fd Coat Protein Structure in Membrane Environments

Complete resolution of the solid-state NMR spectrum of a uniformly 15 N-labeled membrane protein in phospholipid bilayers

“…The large 80 ppm frequency difference between the resonances from Leu-41, near the downfield end of the spectrum, and Leu-14, near the upfield end, demonstrates the effect of protein structure on these spectra. Leu-41, in the trans-membrane helix, has its NOH bond approximately parallel to the field and to 33 of the chemical shift tensor, while Leu-14, in the amphipathic inplane helix, has its NOH bond perpendicular (29).…”

“…15 N-labeled fd coat protein was prepared as described (29), starting with the isolation of bacteriophage particles by polyethylene glycol precipitation from the supernatant, following the growth of fd-infected E. coli (K3300Fϩ) in a medium containing either 15 N ammonium sulfate for uniform labeling, or 15 N Leu (Cambridge Isotope Laboratories, Cambridge, MA) for selective labeling. The coat protein was purified by size exclusion chromatography.…”

“…Newly synthesized copies of the protein are stored in the membrane of infected Escherichia coli bacteria prior to incorporation into virus particles as they are extruded through the membrane. The structure of the membrane bound form of this protein has been determined in micelles by solution NMR spectroscopy (29)(30)(31)(32). It has an amphipathic helix that lies in the plane of the membrane (residues 7-16), and a longer hydrophobic membrane spanning helix (residues 27-44).…”

NMR Studies of the Major Coat Protein of Bacteriophage M13

“…It has been reported as being a dimer with a large fraction of a-helix and P-sheet (Nozaki et al, 1978;Datema et al, 1987) and a helical dimer (Henry and Sykes, 1992), but also as an almost completely a-helical monomer (McDonnell et al, 1993). It has been shown that these differences are brought about by the isolation and purification methods used for the preparation of the samples (Hemminga et al, 1992), and by sample conditions (e.g.…”

“…It has been shown that these differences are brought about by the isolation and purification methods used for the preparation of the samples (Hemminga et al, 1992), and by sample conditions (e.g. detergent/gVIIIp ratio) (McDonnell et al, 1993). We used a high detergent/gVIIIp ratio which resulted in gVIIIp being in the monomeric form (Van de Ven et al, 1993).…”