Vladimir A. Drachev

A mechanism of macroscopic (amorphous) aggregation of the tobacco mosaic virus coat protein

To gain more insight into the mechanisms of heating-induced irreversible macroscopic aggregation of the tobacco mosaic virus (TMV) coat protein (CP), the effects of pH and ionic strength on this process were studied using turbidimetry, CD spectroscopy, and fluorescence spectroscopy. At 42 degrees C, the TMV CP passed very rapidly (in less than 15s) into a slightly unfolded conformation, presumably because heating disordered a segment of the subunit where the so-called hydrophobic girdle of the molecule resides. We suppose that the amino acid residues of this girdle are responsible for the aberrant hydrophobic interactions between subunits that initiate macroscopic protein aggregation. Its rate increased by several thousands of times as the phosphate buffer molarity was varied from 20 to 70 mM, suggesting that neutralization of strong repulsive electrostatic interactions of TMV CP molecules at high ionic strengths is a prerequisite for amorphous aggregation of this protein.

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.

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°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 α-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.

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: I21 → 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°C for U1 and from 38 to 72°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.

Appearance of “β-Like” Circular Dichroism Spectra on Protein Aggregation That Is not Accompanied by Transition to β-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 α-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 “β-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 β-structure in the course of aggregation.

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°C and on the protein structure were studied. It was found that SDS completely inhibits the TMV CP (11.5 μM) 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 α-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.

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°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.

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

The thermal unfolding of duck gizzard tropomyosin dimers, αβ, αα, and ββ, and of a 1:1 mixture of αα and ββ homodimers was studied by differential scanning calorimetry (DSC). Both αα and ββ homodimers demonstrated a broad thermal transition with maxima at 37.4°C and 44.6°C, respectively. However, a sharp cooperative thermal transition at 41.5°C characteristic for αβ 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.

Comparative structural stability of subunits of the potato virus X coat protein in solution and in virus particles

Several optical methods and differential scanning calorimetry were used to study the structure and stability of free coat protein (CP) molecules and CP molecules in the virion of the potato virus X (PVX), a filamentous plant virus. All criteria suggest that PVX CP (hereinafter, CP) subunits in solution at room temperature display a certain preserved tertiary structure; however, this structure is very unstable and already denatures at 35°C. Very low concentrations of sodium dodecylsulfate or cetyltrimethylammonium bromide also disrupt the CP tertiary structure, three-five molecules of these detergents per one protein molecule being sufficient. However, the secondary structure of CP molecules does not change under the same conditions. Once included into the virion, CP subunits become considerably more stable towards increased temperature and detergents. This combination of a highly labile tertiary structure and a fairly stable secondary structure of free CP can be a structural basis for the recently discovered ability of PVX CP to assume two distinct functional states within the virion.

An “all-speed” autocalibration method for sedimentation equilibrium in dilute homogeneous and multicomponent solutions: II. Determination of molecular weights of proteins

The equilibrium ultracentrifugation of homogeneous and heterogeneous protein solutions carried out by the “all-speed” autocalibration technique described in the companion article (V. Ya. Chernyak and N. N. Magretova, (1982) Anal. Biochem.123, 101–109) is reported. These experiments corroborate conclusions made on the basis of the computer-simulated “runs” and the proposed computational procedure. The molecular weights of proteins as determined here concur with the reference data down to ±1% and about ±6% in experiments with homogeneous and multidisperse (two- and three-species) systems, respectively. The suggested method also allows one to use interference optics at a relatively low rotor speed and at a sufficiently small concentration (<1.5 mg/ml), without an auxiliary synthetic boundary run or any artificial device. The approximate findings about the weight ratio of the components in an unperturbed solution may be computed under the assumption that conservation of mass takes place.