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Sedimentation and proton binding studies on the endothermic self-association of tobacco mosaic virus (TMV) protein indicate that the so-called "20S" sedimenting protein is an interaction system involving at least the 34-subunit two-turn yield cylindrical disk aggregate and the 49-subunit three-turn helical rod. The pH dependence of this overall equilibrium suggests that disk formation is proton-linked through the binding of protons to the two-turn helix which is not present as significant concentrations near pH 7. There is a temperature-induced intramolecular conformation change in the protein leading to a difference spectrum which is complete in 5 x 10(-6) s at pH 7 and 20 degrees C and is dominated at 300 nm by tryptophan residues. Kinetics measurements of protein polymerization, from 10(-6) to 10(3) s, reveal three relaxation processes at pH 7.0, 20 degrees C, 0.10 M ionic strength K (H) PO4. The fastest relaxation time is a few milliseconds and represents reactions within the 4S protein distribution. The second fastest relaxation is 50-100 x 10(-3) s and represents elementary polymerization steps involved in the formation of the approximately 20 S protein. Analysis of the slowest relaxation, approximately 5 x 10(4) s, suggests that this very slow formation of approximately 20 S protein may be dominated by some first order process in the overall dissociation of approximately 20S protein. Sedimentation measurements of the rate of TMV reconstitution, under the same conditions, show by direct measurements of 4S and approximately 20S incorporation at various 4S to approximately 20S weight ratios that the relative rate of approximately 20S incorporation decreases almost linearly, from 0 to 50% 4S. There appears to be one or more regions of TMV-RNA, approximately 1-1.5 kilobases long, which incorporates approximately 20S protein exclusively. Solutions of approximately 95-100% approximately 20S protein have been prepared for the first time and used for reconstitution with RNA. Such protein solutions yield full size TMV, but at a slower rate than if 4S protein is added. Thus the elongation reaction in TMV assembly, following nucleation with approximately 20S protein, is not exclusively dependent upon the presence of either 4S or approximately 20S protein aggregates. The initial, maximum, rate of reconstitution increases about threefold when the protein composition is changed from 5% to 30% 4S protein, at constant total protein concentration at pH 7.0, 20 degrees C in 0.10 M ionic strength K (H)PO4. The probable binding frame at the internal assembly nucleation site of TMV-RNA has been determined by measuring the association constants for the binding of various trinucleoside diphosphates to helical TMV protein rods. The -CAG-AAG-AAG-sequence at the nucleation site is capable of providing at least 10-14 kcal/mol of sites of binding free energy for the nucleation event in TMV self-assembly.

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Desmin protofilaments and the proteolytically derived a-helical rod domain have been characterized by highresolution gel permeation chromatography (GPC) using columns calibrated for the determination of viscosity radii. Additional characterization by chemical cross-linking and the determination of sedimentation values allowed the calculation of the molecular dimensions of the molecular species isolated. In dilute buffers GPC separated desmin rod preparations into two complexes: a dimer species (single coiled coil) with a length of 50 & 5 nm and a tetramer species (two coiled coils) with a length of 65 & 5 nm. Thus the two coiled coils in the tetramer are staggered by approximately 15 nm. The hydrodynamically derived lengths of the rod dimer and tetramer are supported by electron microscopy after metal shadowing. The hydrodynamic properties of desmin protofilaments follow that of the rod tetramer. The rod is organized from double-stranded coiled coils [4] in which the participating a-helical chains are aligned in parallel and in register [5, 61. Usually two coiled coils are found tightly associated in the tetrameric protofilament unit, which is the building block of the intermediate filaments [4, 7-101. Two main questions arise about the structure of the tetramer: are the two coiled coils of the tetramer arranged in parallel or antiparallel manner and are the coiled coils aligned in register or with a stagger? Protofilaments and the isolated rod, lacking the head and tail domains due to limited proteolysis, have been extensively studied by electron microscopy [lo-151. Molecules displaying the unit length of the coiled coil (about 40 -50 nm) as well as particles with the one and a half fold length (about 70 nm) have been recognized in these studies. Because a firm correlation of the observed length variants with the number of chains per particle was not reached, the mode of alignment remained unresolved.We have now re-investigated the shape of the tetramer by hydrodynamic identification of distinct oligomer species. We show that the chymotryptically derived rod domain of chicken desmin (residues 70-415) is present in dilute buffers as a mixture of dimers (single coiled coils) and tetramers (double coiled coils), which can be separated by gel permeation chromatography (GPC). The chromatographically derivedCorrespondence to