Proteolytic dissection of turnip crinkle virus subunit in solution.

Turnip crinkle virus (TCV) can be dissociated under mild conditions, yielding dimers of the protein subunit, as shown by chemical cross-linking and by gel filtration. These dimers have defined regions of proteolytic sensitivity, correDeterminat ion of the complete three-dimensional structure of tomato bushy stunt virus (TBSV) has revealed a number of remarkable features with implications for mechanisms of assembly (Harrison et al., 1978; Harrison, 1980). The related turnip crinkle virus (TCV) has also been visualized by highresolution X-ray diffraction, and the structure of its protein shell is, as expected, substantially the same as that of TBSV (J. Hogle and S . C. Harrison, unpublished results). Success in reversible disassembly of TCV makes it a better choice than TBSV for mapping an assembly pathway. As an initial step toward this mapping, we have carried out chemical crosslinking and proteolytic dissection studies of the isolated TCV subunit. The rationale of these experiments depends on the general architecture of TBSV and TCV (shown diagrammatically in Figure 1). The particle contains 180 coat protein subunits From the Department of Biochemistry and Molecular Biology, Harvard University, Cambridge, Massachusetts 02 138. Received January 14, 1982. This work was supported in part by the National Institutes of Health (Grant CA-13202), by the National Science Foundation (Grant PCM 79-22159), and by an Alfred R. Sloan fellowship to S.C.H. The experiments form part of a Senior Honors Thesis in Biochemical Sciences, Harvard University. 0006-2960/82/0421-3862$01.25/0 sponding to the arm and hinge of the folded polypeptide chain. The results are discussed in terms of the known high-resolution structure of TCV and with regard to implications for the pathway of viral assembly. (M, -40000), probably one chain of a M, 80000 protein, and a molecule of singlestranded RNA (4800 nucleotides) (Ziegler et al., 1974). The coat subunit, containing about 390 amino acids, folds into distinct regions: a projecting domain (P), a domain forming a tightly connected shell (S), an arm (a), and an internal domain (R). The three symmetrically distinct environments for this subunit are labeled A, B, and C. The polypeptide accommodates to these three packing modes by flexion at the hinge (h) between S and P and by an ordering or disordering of the arm, a. Units at positions A and B (60 of each) have one hinge configuration, and the entire N-terminal region (R and a) appears to be spatially disordered. Subunits at positions C (60 in all) have another hinge position, and the arm is folded in an ordered way along the inner side of the S domain. The R region is spatially disordered in C subunits as well as in A and B. “Spatially disordered” is used here to mean merely that the segment is not fixed with respect to the outer shell. The R domain itself may be precisely folded but flexibly tethered to the rest of the subunit such that it can adopt a variety of positions vis-8-vis the S domain and hence present no strong features in a high-resolution electron density map. The entire RNA molecule is spatially disordered in this sense.