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.
Highlights
Two siphophages, p2 and TP901–1, have been shown to infect different strains of the low GC content gramϩ lactic acid bacterium Lactococcus lactis
Lactococcal Phage Baseplate Assembly spectrometry (ESI-MS). Their size and sometimes fragile nature can make their transfer to the gas phase difficult and their subsequent mass measurement is complicated because of the greater likelihood of heterogeneity and the presence of small molecules and ions trapped within their quaternary structures, which can cause a significant but variable increase in molecular mass
Recent instrumental developments and sample ionization optimization have made the analysis of noncovalently bound MDa complexes a reality and ESI-MS has become a powerful tool for studying protein interactions within macromolecular assemblies [15,16,17,18,19,20]
Summary
P2 and TP901–1, have been shown to infect different strains of the low GC content gramϩ lactic acid bacterium Lactococcus lactis They possess a large multiprotein organelle (1–2 MDa) at their distal tail end, termed the baseplate, which is in charge of specific host recognition, attachment, and initiation of infection. Recent instrumental developments and sample ionization optimization have made the analysis of noncovalently bound MDa complexes a reality and ESI-MS has become a powerful tool for studying protein interactions within macromolecular assemblies [15,16,17,18,19,20] This rapid and highly sensitive analytical method can provide mass measurements from which stoichiometries can be confidently ascertained for individual biomolecular complexes within a heterogeneous mix. The marriage of IMS with MS can be considered the ideal companion to complement three-dimensional structural studies
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