Analysis of rapid, large-scale protein quaternary structural changes: Time-resolved X-ray solution scattering of Nudaurelia capensis omega virus (N omega V) maturation

Time-resolved small-angle X-ray scattering (TR-SAXS) was used to study the kinetics of a large conformational change that occurs during the maturation of an icosahedral virus. Virus-like particles (VLPs) of the T = 4 non-enve loped RNA virus Nudaurelia capensis omega virus (N omegaV) were shown to un dergo a large pH-dependent conformational change. Electron cryo-microscopy (cryoEM) and X-ray solution scattering were used to show that the precursor VLP (procapsid) was 16% larger in diameter, than the resulting capsid, whi ch was shown by the cryoEM study to, closely resemble the infectious mature virion. The procapsid. form of the VLPs was observed at pH 7.5 and was con verted to the capsid form at pH 5.0. Static SAXS measurements of the VLPs i n solutions ranging between these pH values determined that the half-titrat ion point of the transition was pH 6.0. Time-resolved SAXS experiments were performed on VLP solutions by initiating a pH change from 7.5 to 5.0 using a stopped-flow device, and the time-scale of the conformational change occ urred in the subsecond range. Using a less drastic pH change (lowering the pH to 5.8 or 5.5), the conformational change occurred more slowly, on the s ubminute or minute time-scale, with the detection of a fast-forming interme diate in the transition. Further characterization using static SAXS measure ments showed that the conformational change was initially reversible but be came irreversible after autoproteolytic maturation was about 15% complete. In addition to characterizing the large quaternary conformational change, w e have been able for the first time to demonstrate that it takes place on t he subsecond time-scale, a regime comparable to that observed in other mult isubunit assemblies. (C) 2001 Academic Press.