title: Dynamic motions of free and bound O29 scaffolding protein identified by hydrogen deuterium exchange mass spectrometry.
abstract: In the double-stranded DNA containing bacteriophages, hundreds of copies of capsid protein subunits polymerize to form icosahedral shells, called procapsids, into which the viral genome is subsequently packaged to form infectious virions. High assembly fidelity requires the assistance of scaffolding protein molecules, which interact with the capsid proteins to insure proper geometrical incorporation of subunits into the growing icosahedral lattices. The interactions between the scaffolding and capsid proteins are transient and are subsequently disrupted during DNA packaging. Removal of scaffolding protein is achieved either by proteolysis or alternatively by some form of conformational switch that allows it to dissociate from the capsid. To identify the switch controlling scaffolding protein association and release, hydrogen deuterium exchange was applied to Bacillus subtilis phage Ø29 scaffolding protein gp7 in both free and procapsid-bound forms. The H/D exchange experiments revealed highly dynamic and cooperative opening motions of scaffolding molecules in the N-terminal helix-loop-helix (H-L-H) region. The motions can be promoted by destabilizing the hydrophobic contact between two helices. At low temperature where high energy motions were damped, or in a mutant in which the helices were tethered through the introduction of a disulfide bond, this region displayed restricted cooperative opening motions as demonstrated by a switch in the exchange kinetics from correlated EX1 exchange to uncorrelated EX2 exchange. The cooperative opening rate was increased in the procapsid-bound form, suggesting this region might interact with the capsid protein. Its dynamic nature might play a role in the assembly and release mechanism.
authors.affiliation: Department of Biochemistry and Molecular Genetics, University of Alabama at Birmingham, 35294, USA.
authors.forename: Chi-Yu
authors.initials: CY
authors.lastname: Fu
authors.forename: Peter E
authors.initials: PE
authors.lastname: Prevelige
mesh_terms: D017103:Bacillus Phages / Q000737:chemistry* / Q000378:metabolism; D036022:Capsid Proteins / Q000737:chemistry / Q000378:metabolism; D041961:Deuterium Exchange Measurement / Q000379:methods*; D018257:Helix-Loop-Helix Motifs; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D013058:Mass Spectrometry / Q000379:methods*; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D015678:Viral Structural Proteins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D036022:Capsid Proteins; D011994:Recombinant Proteins; D015678:Viral Structural Proteins; C111088:scaffolding protein, bacteriophage P22