M13 coat protein

The conformations of bound peptides were also studied using trCCR by Shimada and co-workers [30]. In this case peptides were labelled in E. coli as fusion proteins with the M13 coat protein, and were expressed in N, C... 

Cleavage of the signal sequence in prokaryotes can occur either cotranslationally (Josefsson and Randall, 1981) or posttranslationally (Wu et al., 1983). Cleavage does not appear to be necessary for protein export, as mutants deficient in the cleavage of lipoprotein (Lin etal., 1978), MBP (Ryan et al., 1986a), or the M13 coat protein (Russell and Model, 1981) signal sequences are localized properly. 

One can dso obtain NMR spectra for proteins in micelles, which may allow the study of membrane protein structure in an environment approximating their native one. A combination of labels (l N,13c) was used for NMR studies of detergent-solubilized M13 coat protein. Although most of die resonances in the spectrum have not been assigned, there was clear indication that many of the protein residues had two distinct resonances of equal intensity. This was interpreted to mean (in combination with the results of sedimentation equilibrium, Raman and CD studies) that the protein was present in two conformers that represent the non-equivalent monomers of an asymmetric dimer. NMR has also been used to determine the spatial structures of gramicidin A and -labeled bacteriorhodopsin fragments in a membrane-like milieu b... 

To achieve surface display, five of the M13 coat proteins have been used in fusion to foreign protein fragments. In the most widespread system the antibody is coupled to the N-terminus or second domain of the minor coat protein pill (11,12,14). The naive function of the three to five copies of the pill, in particular their N-terminal domain, is to provide binding of the phage to the f-pili of E. coli to initiate infection (39). The major coat protein (pVIII) has been used as an alternative fusion partner, with only very few successes reported in the past decade (40). This fusion technique is more useful for the display of short peptides (41,42). Fusions to pVI have been tried, but not yet with antibodies (43). pVII and pIX were... 

Henry GD, Sykes BD (1992) Assignment of amide H and N NMR resonances in detergent-solubilized M13 coat protein a model for the coat protein dimer. Biochemistry... 

Papavoine CH, Konings RN, Hilbers CW, Van de Ven FJ (1994) Location of M13 coat protein in sodium dodecyl-sulfate micelles as determined by NMR. Biochemistry 33 12990-12997... 

In wt another NMR example, Sykes et a1. used a DISPA analysis of the 1"F NMR spectrum for M13 coat protein the DISPA data points were located on the reference circle, within experimental error, showing that the two fluorotyrosines exhibited the same chemical shift.22 Subsequent experiments based on solvent shifts confirmed that both fluorinated amino acid residues were "buried" and not accessible to solvent. 

Phospholipid membranes have been further studied by incorporation of various fluorinated compounds. Thus 8,8-difluoromyristic acid incorporated into the membrane of two strains of E. coli, fluorotyrosyl residues of M13 coat protein reconstituted in synthetic phospholipid vesicles, and... 

Belcher and coworkers have also exploited the M13 phage as a biotemplate to synthesize qnantnm dot nano-wires. Specifically, genetically engineered M13 phage were employed to display a previonsly determined peptide sequence in the pVIII M13 coat protein that specifically facilitates the nncleation and growth of ZnS or CdS... 

Figure 5.16 Illustration of the manner by which the virion of a filamentous single-stranded phage (such as M13 or fd) leaves an infected cell without lysis. The A protein passes first through the membrane at a site on the membrane where coat protein molecules have first become imbedded. The intracellular circular DNA is coated with dimers of another protein, gp5, which is displaced by coat protein as the DNA passes through the intact membrane.

Kretzschmar T, Geiser M, Evaluation of antibodies fused to minor coat protein III and major coat protein VIII of bacteriophage M13, Gene, 155 61-65, 1995. 

Markland W, Roberts BL, Saxena MJ, Guterman SK, Ladner RC, Design, construction and function of a multicopy display vector using fusion to the major coat protein of bacteriophage M13, Gene, 109 13-19, 1991. 

Cotiphage strains M13, fd, Pfl, Xf ssDNA-binding protein pV (Skinner et at, 1994), m or coat protein pVlII (Marvin et at, 1994 McDonnell et at, 1993 Nambudripad et at, 1991a)... 

The genetic evidence presented above makes it clear that E. coli, and possibly other bacteria, possess a complex set of proteins that act in the protein-secretion process. Although it appears that at least one protein, the M13 phage coat protein, can be localized and processed in the absence of proteins other than signal peptidase (Section V,B) (Silver et al., 1981 Ohno-Iwashita and Wickner, 1983 Watts et al., 1981), most proteins of the bacterial cell envelope require the participation of a secretion apparatus for proper localization. Whether the bacterial secretion process is analogous to the eukaryotic process remains to be seen. The recent development of in vitro translocation systems derived from E. coli should facilitate research in this area (Rhoads et al., 1984 Muller and Blobel, 1984b). 

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