Detection and characterization of an intermediate conformation during the divalent ion-dependent swelling of tomato bushy stunt virus

Abstract

We have used time-resolved small-angle X-ray scattering (SAXS) in solution to study the swelling reaction of TBSV upon chelation of its constituent calcium at mildly basic pH. The reaction was initiated by rapid mixing of a virus solution with the same buffer containing a variable amount of EDTA. The X-ray scattering data sets recorded after mixing were submitted to a singular value decomposition analysis which demonstrated the existence of an intermediate state in addition to the compact and fully swollen forms of the virion. The kinetics of the reaction display an initial lag, and a linear combination of three exponential terms is required for a satisfactory analytical fit. Accordingly, a model is put forward involving three sequential irreversible processes between four species. Beyond the three structural species mentioned above, the fourth one, which is the second species along the time sequence, is proposed to represent those viruses which, although partially deprived of Ca2+ ions, are still in the compact conformation. Using the combination of the kinetic model and the structural data, an estimate of the intermediate scattering pattern can be derived from each time resolved frame. These patterns are all very similar after a slight drift towards the swollen pattern over the first 2 min. The curve presents well-resolved minima and maxima, corresponding to an isometric particle with an outer radius of about 172 A for the intermediate conformation.