title: A minimized M13 coat protein defines the requirements for assembly into the bacteriophage particle.
abstract: The M13 filamentous bacteriophage coat is a symmetric array of several thousand alpha-helical major coat proteins (P8) that surround the DNA core. P8 molecules initially reside in the host membrane and subsequently transition into their role as coat proteins during the phage assembly process. A comprehensive mutational analysis of the 50-residue P8 sequence revealed that only a small subset of the side-chains were necessary for efficient incorporation into a wild-type (wt) coat. In the three-dimensional structure of P8, these side-chains cluster into three functional epitopes: a hydrophobic epitope located near the N terminus and two epitopes (one hydrophobic and the other basic) located near the C terminus on opposite faces of the helix. The results support a model for assembly in which the incorporation of P8 is mediated by intermolecular interactions involving these functional epitopes. In this model, the N-terminal hydrophobic epitope docks with P8 molecules already assembled into the phage particle in the periplasm, and the basic epitope interacts with the acidic DNA backbone in the cytoplasm. These interactions could facilitate the transition of P8 from the membrane into the assembling phage, and the incorporation of a single P8 would be completed by the docking of additional P8 molecules with the second hydrophobic epitope at the C terminus. We constructed a minimized P8 that contained only nine non-Ala side-chains yet retained all three functional epitopes. The minimized P8 assembled into the wt coat almost as efficiently as wt P8, thus defining the minimum requirements for protein incorporation into the filamentous phage coat. The results suggest possible mechanisms of natural viral evolution and establish guidelines for the artificial evolution of improved coat proteins for phage display technology.
authors.affiliation: Department of Protein Engineering, Genentech Inc., South San Francisco, CA 94080, USA.
authors.forename: Tomer A
authors.initials: TA
authors.lastname: Roth
authors.forename: Gregory A
authors.initials: GA
authors.lastname: Weiss
authors.forename: Charles
authors.initials: C
authors.lastname: Eigenbrot
authors.forename: Sachdev S
authors.initials: SS
authors.lastname: Sidhu
mesh_terms: D017104:Bacteriophage M13 / Q000737:chemistry* / Q000502:physiology*; D005075:Biological Evolution; D002213:Capsid / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D036022:Capsid Proteins; D019020:Directed Molecular Evolution; D004797:Enzyme-Linked Immunosorbent Assay; D000939:Epitopes; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D008958:Models, Molecular; D016296:Mutagenesis / Q000235:genetics; D019151:Peptide Library; D011485:Protein Binding; D011487:Protein Conformation; D015202:Protein Engineering; D019065:Virus Assembly
chemical_list: D036022:Capsid Proteins; D000939:Epitopes; D008565:Membrane Proteins; D019151:Peptide Library; C025668:coat protein, Bacteriophage M13