RESUMO
The increased ultrasonic absorption brought about by self-assembly of biomolecules is analyzed for the assembly process from the 20S aggregate to the helical rod of tobacco mosaic virus protein in solution, designated here as the 20S --> P-helix transition. The analysis is based on theoretical developments in ultrasonic relaxation spectrometry presented previously and illustrates the possibility that this technique can be used for characterizing fluctuations. The analysis makes use of NMR data for the system in solution and of x-ray diffraction data for the closely related transition from the two-ring disk to the virion. These x-ray data comprise the high-resolution structures and the Debye-Waller temperature factors of the main chain atoms of both the two-ring disk in crystals and the virion in oriented gel form. First, reduced ultrasonic spectra are obtained for the 4S, 20S, and helical rod aggregates. The fluctuation-enhancement factor for the helical rod is determined independently of any deconvolution into normal modes of relaxation and is shown not to depend on the particular procedure of reduction employed. The increase of ultrasonic absorption in the 20S --> P-helix transition primarily reveals enhancement of the relaxing system's normal-mode volume fluctuations. The observed relaxations probably involve one conformational process per subunit. The normal-mode volume fluctuations are then estimated from a bimodal least-squares best fit to the data, and a lower bound for the reaction volume associated with the fast steps is obtained. Two mechanisms are considered as follows: (i) a destabilization process in which the free-energy difference between two states is reduced and (ii) an increase in reaction volumes of local conformation changes in the helical aggregate, resulting from the formation of a "carboxyl cage-like" structure and from the change in environment produced inside the cage. Increased reaction volumes would not be detected with x-ray diffraction. The possible occurrence of fluctuations at the RNA binding site raises the question of whether a quaternary structure that exhibits significant conformational fluctuations must be present for the binding of the nucleic acid.
RESUMO
The structural fluctuations specific to self-assembled biological systems have been investigated further with ultrasonic techniques by using two strains of tobacco mosaic virus (TMV), as well as the helical aggregate of the common strain protein and subassemblies of it. We confirmed our earlier conclusion that protein assemblies exhibit specific structural fluctuations detected in ultrasonic experiments. As in spherical viruses, the fluctuations exhibited by the protein aggregates having a quaternary structure similar to that of the virion were modified in the virus by interaction with the RNA strand. It is unlikely that the origin for the observed effect is due either to: (1) the difference in local mobility of the segment 89 to 113 of the polypeptide chain in TMV and in the helical aggregate on the one hand, and in smaller aggregates, on the other hand; or (2) a local fluctuation associated with proton transfer reactions or ion-pair interactions. The most remarkable feature in the TMV system is the fact that the two-ring disk showed no excess of ultrasonic absorption with respect to the A-protein oligomer, while a large increase of ultrasonic absorption was observed in the rod-like aggregate that had undergone the disk-helix transition.
