this antigen for mice this permits to recommend it for immunogenic studies.