Structure and Synthesis of a Lipid‐Containing Bacteriophage

Abstract

The nucleocapsid proteins of bacteriophage PM2 and the inner lamella of the lipid bilayer, containing most of the phosphatidylethanolamine residues, were selectively cross-linked in the presence of 0.1 -0.5 % glutaraldehyde, 5 mM dimethylsuberimidate, or 0.05 % toluene 2,4-diisocyanate. The biological activity (p.f.u.) of PM2 modified by these reagents decreased 106-fold in all cases. The spike and coat proteins were selectively cross-linked in the presence of 7.5 mM N,N'-p-phenylenedimaleimide. The biological activity of virus modified by this reagent was unaffected. The electron paramagnetic resonance spectra of fatty acid spin labels incorporated into native and chemically modified viral membranes were qualitatively similar but show quantitative differences. Fixation with glutaraldehyde increased the rigidity of the membrane while Triton X-100 induced a more flexible structure. There was no change in the electron paramagnetic resonance spectrum of virus treated with N,N'-pphenylenedimaleimide, however.Physical interactions between lipids and proteins stabilize natural membrane structures [l]. The lipidcontaining bacteriophage PM2 represents a simple model to study such physical interactions in one type of biological membrane [2]. The bacteriophage contains superhelical DNA, a lipid bilayer, and four structural proteins. Protein I forms the spikes, protein I1 the outer icosahedral shell, and protein I11 the inner icosahedral shell around the DNA [3]. Protein IV is closely associated with the DNA [3]. The phospholipid bilayer lies between the two protein shells. The bilayer is asymmetric; most of the phosphatidylglycerol residues form the outer lipid layer, most of the phosphatidylethanolamine residues the inner lipid layer [4]. Electrostatic interactions between phosphatidylglycerol and protein 11, which is basic (PI 12.3), play a major role in the structural integrity, as well as in the assembly, of the bacteriophage [4,5]. To study the protein-lipid interactions further, the virus was modified with cross-linking reagents, resulting in either protein-lipid or protein-protein crosslinking. Electron spin resonance was then used to investigate the movement of the hydrocarbon chains in the bilayer of such modified viruses. Several stearic This is paper number XXI in the series.Abbreviations. EPR, electron paramagnetic resonance; p.f.u., plaque-forming units.acid analogs with the nitroxide group at different positions were used as spin label in these experiments ~~7 1 . MATERIALS AND METHODS GeneralThe preparation of highly purified bacteriophage PM2 was described by Hinnen et al. [3]. Standard techniques such as analytical polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate, quantitative protein determination, plaque test and determination of the radioactivity by liquid scintillation counting have been described earlier [3,4]. Virus labeled with [G-3H]glycerol was prepared and purified as described by Tsukagoshi and Franklin [8]. Lipid was extracted from the virus as described by Frankli...