Hydrogen-deuterium exchange as a probe of folding and assembly in viral capsids

The dynamics of proteins within large cellular assemblies are important in the molecular transformations that are required for macromolecular synthesi s, transport, and metabolism. The capsid expansion (maturation) accompanyin g DNA packaging in the dsDNA bacteriophage P22 represents an experimentally accessible case of such a transformation. A novel method, based on hydroge n-deuterium exchange was devised to investigate the dynamics of capsid expa nsion. Mass spectrometric detection of deuterium incorporation allows for a sensitive and quantitative determination of hydrogen-deuterium exchange dy namics irrespective of the size of the assembly. Partial digestion of the e xchanged protein with pepsin allows for region-specific assignment of the e xchange. Procapsids and mature capsids were probed under native and slightl y denaturing conditions. These experiments revealed regions that exhibit di fferent degrees of flexibility in the procapsid and in the mature capsid. I n addition, exchange and deuterium trapping during the process of expansion itself was observed and allowed for the identification of segments of the protein subunit that become buried or stabilized as a result of expansion. This approach may help to identify residues participating in macromolecular transformations and uncover novel patterns and hierarchies of interactions that determine functional movements within molecular machines. (C) 2001 Ac ademic Press.