NMR studies of the structure and dynamics of filamentous bacteriophage fd coat proteins

Abstract

The structure and dynamics of the coat proteins from the bacteriophage fd were determined using multidimensional high resolution solution NMR spectroscopy. The coat protein is a 50 amino acid protein which resides in the E. coli bacterial inner membrane during its lifecycle. The coat protein is synthesized in the cytoplasm of its host as a precursor procoat protein which has a 23 residue signal sequence. The characterization of the structure and dynamics of both coat and procoat proteins is essential to gain an understanding of the molecular events associated with phage assembly, protein translocation across a membrane and the role of the signal sequence. Multidimensional heteronuclear (\sp{15}N) solution NMR was used to determine the structure and dynamics of coat protein/micelle complexes. The correlation times of the coat protein/micelle complexes are approximately 10 nanoseconds, making the spectroscopic studies much more difficult than would be expected for 50 and 73 amino acid proteins. The incorporation of \sp{15}N into the proteins, the use of inverse detected experiments and the use of three dimensional NMR experiments allowed for the characterization of the secondary structure of fd coat protein in micelles. The structure of fd coat protein is almost entirely $\alpha$ helical, except for loosely structured, mobile termini. The backbone dynamics of both coat and procoat proteins indicate residues that are mobile on the nanosecond timescale, when compared to the overall correlation time of the proteins. Mobile termini are present in both procoat and coat protein and procoat protein has additional mobility in residues near the cleavage protein is almost entirely $\alpha$ helical, except for loosely structured, mobile termini. The backbone dynamics of both coat and procoat proteins indicate residues that are mobile on the nanosecond timescale, when compared to the overall correlation time of the proteins. Mobile termini are present in both procoat and coat protein and procoat protein has additional mobility in residues near the cleavage site ($-$1/+1). The structure and dynamics of fd coat protein are similar to to other membrane proteins because they are composed $\alpha$ helices and have mobile termini and loops