Capsid structure and dynamics of a human rhinovirus probed by hydrogen exchange mass spectrometry.
Viral capsids are dynamic protein assemblies surrounding viral genomes. Despite the high-resolution structures determined by X-ray crystallography and cryo-electron microscopy, their in-solution structure and dynamics can be probed by hydrogen exchange. We report here using hydrogen exchange combined with protein enzymatic fragmentation and mass spectrometry to determine the capsid structure and dynamics of a human rhinovirus, HRV14. Capsid proteins (VP1-4) were labeled with deuterium by incubating intact virus in D(2)O buffer at neutral pH. The labeled proteins were digested by immobilized pepsin to give peptides analyzed by capillary reverse-phase HPLC coupled with nano-electrospray mass spectrometry. Deuterium levels incorporated at amide linkages in peptic fragments were measured for different exchange times from 12 sec to 30 h to assess the amide hydrogen exchange rates along each of the four protein backbones. Exchange results generally agree with the crystal structure of VP1-4,with extended, flexible terminal and surface-loop regions in fast exchange and folded helical and sheet structures in slow exchange. In addition, three alpha-helices, one from each of VP1-3, exhibited very slow exchange, indicating high stability of the protomeric interface. The beta-strands at VP3 N terminus also had very slow exchange, suggesting stable pentamer contacts. It was noted, however, that the interface around the fivefold axis had fast and intermediate exchange, indicating relatively more flexibility. Even faster exchange rates were found in the N terminus of VP1 and most segments of VP4, suggesting high flexibilities, which may correspond to their potential roles in virus uncoating.
Wang
Lintao
L
Department of Chemistry, University of Nebraska, Lincoln 68588-0304, USA. lintao.wang@immunogen.com
Smith
David L
DL
D002213:Capsid / Q000737:chemistry*; D036022:Capsid Proteins / Q000737:chemistry*; D006801:Humans; D046912:Multiprotein Complexes / Q000737:chemistry*; D020836:Protein Structure, Quaternary; D012229:Rhinovirus / Q000737:chemistry*
D036022:Capsid Proteins; D046912:Multiprotein Complexes