| The structure of the major coat protein from the filamentous bacteriophage fd was determined by Solid-State NMR. The fifty aminoacids protein was studied as part of the 1.6 × 107 Da virus particle, oriented in the magnetic field and isotopically labeled with 15N in the amide sites. Two-dimensional spectra obtained with the PISEMA (polarization inversion and Spin Exchange at the Magic Angle) sequence were analyzed with a combination of PISA wheels and Dipolar waves calculations, and the structure calculated with a structural fitting routine. A model of the virus capsid was built based on the coat protein structure and information from deuterium exchange experiments, considering the symmetry derived from fiber diffraction studies.; Combined with the structure of the coat protein in lipid bilayers, previously determined by Solid-State NMR, the technique provides insights into the virus assembly process. This establishes Solid-State NMR as one of the three main tools in structural biology, alongside X-ray crystallography and Solution NMR. The absence of size limitations and the non-invasive character of the technique are very attractive aspects of the methodology developed, and it is highly suitable for studies of large supramolecular systems and membrane proteins.; The bacteriophage fd is a very important tool in biotechnology, and the technique of phage display has been used extensively in combinatorial chemistry and in the identification of immunogenic peptides. Short peptides have been shown to induce a higher antibody titer when presented as part of the bacteriophage than when free in solution. We are developing methods to study the structure of peptides displayed as part of the major coat protein, and initial results are shown from three different approaches. Low temperature experiments are being developed that will provide an increase in the sensitivity of the experiments and preserve the samples from excessive radio-frequency heat. |
