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At least 127 records · Page 7Linked to original sources
PubMed1994

Molecular models and structural comparisons of native and mutant class I filamentous bacteriophages Ff (fd, f1, M13), If1 and IKe.

The filamentous bacteriophages are flexible rods about 1 to 2 microns long and 6 nm in diameter, with a helical shell of protein subunits surrounding a DNA core. The approximately 50-residue coat protein subunit is largely alpha-helix and the axis of the alpha-helix makes a small angle with the axis of the virion. The protein shell can be considered in three sections: the outer surface, occupied by the N-terminal region of the subunit, rich in acidic residues that interact with the surrounding solvent and give the virion a low isoelectric point; the interior of the shell, including a 19-residue stretch of apolar side-chains, where protein subunits interact mainly with each other; and the inner surface, occupied by the C-terminal region of the subunit, rich in basic residues that interact with the DNA core. The fact that virtually all protein side-chain interactions are between different subunits in the coat protein array, rather than within subunits, makes this a useful model system for studies of interactions between alpha-helix subunits in a macromolecular assembly. We describe molecular models of the class I filamentous bacteriophages. This class includes strains fd, f1, M13 (these 3 very similar strains are members of the Ff group), If1 and IKe. Our model of fd has been refined to fit quantitative X-ray fibre diffraction data to 30 A resolution in the meridional direction and 7 A resolution in the equatorial direction. A simulated 3.3 A resolution diffraction pattern from this model has the same general distribution of intensity as the experimental diffraction pattern. The observed diffraction data at 7 A resolution are fitted much better by the calculated diffraction pattern of our molecular model than by that of a model in which the alpha-helix subunit is represented by a rod of uniform density. The fact that our fd model explains the fd diffraction data is only part of our structure analysis. The atomic details of the model are supported by non-diffraction data, in part previously published and in part newly reported here. These data include information about permitted or forbidden side-chain replacements, about the effect of chemical modification, and about spectroscopic experiments.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1997

Oligonucleotide-directed mutagenesis and subsequent expression of the corresponding recombinant proteins without changing the bacterial vector system.

A new bacterial vector was constructed that combines the attractive features of gene fusion vectors and phagemids. A gene of interest cloned into this new vector can either be expressed as fusion protein or be prepared as single-stranded template DNA within the same system. Thus, time consuming subcloning procedures changing the bacterial vector according to the required method are avoided. As an example sequencing, expression and subsequent purification of site-directed mutants of herpes simplex virus type 1 thymidine kinase are discussed.

Cloning, Molecular↗
PubMed1999

Inhibiting HIV-1 entry: discovery of D-peptide inhibitors that target the gp41 coiled-coil pocket.

The HIV-1 gp41 protein promotes viral entry by mediating the fusion of viral and cellular membranes. A prominent pocket on the surface of a central trimeric coiled coil within gp41 was previously identified as a potential target for drugs that inhibit HIV-1 entry. We designed a peptide, IQN17, which properly presents this pocket. Utilizing IQN17 and mirror-image phage display, we identified cyclic, D-peptide inhibitors of HIV-1 infection that share a sequence motif. A 1.5 A cocrystal structure of IQN17 in complex with a D-peptide, and NMR studies, show that conserved residues of these inhibitors make intimate contact with the gp41 pocket. Our studies validate the pocket per se as a target for drug development. IQN17 and these D-peptide inhibitors are likely to be useful for development and identification of a new class of orally bioavailable anti-HIV drugs.

Amino Acid Sequence↗
PubMed1997

The C-terminal domain of TolA is the coreceptor for filamentous phage infection of E. coli.

Filamentous bacteriophages infecting gram-negative bacteria display tropism for a variety of pilus structures. However, the obligatory coreceptor of phage infection, postulated from genetic studies, has remained elusive. Here we identify the C-terminal domain of the periplasmic protein TolA as the coreceptor for infection of Escherichia coli by phage fd and the N-terminal domain of the phage minor coat protein g3p as its cognate ligand. The neighboring g3p domain binds the primary receptor of phage infection, the F pilus, and blocks TolA binding in its absence. Contact with the pilus releases this blockage during infection. Our findings support a sequential two-way docking mechanism for phage infection, analogous to infection pathways proposed for a range of eukaryotic viruses including herpes simplex, adenoviruses, and also lentiviruses like HIV-1.

Amino Acid Sequence↗
PubMed2002

Expression of MHC class I molecules together with antigenic peptides on filamentous phages.

The availability of combinatorial T cell epitope libraries made using a phage display system would be useful for identifying the antigens recognized by T cells of unknown specificity. To this end, we have investigated here whether single chain-MHC class I molecules (scMHC-I) could be expressed together with antigenic peptides on filamentous phages. The results show that filamentous phages can express scMHC-I. Moreover, the expressed scMHC-I was able to bind antigenic peptide. These data support the use of combinatorial scMHC/T cell epitope libraries for screening potential T cell antigens.

Antigens↗
PubMed2003

Reduction of beta-amyloid plaques in brain of transgenic mouse model of Alzheimer's disease by EFRH-phage immunization.

Antibodies to the epitope EFRH, representing residues 3-6 within the beta-amyloid (Abeta) sequence, were previously shown to affect the solubility and disaggregation of Abeta fibrils in vitro. Here, we describe a novel method of immunization, using as antigen the EFRH peptide displayed on the surface of the filamentous phage. The EFRH phage evoked effective auto-immune antibodies in amyloid precursor protein [V717I] (APP[V717I]) transgenic mice that recapitulate the amyloid plaques and vascular pathology of Alzheimer's disease (AD). The immunization provoked a considerable reduction in the number of Abeta amyloid plaques in the brain of the transgenic mice and may serve as the basis for anti-Abeta vaccine.

Alzheimer Disease↗
PubMed1998

Effects of relative humidity and applied force on atomic force microscopy images of the filamentous phage fd.

The filamentous phage fd was studied by both contact- and tapping-mode atomic force microscopy under conditions of controlled variations in relative humidity and changes in the applied tip force. By spin-coating freshly cleaved mica with phage containing solutions having very low salt content followed by rapid humidity control, stable and reliable sample preparation was achieved. The apparent height of the phage varied by about 10-fold with a quadratic dependence on the stabilized relative humidity, extrapolating to 73% of the accepted X-ray diffraction-based height at 0% relative humidity. The variation in measured height with relative humidity largely reconciles previous widely varying atomic force microscopy estimates of this dimension for the filamentous phage. Our finding that contact-mode images of phage are more difficult to analyze than those acquired in tapping mode are consistent with previously published results on other biological specimens such as DNA.

Humidity↗
PubMed2000

Locating the active site for angiogenesis and cell proliferation due to fibrin fragment E with a phage epitope display library.

The plasmin-mediated lysis of fibrin present in a wound, or in chronic inflammatory disease, results in the release of fibrin degradation products. One of the two major products is fibrin fragment E, which has been shown to stimulate cell proliferation in many cell types including endothelium, fibroblasts, and smooth muscle cells, and to be angiogenic in the chick chorioallantoic membrane (CAM) system. The activity of fibrin fragment E is dependent on N-terminus thrombin action. Antibodies against fibrin E, which block the cell proliferative activity, were used to locate the active site. Phage epitope display libraries were used to identify the sequence of a peptide, which resembles a region of the N terminus structure. The equivalent synthetic peptide (WTM110) has optimal stimulatory properties at equimolar concentrations to the parent molecule. Such peptide information has therapeutic potential for both stimulating and suppressing angiogenesis and cell proliferation.

Amino Acid Sequence↗
PubMed1997

Extending filamentous phage host range by the grafting of a heterologous receptor binding domain.

fd and IKe are two similar filamentous phage which infect their hosts by means of pili found on the host membrane: fd infects bacteria bearing F pili, whereas IKe infects bacteria bearing N or I pili. Infection is mediated by the gene 3 protein (g3p), which of the nine proteins found in both phage is the most diverse. Previous attempts to incorporate g3p from one phage into the other by complementation have been unsuccessful [Bross et al. (1988) J. Gen. Microbiol. 134, 461-471]. Here we have grafted different parts of IKe g3p to the end of fd g3p and so augmented the host range of fd phage. We show that phage bearing such chimeric g3p are able to infect bacteria bearing both N and F pili providing they contain at least the receptor domain of IKe g3p, the infection of N bearing bacteria occurring at a level 70,000 times greater than background. This level of infection can be increased tenfold by including the glycine-rich domain as well. Addition of the penetration domain does not improve the level of infection above that of the receptor domain alone, indicating that the fd penetration domain is functional in the infection of bacteria bearing either N or F pili. Similarly derived fd phagemid also show increased infection of bacteria bearing N pili, albeit at much lower levels, suggesting that efficient infection requires more than one functional g3p on the surface of the phage.

Bacteria↗
PubMed1999

A phagemid vector using the E. coli phage shock promoter facilitates phage display of toxic proteins.

Phage display is a powerful tool with which to adapt the specificity of protease inhibitors. To this end, a library of variants of the potato protease inhibitor PI2 was introduced in a canonical phagemid vector. Although PI2 is a natural trypsin inhibitor, we were unable to select trypsin-binding variants from the library. Instead, only mutants carrying deletions or amber stop codons were found. Bacteria carrying these mutations had a much faster growth rate than those carrying the wt PI2-encoding gene, even when the promoter was repressed. To overcome these problems, two new phagemid vectors for g3-mediated phage display were constructed. The first vector has a lower plasmid copy number, as compared to the canonical vector. Bacteria harboring this new vector are much less affected by the presence of the PI2-g3 fusion gene, which appears from a markedly reduced growth retardation. A second vector was equipped with the promoter of the Escherichia coli psp operon, instead of the lac promoter, to control the PI2-g3 gene fusion expression. The psp promoter is induced upon helper phage infection. A phagemid vector with this promoter controlling a PI2-g3 gene fusion did not affect the viability of the host. Furthermore, both new vectors were shown to produce phage particles that display the inhibitor protein and were therefore considered suitable for phage display. The inhibitor library was introduced in both new vectors. Trypsin-binding phages with inhibitory sequences were selected, instead of sequences with stop codons or deletions. This demonstrates the usefulness of these new vectors for phage display of proteins that affect the viability of E. coli.

Amino Acid Sequence↗
PubMed1995

Mutual adaptation of bacteriophage fd, pfd plasmids and their host strains.

The synthetic plasmid pfdC1 with the replication origin of phage fd and fd gene 2 grows autonomously in E. coli cells. DNA sequencing revealed several mutations compared to the fd genome causing reduced expression of viral gene 2 protein, which can be toxic for the host cell. Another adaptation was noticed for E. coli strains with a copy of fd gene 2 on the F-episome and a pfdA-plasmid with a minimal fd replication origin, when maintained at 42 degrees C. The carrier cells adjusted their cellular metabolism to these stress conditions, whereas replication functions of the plasmid or expression of fd gene 2 on the F-episome were not changed. The filamentous bacteriophages tend to reduce their genome size into miniphages, which was also observed for phages with an antibiotic resistance gene. Bacteriophages with a transposon insertion in the viral gene 2 had a tendency to restore the mutated gene by exchange with the functional gene 2 carried in recA-host cells. Mobilization of pfd-plasmids with RP4 transfer functions was reduced due to interference of replication and transfer in the rolling circle mode. The vectors used in these studies can also be applied as cloning vectors, which are compatible with many other plasmid vectors.

Adaptation, Physiological↗