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Capsid structure and dynamics of a human rhinovirus probed by hydrogen exchange mass spectrometry

Department of Chemistry, University of Nebraska, Lincoln, Nebraska 68588-0304, USA

(RECEIVED February 3, 2005; FINAL REVISION March 15, 2005; ACCEPTED March 20, 2005)

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 D₂O 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 -helices, one from each of VP1–3, exhibited very slow exchange, indicating high stability of the protomeric interface. The -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.

Keywords: human rhinovirus; viral capsid; protein structure and dynamics; hydrogen exchange; mass spectrometry

Abbreviations: CID, collision induced dissociation • ESIMS, electrospray ionization mass spectrometry • GudHCl, guanidine hydrogen chloride • HRV, human rhinovirus • HXMS, hydrogen exchange mass spectrometry • NIm, neutralizing immunogenic • TFA, trifluoroacetic acid

Article published online ahead of print. Article and publication date are at http://www.proteinscience.org/cgi/doi/10.1110/ps.051390405.

Reprint requests to: Lintao Wang, ImmunoGen Inc., 128 Sidney Street, Cambridge, MA 02139, USA; e-mail: lintao.wang{at}immunogen.com; fax: (617) 995-2510.

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