Structural studies of filamentous bacteriophage by NMR spectroscopy
Filamentous bacteriophage represent an ideal system for structural studies and comparisons of biologically important molecules by solid state NMR spectroscopy. In addition to being cloning vectors and the basis for genetically-encoded combinatorial libraries, phage particles with molecular masses > 10,000 kDa can orient spontaneously in magnetic fields, allowing the techniques of solid state NMR spectroscopy to be applied. The atomic-resolution structures of the E. coli bacteriophage fd and Pseudomonas aeruginosa bacteriophage Pf1 major coat proteins are presented and compared. This adds valuable structural biology insight to the use of phage-display libraries for combinatorial screening of potential epitopes and drugs.
| Phage are grown on E. coli or P. aeruginosa in minimal media. For uniform 15N-labeled samples, the sole N source is 15N-ammonium sulfate. For selective labels, the desired 15N amino acid is added, as well as the other unlabeled (14N) amino acids. Purification involves precipitation by polyethylene glycol followed by cesium chloride density gradient ultracentrifugation. | Growth and Purification |
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| Solid State NMR spectroscopy was performed at 550 and 750 MHz using custom designed and built round coil probes and homebuilt, Tecmag Apollo, and Bruker consoles. Spectrum acquired utilized the PISEMA pulse sequence. | NMR Spectroscopy |
| Selectively 15N-labeled samples are used in conjunction with PISA wheel and dipolar wave analysis to assign resonances in the 15N uniformly labeled spectrum of the major coat protein in the intact phage. D2O exchange experiments are performed and found helpful in confirming assignments and resolving ambiguities. | Assignments based on Selective labels PISA wheels D2O exchange |
| Dipolar wave analysis suggests the boundaries of helical segments, tilt angles, and deviations from ideal helices such as kinks or bends. Structural fitting of both the dipolar coupling and chemical shift data allow calculation of the backbone structure of the major coat protein in the intact phage. | Monomer structure calculation |
| Models for the phage were generated using the structure of the monomer coat protein, symmetry relationships from X-ray fiber diffration data, addition of side chains, and energy minimization. | Virion model assembly |
