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.

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The differential scanning calorimetry (DSC) 'melting curves' for virions and coat proteins (CP) of wild-type tobacco mosaic virus (strain UI) and for its CP ts mutant ts21-66 were measured. Strain UI 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 UI and from 38 to 72°C for ts21-66 CP. In the RNA-free helical viruslike protein assemblies UI 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.

Amino acid substitutions in a majority of tobacco mosaic virus (TMV) coat protein (CP) ts-mutants have previously been mapped to the same region of the CP molecule tertiary structure, located at a distance of about 70 A from TMV virion axis. In the present work some properties of a new TMV CP ts-mutant ts21-66 (two substitutions I21=>T and D66=>G, both in the 70-A region) were studied. Thermal inactivation characteristics, sedimentation properties, circular dichroism spectra, and modification by a lysine-specific reagent, trinitrobenzensulfonic acid, of ts21-66 CP were compared with those of wild-type (U1) TMV CP. It is concluded that the 70-A region represents the most labile portion of the TMV CP molecule. Partial disordering of this region in the mutant CP at permissive temperatures leads to loss of the capacity to form two-layer aggregates of the cylindrical type, while further disordering induced by mild heating results also in the loss of the ability to form ordered helical aggregates.

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