Abstract
TMV vulgare, A 14, Ni 725; Two-and Three-Layer Aggregates, Structural and Mechanistic Differences, Inter-Subunit Interactions, Non-Specific Aggregations The aggregation behaviour of tobacco mosaic virus (TMV) protein vulgare was compared to that of two mutants, A 14 and Ni725, with amino acid exchanges localized in the coat protein at posi tions 107 (Thr → Met, in N i725) and 129 (lie → Thr, in both mutants). This behaviour, as meas ured by sedimentation, hydrogen ion titration, light-scattering, and near-UV absorption difference and circular dichroism (CD) spectroscopy, differs characteristically both in the range of the A-protein (pH 8) and near neutrality, whereas nuclear magnetic resonance (NMR) and far-UV CD point at only subtle, or no structural differences between the three strains.
Near pH 8, the A-proteins of both mutants sediment nearly exclusively as 8 S aggregates, under conditions where vulgare protein forms a 4 S /8 S mixture (two-layer and three-layer aggregates, Vogel etal. in conditions where vulgare 4S aggregates dominate, both mutants sediment as a 4 S /8 S mixture. The average molecular weights of the 8S proteins corre spond to 12 (vulgare) to 15 (mutants) subunits. -Near neutrality both mutants titrate and polyme rize more cooperatively than vulgare protein; additionally, the pK(app.) of Ni 725 is shifted up wards, due to the higher a-helix forming potential of Met against Thr (pos. 107). Both mutants form large aggregates (> 200 S) of obviously helical conformation, by the uptake of one proton per subunit, whereas 20 S-disks constituting, under the same conditions, the stable entities in vul gare protein, are made only in minor amounts. These large mutant aggregates are remarkably more stable than the vulgare "overshoot" aggregates which transiently, too, may approach s-values and turbidities similar to the mutant aggregates; conformational changes, observed prior or in parallel to the formation of vulgare overshoot and disk aggregates, are significantly retarded in the large mutant aggregates. – Raising the ionic strength seems the only way to form mutant disks and stacks of disks (20-30 S) comparable to vulgare, pointing to the different pathways of disk formation, either at neutral pH or high ionic strength. – Evidence is given that the 8S aggregates of both mutant and vulgare proteins may behave similar in aggregation, the differences mainly being inserted by the 4S (two-layer) aggregates present in vulgare protein, which near neutrality seem responsible for the direct formation of (two-layer) disks. -The non-conservative exchange in po sition 129, altering the environment of Trp residues (52+17?), should weaken the "extended salt-bridge system" ("pairing") observed between the two layers of the disk (Bloomer et al., Nature, 1978). A competition is suggested between the strength of this pairing, and the binding of a third layer, regulating the mode of aggregation to two-layer, to three-layer, and to higher aggregates; this is corroborated by comparison with published results on temperature-sensitive (ts I) mutants and chemically modified proteins. – To explain the effects of residue 129 on the titration of the protein we suggest a mechanical analogy, made up of a balance between the charge and state of the "carboxyle cage" (Stubbs et al., Nature, 1977), as regulatory site, and the strength of the
BeStSel: From Secondary Structure Analysis to Protein Fold Prediction by Circular Dichroism Spectroscopy