Current Approaches on Viral Infection: Proteomics and Functional Validations

Zheng, Jie; Tan, Boon Huan; Sugrue, Richard; Tang, Kai

Hydrogen/deuterium exchange combined with MS is able to investigate the protein structures and dynamics by studying their conformational alternations. Some amide hydrogens at the backbone could be readily exchanged when incubated in a deuterated environment while some amide hydrogens hidden in the interior of the protein or involved in hydrogen bonding have restricted access to deuterium. A quench condition (0ËšC, pH 2.5) is used to stop the exchange reaction followed by pepsin digestion of proteins prior to MS analysis. Therefore, the deuterium labeling induced mass shift and H/D exchange rate could reflect the protein conformational information and hydrogen bond interactions (Hamuro et al., 2003; Engen, 2009 ). HDX combined with high resolution MS was capable of studying structural information such as protein-nucleic acid bindings, protein-protein interplays, and protein maturation rearrangements. Viral molecular motors, e.g., helicases or packaging factors, are associated with nucleic acid binding and hydrolysis functions. The hexameric packaging motor (P4) of cystovirus enabled to bind viral RNAs through its RNA binding channel coupled with ATPase activities. The conformational dynamics of P4 in the presence and absence of RNA were examined by HDX coupled with MS. The HDX kinetics revealed distinctive states for different domains of P4 in response to nucleotide-binding, RNA loading, and translocation as well as ATPase activities, and thus provided a comprehensive understanding to P4 molecular architecture in different biological states (Lisal et al., 2005) . Assembly of MS2 viral coat protein is initiated by binding with a RNA stem-loop, resulting in a conformational switch from a symmetric dimer to an asymmetric structure. In this circumstance, detailed structural information was characterized by HDX and MS that some known RNA binding regions showed a more fluctuated HDX kinetics (Morton et al., 2010) . PPI dynamics could also be studied by HDX and MS. For instance, Kong et al. (2010) comparatively studied the local conformational rearrangements within HIV-1 gp120 in the presence or absence of CD4. Monroe et al. (2010) also applied HDX combined with LTQ-FT MS to study the immature, mature, and mutant Gag polyprotein to further unravel the capsid assembly.