Dynamics of fd coat protein in lipid bilayers

The dynamics of backbone and side-chain sites of the membrane-bound form of fd coat protein are described with solid-state 2H and 15N NMR experiments. The samples were isotopically labeled coat protein in phospholipid bilayers in excess water. The protein itself is immobile and does not undergo rapid rotation within the bilayer. Like the structural form of the protein, the membrane-bound form has four mobile residues at the N-terminus. The membrane-bound form differs from the structural form in having several mobile residues at the C-terminus. Many of the side chains of residues with immobile backbone sites undergo large amplitude jump motions. The dynamics are generally similar in both the structural and membrane-bound forms of the protein.     

Association of the major coat protein of fd bacteriophage with phospholipid vesicles

The association of the major coat protein of fd bacteriophage with a phospholipid bilayer was investigated by analyzing the protein's susceptibility to proteolysis and its circular dichroism spectrum when incorporated into single-walled phospholipid vesicles. In the limits tested, this association appeared to be independent of the mass ratio of protein to lipid and of vesicle size, phospholipid composition, and method of preparation. The circular dichroism data are consistent with a similar "membrane-bound" conformation for all cases of vesicle-associated coat protein and for deoxycholate micelle-associated coat protein. Proteolysis of coat protein associated with deoxycholate micelles and with phospholipid vesicles defined the central hydrophobic core presumed to represent that portion of the protein which associates with membrane bilayers in vivo. The isolated core, which assumed a predominantly beta-type conformation in detergent solution, maintained a beta conformation when associated with a vesicle phospholipid bilayer.     

Effect of coat protein mutations in bacteriophage fd studied by sedimentation analysis

(a) Bacteriophage fd is a filamentous virus that has previously been well characterized. (b) Earlier work using point mutagenesis indicated that a lysine residue at position 48 in the major coat protein plays a crucial role in interacting with the DNA and governing the assembly into an intact virion. (c) In this study the sedimentation properties (sedimentation velocity and equilibrium) of wild-type fd and two mutants substituted at lysine-48 (K48Q and K48A) were compared. (d) Both mutants are similar to each other [M_r (19.5 ± 1.5) × 10⁶] but somewhat bigger than the wild-type [M_r (15.1 ± 1.5) × 10⁶]. The value for the wild-type is consistent with earlier published values. (e) By combining these data with sedimentation coefficient data, it is possible to compare the contour lengths and relative flexibilities of the mutants with those of the wild-type virion. (f) The mutants are shown hydrodynamically to have larger contour lengths (as also observed by electron microscopy): the ~20% difference in values obtained assuming rigid particle hydrodynamics with those obtained from electron microscopy is strongly suggestive of some difference in flexibility between the wild-type and mutants.     

NMR of fd coat protein

The conformations of the major coat protein of a filamentous bacteriophage can be described by nuclear magnetic resonance spectroscopy of the protein and the virus. The NMR experiments involve detection of the ¹³C and ¹H nuclei of the coat protein. Both the ¹³C and ¹H nuclear magnetic resonance (NMR) spectra show that regions of the polypeptide chain have substantially more motion than a typical globular protein. The fd coat protein was purified by gel chromatography of the SDS solubilized virus. Natural abundance ¹³C NMR spectra at 38 MHz resolve all of the nonprotonated aromatic carbons from the three phenylalanines, two tyrosines, and one tryptophan of the coat protein. The α carbons of the coat protein show at least two different classes of relaxation behavior, indicative of substantial variation in the motion of the backbone carbons in contrast to the rigidity of the α carbons of globular proteins. The ¹H spectrum at 360 MHz shows all of the aromatic carbons and many of the amide protons. Titration of a ¹H spectra gives the pKas for the tyrosines.     

A coat protein from bacteriophage fd: I. Hydrodynamic measurements and biological characterization

A coat protein has been isolated from highly purified fd bacteriophage disintegrated in phenol. Dissociation of the isolated protein to the monomeric state has only been achieved in 1% sodium dodecyl sulphate. At lower concentrations of the detergent as well as in several other solvents, the protein exists in the form of uniformly sized oligomers, probably heptamers or octamers. Protein dissolved in concentrated urea and dialysed versus neutral buffer aggregates to give rod-shaped particles with approximately the diameter of virus particles, but otherwise unlike virus in appearance. Such protein functions in blocking fd receptive sites of the bacterial host and in inactivating fd antiserum, although without complete exhaustion. The implications of these findings for the structure of the aggregated protein are discussed.     

Protein dynamics by solid-state nuclear magnetic resonance spectroscopy: Peptide backbone of the coat protein in fd bacteriophage

¹³C and ¹⁵N chemical shift anisotropy and ¹⁵N¹H dipolar powder patterns from backbone sites of the coat protein in fd bacteriophage are not averaged by motion. This means that the polypeptide backbone of the protein has no large amplitude motions rapid compared to 10⁴ Hz. Relaxation studies on the ¹³C_α and ¹⁵N amide resonances indicate the presence of motions on the 10⁹ Hz timescale. These results are reconciled with a model where an otherwise rigid backbone undergoes small amplitude, rapid motions.     

Chemical modification of the coat protein in bacteriophage fd and orientation of the virion during assembly and disassembly.

The major (gene VIII) coat protein of bacteriophage fd was radiolabelled by treating the virus with methyl[3H]acetimidate without causing any loss of infectivity. Complete amidination of lysine-8 in the amino acid sequence of the protein was achieved but little or no modification of the lysine residues near the C terminus was observed. This supports the assumption that the coat protein is oriented in the viral filament with its N terminus on the outside and its C-terminal region abutting the DNA. Escherichia coli was co-infected with radiolabelled bacteriophage and with unlabelled miniphage, a shorter defective form of phage fd. Radiolabel was detected in the progeny miniphage, proving that individual coat protein subunits can be recycled and assembled onto progeny miniphage DNA. About 35% of the coat protein subunits of phage particles infecting E. coli were recycled in 1 h. These facts support a model of the assembly and disassembly of the virion at the bacterial membrane in which the end of the particle containing the minor adsorption (gene III) protein, which is presumably the first to disassemble during infection, is the last to assemble during morphogenesis.     

Protein structure by solid state nuclear magnetic resonance. Residues 40 to 45 of bacteriophage fd coat protein

The three-dimensional structure of part of the coat protein in the filamentous bacteriophage fd is described by nuclear magnetic resonance (n.m.r.). Residues 40 to 45 are in a somewhat distorted alpha-helix. This n.m.r. approach for determining protein structure relies on the spectral manifestations of chemical shift and heteronuclear dipolar couplings in a symmetrical assembly of protein subunits oriented parallel to the applied magnetic field. The angles between individual peptide linkages and the filament axis of the virion constitute the basic source of structural information. These angles are directly related to x, y, z co-ordinates for describing the protein structure.     

Epitope structures recognised by antibodies against the major coat protein (g8p) of filamentous bacteriophage fd (Inoviridae).

To map the accessible surface of filamentous bacteriophage fd particles, the epitope structures of polyclonal rabbit serum and three mouse monoclonal antibodies raised against complete phage were analysed. Western blot analysis confirmed the major coat protein, gene VIII product (g8p or pVIII), to be the antigen. Overlapping peptides were synthesised by spot synthesis on cellulose membranes, covering the whole sequence of g8p. Each of the three tested monoclonal antibodies, B62-FE2, B62-GF3/G12 and B62-EA11, reacted with a core epitope covering ten amino acid residues at or near the amino terminus of g8p. The epitope recognised by B62-FE2 consists of the ten N-terminal amino acid residues of g8p. Extension of the amino terminus by various sequences did not inhibit binding, indicating that a terminal amino group is not essential for the interaction. Both B62-GF3/G12 and B62-EA11 recognise internal epitopes covering amino acid residues 3 to 12 of g8p. The epitopes of the polyclonal rabbit serum were also confined to the 12 N-terminal amino acid residues. The contribution of individual amino acid residues to the binding was analysed by a set of peptides containing individual amino acids exchanged by glycine. Accessible residues were Glu2, Asp4, Asp5, Pro6, Lys8, Phe11 and Asp12. The positions of the essential amino acid residues within the epitope are in accordance with a helical conformation of the amino-terminal region of g8p. Further, the results suggest new designs of phage display screening vectors to improve their performance in analysing non-linear epitopes.     

Effects of temperature and Y21M mutation on conformational heterogeneity of the major coat protein (pVIII) of filamentous bacteriophage fd

Solid-state NMR spectroscopy was used to analyze the conformational heterogeneity of the major coat protein (pVIII) of filamentous bacteriophage fd. Both one and two-dimensional solid-state NMR spectra of magnetically aligned samples of fd bacteriophage reveal that an increase in temperature and a single site substitution (Tyr21 to Met, Y21M) reduce the conformational heterogeneity observed throughout wild-type pVIII. The NMR results are consistent with previous studies indicating that conformational flexibility in the hinge-bend segment that links the amphipathic and hydrophobic helices in the membrane-bound form of the protein plays an essential role during phage assembly, which involves a major change in the tertiary, but not secondary, structure of the coat protein.     

Membrane assembly of the bacteriophage Pf3 major coat protein

The Pf3 major coat protein of the Pf3 bacteriophage is stored in the inner membrane of the infected cell during the reproductive cycle. The protein consists of 44 amino acids, and contains an acidic amphipathic N-terminal domain, a hydrophobic domain, and a short basic C-terminal domain. The mainly alpha-helical membrane-bound protein traverses the membrane once, leaving the C-terminus in the cytoplasm and the N-terminus in the periplasm. A cysteine-scanning approach was followed to measure which part of the membrane-bound Pf3 protein is inside or outside the membrane. In this approach, the fluorescence probe N-[(iodoacetyl)amino]ethyl-1-sulfonaphthylamine (IAEDANS) was attached to single-cysteine mutants of the Pf3 coat protein. The labeled mutant coat proteins were reconstituted into the phospholipid DOPC/DOPG (80/20 molar ratio) and DOPE/DOPG (80/20 molar ratio) model membranes. We subsequently studied the fluorescence characteristics at the different positions in the protein. We measured the local polarity of the environment of the probe, as well as the accessibility of the probe to the fluorescence quencher acrylamide. The results of this study show a single membrane-spanning protein with both the C- and N-termini remaining close to the surface of the membrane. A nearly identical result was seen previously for the membrane-bound M13 coat protein. On the basis of a comparison between the results from both studies, we suggest an L-shaped membrane-bound model for the Pf3 coat protein. DOPE-containing model membranes revealed a higher polarity, and quenching efficiency at the membrane/water interface. Furthermore, from the outside to the inside of the membrane, a steeper polarity gradient was measured at the PE/PG interface as compared to the PC/PG interface. These results suggest a thinner interface for DOPE/DOPG than for DOPC/DOPG membranes.     

Refined structure of the gene 5 DNA binding protein from bacteriophage fd

The three-dimensional structure of the gene 5 DNA binding protein (G5BP) from bacteriophage fd has been determined from a combination of multiple isomorphous replacement techniques, partial refinements and deleted fragment difference Fourier syntheses. The structure was refined using restrained parameter least-squares and difference Fourier methods to a final residual of R = 0.217 for the 3528 statistically significant reflections present to 2.3 A resolution. In addition to the 682 atoms of the protein, 12 solvent molecules were included. We describe here the dispositions and orientations of the amino acid side-chains and their interactions as visualized in the G5BP structure. The G5BP monomer of 87 peptide units is almost entirely in the beta-conformation, organized as a three-stranded sheet, a two-stranded beta-ribbon and a broad connecting loop. There is no alpha-helix present in the molecule. Two G5BP monomers are tightly interlocked about an intermolecular dyad axis to form a compact dimer unit of about 55 A X 45 A X 36 A. The dimer is characterized by two symmetry-related antiparallel clefts that traverse the monomer surfaces essentially perpendicular to the dyad axis. From the three-stranded antiparallel beta-sheet, formed from the first two-thirds of the sequence, extend three tyrosine residues (26, 34, 41), a lysine (46) and two arginine residues (16, 21) that, as indicated by other physical and chemical experiments, are directly involved in DNA binding. Other residues likely to share binding responsibility are arginine 80 extending from the beta-ribbon and phenylalanine 73 from the tip of this loop, but as provided, however, by the opposite monomer within each G5BP dimer pair. Thus, both symmetry-related DNA binding sites have a composite nature and include contributions from both elements of the dimer. The gene 5 dimer is clearly the active binding species, and the two monomers within the dyad-related pair are so structurally contiguous that one cannot be certain whether the isolated monomer would maintain its observed crystal structure. This linkage is manifested primarily as a skeletal core of hydrophobic residues that extends from the center of each monomer continuously through an intermolecular beta-barrel that joins the pair. Protruding from the major area of density of each monomer is an elongated wing of tenuous structure comprising residues 15 through 32, which is, we believe, intimately involved in DNA binding. This wing appears to be dynamic and mobile, even in the crystal and, therefore, is likely to undergo conformational change in the presence of the ligand.