No abstract

No abstract

The kinetics of thermal aggregation of coat protein (CP) of tobacco mosaic virus (TMV) have been studied at 42 and 52 degrees C in a wide range of protein concentrations, [P]0. The kinetics of aggregation were followed by monitoring the increase in the apparent absorbance (A) at 320 nm. At 52 degrees C the kinetic curves may be approximated by the exponential law in the range of TMV CP concentrations from 0.02 to 0.30 mg/ml, the first order rate constant being linearly proportional to [P]0 (50 mM phosphate buffer, pH 8.0). The analogous picture was observed at 42 degrees C in the range of TMV CP concentrations from 0.01 to 0.04 mg/ml (100 mM phosphate buffer, pH 8.0). At higher TMV CP concentrations the time of half-conversion approaches a limiting value with increasing [P]0 and at sufficiently high protein concentrations the kinetic curves fall on a common curve in the coordinates [A/A(lim); t] (t is time and A(lim) is the limiting value of A at t --> infinity). According to a mechanism of aggregation of TMV CP proposed by the authors at rather low protein concentrations the rate of aggregation is limited by the stage of growth of aggregate, which proceeds as a reaction of the pseudo-first order, whereas at rather high protein concentrations the rate-limiting stage is the stage of protein molecule unfolding.

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.

Post-mitotic reassembly of nuclear envelope (NE) and the endoplasmic reticulum (ER) has been reconstituted in a cell-free system based on interphase Xenopus egg extract. To evaluate the relative contributions of cytosolic and transmembrane proteins in NE and ER assembly, we replaced a part of native membrane vesicles with ones either functionally impaired by trypsin or N-ethylmaleimide treatments or with protein-free liposomes. Although neither impaired membrane vesicles nor liposomes formed ER and nuclear membrane, they both supported assembly reactions by fusing with native membrane vesicles. At membrane concentrations insufficient to generate full-sized functional nuclei, addition of liposomes and their fusion with membrane vesicles resulted in an extensive expansion of NE, further chromatin decondensation, restoration of the functionality, and spatial distribution of the nuclear pore complexes (NPCs), and, absent newly delivered transmembrane proteins, an increase in NPC numbers. This rescue of the nuclear assembly by liposomes was inhibited by wheat germ agglutinin and thus required active nuclear transport, similarly to the assembly of full-sized functional NE with membrane vesicles. Mechanism of fusion between liposomes and between liposomes and membrane vesicles was investigated using lipid mixing assay. This fusion required interphase cytosol and, like fusion between native membrane vesicles, was inhibited by guanosine 5-3-O-(thio)triphosphate, soluble N-ethylmaleimide-sensitive factor attachment protein, and N-ethylmaleimide. Our findings suggest that interphase cytosol contains proteins that mediate the fusion stage of ER and NE reassembly, emphasize an unexpected tolerance of nucleus assembly to changes in concentrations of transmembrane proteins, and reveal the existence of a feedback mechanism that couples NE expansion with NPC assembly. The nuclear envelope (NE)2 prevents free diffusion of macromolecules between the nucleus and cytoplasm and therefore separates processes of gene transcription and translation in the cell. In species with an open mitosis, the NE breaks and reassembles during each cell cycle. NE reassembly starts during anaphase and involves formation of a double membrane around segregated chromosomes, insertion of multiprotein nuclear pore complexes (NPCs), and further NE expansion. The outer and inner membranes of NE are connected at the sites of NPCs, which provide selective nucleocytoplasmic transport. The space between nuclear membranes (perinuclear space) is continuous with the endoplasmic reticulum (ER) lumen. The inner nuclear membrane has a specific protein composition and contacts with chromatin and, in metazoans, with lamina.NE formation has been intensively studied in vitro with demembraned chromatin and fractionated Xenopus laevis egg extract (1-5). The fractionation step results in ER disruption and yields membrane-free cytosolic extract along with distinct populations of membrane vesicles (MVs) involved in ER and NE formation (6 -13). It was proposed that NE assembly i...

All identified membrane fusion proteins are transmembrane proteins. In the present study, we explored the post-mitotic reassembly of the NE (nuclear envelope). The proteins that drive membrane rearrangements in NE assembly remain unknown. To determine whether transmembrane proteins are prerequisite components of this fusion machinery, we have focused on nuclear reconstitution in a cell-free system. Mixing of soluble interphase cytosolic extract and MV (membrane vesicles) from amphibian eggs with chromatin results in the formation of functional nuclei. We replaced MV and cytosol with protein-free phosphatidylcholine LS (liposomes) that were pre-incubated with interphase cytosol. While later stages of NE assembly yielding functional nucleus did not proceed without integral proteins of MV, LS-associated cytosolic proteins were sufficient to reconstitute membrane targeting to the chromatin and GTP-dependent lipid mixing. Binding involved LS-associated A-type lamin, and fusion involved Ran GTPase. Thus in contrast with post-fusion stages, fusion initiation in NE assembly, like membrane remodelling in budding and fission, does not require transmembrane proteins.

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.

In recent years, the number of works devoted to protein aggregation increases nearly in geometrical progression. This high intensity is accounted for by the fact that now it is clear that many serious diseases of humans and animals (Parkinson's and Alzheimer's diseases, prion infections, and many others) are related to accumulation of various protein aggregates in the body. Earlier, it was believed that specific ordered (the socalled amyloid) aggregates are responsible for the development of these diseases [1,2]. However, ample data accumulated in the past years indicate that pathogenicity is exhibited by unordered amorphous aggregates [3,4]. The amorphous protein aggregation is a topical problem of contemporaneous biotechnology. However, structural studies of amorphous aggregates are significantly hampered by their large sizes, instability, and heterogeneity.The coat protein (CP) of tobacco mosaic virus (TMV) is a classic model to study the ordered aggregation ("polymerization") [5]. We discovered that this protein is a very convenient model for studying the amorphous aggregation as well [6][7][8]. Unordered heatinduced aggregation of TMV CP at 52°ë is a well reproducible process (which is not characteristic of systems of this type), and its rate may be easily regulated by varying the ionic strength of solution, protein concentration, and temperature. In the previous study [8], we reported that heat-induced unordered aggregation of TMV CP was completely inhibited by low (approximately 175 µ M) concentrations of sodium dodecyl sulfate (SDS). In this study, we showed that lower concentrations (less than 20 µ M) of the cationic detergent cetyltrimethylammonium bromide (CTAB) induce a rapid amorphous aggregation of TMV CP at micromolar concentrations in phosphate buffer, pH 8.0, already at room temperature ( 25°ë ).In the past years, CTAB is widely used as an artificial chaperon-an agent facilitating aggregation and proper folding of denatured and aggregated proteins [9, 10]. In those works, CTAB was used at concentrations of approximately 600 µ M.The methods of obtaining TMV CP preparations and recording its amorphous aggregation on spectrophotometers by an increment in turbidity (absorption at 313 nm caused by an increase in light scattering) have been described in detail in our previous articles [6][7][8].Curves illustrating an increase in turbidity for 11.5 µ M TMV CP (200 µ g/ml) in 10 mM phosphate buffer, pH 8.0, in the presence of various concentrations of CTAB are shown in Fig. 1. It can be seen that, even at a CTAB concentration of only 34.5 µ M (detergent : protein molar ratio, 3 : 1), a rapid (without a lag [7]) increase in turbidity is observed. Approximately 10-15 min later, the value of A 313 reaches plateau, which is followed by protein precipitation. At higher concentrations of CTAB, protein aggregation proceeded even more rapidly: its maximal rate for 11.5 µ M CP was observed at a detergent-to-protein ratio of 13 : 1. However, further increase in the concentration of CTAB resulted in a decrease in t...