title: Unraveling lactococcal phage baseplate assembly by mass spectrometry.
abstract: Bacteriophages belonging to the Caudovirales order possess a tail acting as a molecular machine used during infection to recognize the host and ensure high-efficiency genome delivery to the cell cytoplasm. They bear a large and sophisticated multiprotein organelle at their distal tail end, either a baseplate or a tail-tip, which is the control center for infectivity. We report here insights into the baseplate assembly pathways of two lactoccocal phages (p2 and TP901-1) using electrospray ionization-mass spectrometry. Based on our "block cloning" strategy we have expressed large complexes of their baseplates as well as several significant structural subcomplexes. Previous biophysical characterization using size-exclusion chromatography coupled with on-line light scattering and refractometry demonstrated that the overproduced recombinant proteins interact with each other to form large (up to 1.9 MDa) and stable assemblies. The structures of several of these complexes have been determined by x-ray diffraction or by electron microscopy. In this contribution, we demonstrate that electrospray ionization-mass spectrometry yields accurate mass measurements for the different baseplate complexes studied from which their stoichiometries can be discerned, and that the subspecies observed in the spectra provide valuable information on the assembly mechanisms of these large organelles.
authors.affiliation: Astbury Centre for Structural Molecular Biology, University of Leeds, Leeds LS2 9JT, UK.
authors.forename: Dale A
authors.initials: DA
authors.lastname: Shepherd
authors.forename: David
authors.initials: D
authors.lastname: Veesler
authors.forename: Julie
authors.initials: J
authors.lastname: Lichière
authors.forename: Alison E
authors.initials: AE
authors.lastname: Ashcroft
authors.forename: Christian
authors.initials: C
authors.lastname: Cambillau
mesh_terms: D003001:Cloning, Molecular; D004926:Escherichia coli; D013294:Lactococcus lactis / Q000821:virology; D008854:Microscopy, Electron; D008958:Models, Molecular; D046912:Multiprotein Complexes / Q000737:chemistry* / Q000378:metabolism; D016366:Open Reading Frames; D010957:Plasmids; D011485:Protein Binding / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017906:Siphoviridae / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*; D017376:Viral Tail Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D019065:Virus Assembly / Q000235:genetics
chemical_list: D046912:Multiprotein Complexes; D011994:Recombinant Proteins; D017376:Viral Tail Proteins