Analysis of rapid, large-scale protein quaternary structural changes: time-resolved X-ray solution scattering of Nudaurelia capensis omega virus (NomegaV) 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-enveloped RNA virus Nudaurelia capensis omega virus (NomegaV) were shown to undergo 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, which 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 converted to the capsid form at pH 5.0. Static SAXS measurements of the VLPs in solutions ranging between these pH values determined that the half-titration 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 occurred 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 subminute or minute time-scale, with the detection of a fast-forming intermediate in the transition. Further characterization using static SAXS measurements showed that the conformational change was initially reversible but became irreversible after autoproteolytic maturation was about 15 % complete. In addition to characterizing the large quaternary conformational change, we have been able for the first time to demonstrate that it takes place on the subsecond time-scale, a regime comparable to that observed in other multisubunit assemblies.
Canady
M A
MA
Department of Molecular Biology, The Scripps Research Institute, La Jolla, CA 92037, USA.
Tsuruta
H
H
Johnson
J E
JE
D002213:Capsid / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D036022:Capsid Proteins; D020285:Cryoelectron Microscopy; D006863:Hydrogen-Ion Concentration; D007304:Insect Viruses / Q000737:chemistry* / Q000378:metabolism* / Q000648:ultrastructure; D007700:Kinetics; D008958:Models, Molecular; D011498:Protein Precursors / Q000737:chemistry / Q000378:metabolism; D020836:Protein Structure, Quaternary; D012542:Scattering, Radiation; D019065:Virus Assembly; D014965:X-Rays
D036022:Capsid Proteins; D011498:Protein Precursors; C092202:coat protein, Nudaurelia capensis omega virus