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 synthesis, transport, and metabolism. The capsid expansion (maturation) accompanying DNA packaging in the dsDNA bacteriophage P22 represents an experimentally accessible case of such a transformation. A novel method, based on hydrogen-deuterium exchange was devised to investigate the dynamics of capsid expansion. Mass spectrometric detection of deuterium incorporation allows for a sensitive and quantitative determination of hydrogen-deuterium exchange dynamics irrespective of the size of the assembly. Partial digestion of the exchanged protein with pepsin allows for region-specific assignment of the exchange. Procapsids and mature capsids were probed under native and slightly denaturing conditions. These experiments revealed regions that exhibit different degrees of flexibility in the procapsid and in the mature capsid. In 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.
Tuma
R
R
Department of Microbiology, The University of Alabama at Birmingham, Birmingham, AL, 35294-2170, USA.
Coward
L U
LU
Kirk
M C
MC
Barnes
S
S
Prevelige
P E
PE
D000595:Amino Acid Sequence; D017100:Bacteriophage P22 / Q000737:chemistry* / Q000502:physiology; D001665:Binding Sites; D002213:Capsid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D003903:Deuterium / Q000378:metabolism*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010434:Pepsin A / Q000378:metabolism; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D021122:Protein Subunits; D012680:Sensitivity and Specificity; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D019065:Virus Assembly
D021122:Protein Subunits; D003903:Deuterium; D010434:Pepsin A