Structure of the filamentous phage pIV multimer by cryo-electron microscopy

The homo-multimeric pIV protein constitutes a channel required for the assembly and export of filamentous phage across the outer membrane of Escherichia coli. We present a 22 A-resolution three-dimensional reconstruction of detergent-solubilized pIV by cryo-electron microscopy associated with image analysis. The structure reveals a barrel-like complex, 13.5 nm in diameter and 24 nm in length, with D14 point-group symmetry, consisting of a dimer of unit multimers. Side views of each unit multimer exhibit three cylindrical domains named the N-ring, the M-ring and the C-ring. Gold labeling of pIV engineered to contain a single cysteine residue near the N or C terminus unambiguously identified the N-terminal region as the N-ring, and the C-terminal region was inferred to make up the C-ring. A large pore, ranging in inner diameter from 6.0 nm to 8.8 nm, runs through the middle of the multimer, but a central domain, the pore gate, blocks it. Moreover, the pore diameter at the N-ring is smaller than the phage particle. We therefore propose that the pIV multimer undergoes a large conformational change during phage transport, with reorganization of the central domain to open the pore, and widening at the N-ring in order to accommodate the 6.5 nm diameter phage particle.     

Characterization of sodium dodecyl sulfate-resistant proteolytic activity in the hyperthermophilic archaebacterium Pyrococcus furiosus.

Cell extracts from Pyrococcus furiosus were found to contain five proteases, two of which (S66 and S102) are resistant to sodium dodecyl sulfate (SDS) denaturation. Cell extracts incubated at 98 degrees C in the presence of 1% SDS for 24 h exhibited substantial cellular proteolysis such that only four proteins could be visualized by amido black-Coomassie brilliant blue staining of SDS-polyacrylamide gels. The SDS-treated extract retained 19% of the initial proteolytic activity as represented by two proteases, S66 (66 kilodaltons [kDa]) and S102 (102 kDa). Immunoblot analysis with guinea pig sera containing antibodies against protease S66 indicated that S66 is related neither to S102 nor to the other proteases. The results of this analysis also suggest that S66 might be the hydrolysis product of a 200-kDa precursor which does not have proteolytic activity. The 24-h SDS-treated extract showed unusually thermostable proteolytic activity; the measured half-life at 98 degrees C was found to be 33 h. Proteases S66 and S102 were also resistant to denaturation by 8 M urea, 80 mM dithiothreitol, and 5% beta-mercaptoethanol. Purified protease S66 was inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate but not by EDTA, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, or iodoacetic acid. These results indicate that S66 is a serine protease. Amino acid ester hydrolysis studies showed that protease S66 was hydrolytically active towards N-benzoyl-L-arginine ethyl ester.     

Characterization of sodium dodecyl sulfate-resistant proteolytic activity in the hyperthermophilic archaebacterium Pyrococcus furiosus

Cell extracts from Pyrococcus furiosus were found to contain five proteases, two of which (S66 and S102) are resistant to sodium dodecyl sulfate (SDS) denaturation. Cell extracts incubated at 98 degrees C in the presence of 1% SDS for 24 h exhibited substantial cellular proteolysis such that only four proteins could be visualized by amido black-Coomassie brilliant blue staining of SDS-polyacrylamide gels. The SDS-treated extract retained 19% of the initial proteolytic activity as represented by two proteases, S66 (66 kilodaltons [kDa]) and S102 (102 kDa). Immunoblot analysis with guinea pig sera containing antibodies against protease S66 indicated that S66 is related neither to S102 nor to the other proteases. The results of this analysis also suggest that S66 might be the hydrolysis product of a 200-kDa precursor which does not have proteolytic activity. The 24-h SDS-treated extract showed unusually thermostable proteolytic activity; the measured half-life at 98 degrees C was found to be 33 h. Proteases S66 and S102 were also resistant to denaturation by 8 M urea, 80 mM dithiothreitol, and 5% beta-mercaptoethanol. Purified protease S66 was inhibited by phenylmethylsulfonyl fluoride and diisopropyl fluorophosphate but not by EDTA, ethylene glycol-bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid, or iodoacetic acid. These results indicate that S66 is a serine protease. Amino acid ester hydrolysis studies showed that protease S66 was hydrolytically active towards N-benzoyl-L-arginine ethyl ester.     

Nicking-closing enzyme is associated with SV40 DNA in vivo as a sodium dodecyl sulfate-resistant complex.

A fraction of the cellular nicking-closing (NC) enzyme cosediments with SV40 chromatin isolated after Triton X-100 treatment of infected cells nuclei. Extraction of viral DNA according to the Hirt procedure by treatment of infected cells with sodium dodecyl sulfate (SDS) followed by sedimentation in sucrose gradient to separate the DNA from the bulk of detergent also revealed NC activity associated with DNA. Reconstitution experiments showed that only prebinding of the NC enzyme to DNA protects it against irreversible inactivation by SDS. These results suggest that a fraction of the cellular NC activity is indeed associated with the viral chromosome in vivo.     

Mobility of sodium dodecyl sulphate - protein complexes.

Reduced and unreduced lysozyme aggregates formed by formaldehyde cross-linking comprise a set of model compounds for studying the effects of protein conformation on the electrophoretic mobilities of sodium dodecyl sulphate-protein complexes. The reduced aggregates were indistinguisable from normal proteins, but the unreduced aggregates migrated anomalously fast by about 14%. Contrary to expectations, plots of logarithm Rf versus Kr (retardation coefficient) failed to reveal an unusual conformation for the unreduced aggregates. Thus the anomalous mobility caused by several intramolecular disulphide bonds escaped detection by the above two diagnostic plots. Also included in this paper is a discussion of the implications of these results with regard to current models for sodium dodecyl sulphate-protein complexes.     

The use of filamentous phage M13 in protein engineering

M13B1 vector based on the filamentous phage M13 has been constructed. M13B1 phage carries the gene of resistance to ampicillin and contains the unique site of recognition for BamHI restriction endonuclease in gene VIII coding for the major coat protein. BamHI restriction site has been inserted into the gene of the major coat protein by means of oligonucleotide directed mutagenesis. The synthetic DNA fragment coding for the model peptides has been inserted through BamHI site into the M13B1 DNA. The possibility of inserting foreign peptides into the N-terminus at maintaining the viability of hybrid phages has been shown. The differences in specificity of the recombinant phage maturation have been determined by analysing the amino acid sequence of B-protein.     

Pore-forming properties of the adsorption protein of filamentous phage fd

The gene 3-encoded adsorption protein (g3p) of filamentous phage fd has been purified to homogeneity by using high-performance liquid chromatography. Removal of SDS from the SDS-solubilized g3p results in spontaneous oligomerization of the g3p. Reconstitution into artificial lipid bilayer membranes shows that the oligomer forms large aqueous pores that remain open for seconds and are insensitive to changes in membrane potential. The estimated diameter of the pores suggest that they are large enough to allow passage of phage single-stranded DNA. The implications of these findings for phage infection are discussed.     

Interdomain interactions within the gene 3 protein of filamentous phage

A number of disorders related to cystic fibrosis have been described since the cloning of the cystic fibrosis gene, including infertility due to the congenital bilateral absence of the vas deferens. We have identified, in several patients, complex cystic fibrosis transmembrane conductance regulator genotypes like double-mutant alleles. We have now analyzed the structure-function relationships of one of these mutants, R74W-D1270N cystic fibrosis transmembrane conductance regulator, expressed in HeLa cells, to evaluate the contribution of each mutation in the phenotype. We found that R74W cystic fibrosis transmembrane conductance regulator appears to be a polymorphism, while D1270N cystic fibrosis transmembrane conductance regulator could be responsible for the congenital bilateral absence of the vas deferens phenotype. The combination of the two produced a more severe effect on the chloride conductance pathway as well as on the phenotype.     

Electrophoretic behavior of protein dodecyl sulfate complexes in the presence of various amounts of sodium dodecyl sulfate.

This report describes the relationship between the amount of sodium dodecyl sulfate present in a sample solution and the electrophoretic mobility of the protein-dodecyl sulfate complexes. In order to determine the extent of any conformational changes in the proteins and to establish a correlation between any of these structural changes and the electrophoretic behavior, visible absorption spectra and circular dichroism spectra were obtained for heme proteins in the presence of the same amounts of surfactants as used in electrophoresis.From the results obtained, it is apparent that the amount of sodium dodecyl sulfate present in the sample solution must be taken into consideration when performing a separation. Optimum experimental conditions are chosen for attaining enhanced separation and a maximized linear range of molecular weights of proteins that can be accurately determined.     

Recovery of protein from sodium dodecyl sulfate-polyacrylamide gels

An apparatus for extracting small quantities of protein from sodium dodecyl sulfate-polyacrylamide gels is described. It enables protein contained in a slice of polyacrylamide gel to be transferred electrophoretically, into a small volume of buffer solution. The technique is rapid (within 2 h), reproducible, and efficient (up to 90% recoveries).     

Removal of protein interference in the Fiske-Subbarow method by sodium dodecyl sulfate.

In kinetic studies on erythrocyte membrane ATPase, the activity is assayed by following the release of orthophosphate from ATP by the Fiske-SubbaRow method. The deproteinization of the samples, which is essential before assay by this method, is an obstacle to the quick and efficient assay of large numbers of samples. Nonionic detergents, e.g., Lubrol, which are used in the purification of Na⁺-K⁺-ATPase from plasma membranes, also interfere strongly with the Fiske-SubbaRow method.It was found that the addition of sodium dodecyl sulfate to the samples before the orthophosphate assay solubilized much of the membrane protein, allowing orthophosphate to be determined by the Fiske-SubbaRow method without the deproteinization step. Sodium dodecyl sulfate and the solubilized proteins did not interfere with the determination. The addition of sodium dodecyl sulfate was also found to abolish the interference of nonionic detergents with the Fiske-SubbaRow determination. As a result, the assay of erythrocyte membrane ATPase could be greatly simplified.     

Detection of protein kinase activity in sodium dodecyl sulfate-polyacrylamide gels

A procedure is described for identifying protein kinase activity in protein samples following electrophoresis on sodium dodecyl sulfate-polyacrylamide gels. Protein kinase activity is detected by renaturation of the enzymes within the gel followed by phosphorylation with [gamma-32P]ATP of either substrates included in the polyacrylamide gel or of the kinase itself. Then, after removal of the unreacted [gamma-32P]ATP by washing the gel in the presence of an anion-exchange resin, the positions (Mr) of the protein kinase activity are visualized by autoradiography. Studies using a purified catalytic subunit of cAMP-dependent protein kinase indicate that enzyme concentrations as low as 0.01 microgram can easily be detected on gels containing 1 mg/ml casein. The technique is also useful for identifying active subunits of multisubunit enzymes. The active subunit of casein kinase II, for example, can readily be determined by renaturing the dissociated enzyme in gels containing casein. Putative protein kinases present in crude mixtures of proteins can also be detected following separation by gel electrophoresis and can be characterized on the basis of molecular weight and identity of the phosphorylated amino acid. Using this technique, at least three major protein kinases were detected in a mixture of proteins prepared by subfraction of red blood cell membranes.     

Homodimeric peptides displayed by the major coat protein of filamentous phage

Peptide libraries displayed by filamentous bacteriophage have proven a powerful tool for the discovery of novel peptide agonists, antagonists and epitope mimics. Most phage-displayed peptides are fused to the N terminus of either the minor coat protein, pIII, or the major coat protein, pVIII. We report here that peptides containing cysteine residues, displayed as N-terminal fusions to pVIII, can form disulfide-bridged homodimers on the phage coat. Phage clones were randomly selected from libraries containing one or two fixed Cys residues, and surveyed for the presence of peptide-pVIII homodimers by SDS-PAGE analysis that involved pretreatment of the phage with reducing or thiol-modifying agents. For all phage whose recombinant peptide contained a single Cys residue, a significant fraction of the peptide-pVIII molecules were displayed as dimers on the phage coat. The dimeric form was in greater abundance than the monomer in almost all cases in which both forms could be reliably observed. Occasionally, peptides containing two Cys residues also formed dimers. These results indicate that, for a given pVIII-displayed peptide bearing a single Cys residue, a significant fraction of the peptide (>40 %) will dimerize regardless of its sequence; however, sequence constraints probably determine whether all of the peptide will dimerize. Similarly, only occasionally do peptides bearing two Cys residues form intermolecular disulfide bridges instead of intramolecular ones; this indicates that sequence constraints may also determine dimerization versus cyclization. Sucrose-gradient analysis of membranes from cells expressing pVIII fused to a peptide containing a single Cys residue showed that dimeric pVIII is present in the cell prior to its assembly onto phage. A model of the peptide-pVIII homodimer is discussed in light of existing models of the structure and assembly of the phage coat. The unique secondary structures created by the covalent association of peptides on the phage surface suggest a role for homo- and heterodimeric peptide libraries as novel sources of bioactive peptides.     

Antibody engineering by codon-based mutagenesis in a filamentous phage vector system.

A novel codon-based mutagenesis procedure is described that allows rapid and efficient modification of antibody amino acid sequences expressed as F(ab) fragments in M13. The procedure succeeded in generating a library of mutations in the complementarity-determining regions of chimeric L6, an antibody against a tumor-associated Ag. A set of anti-Id antibodies (anti-Id 1, 3, and 7) that bind near the L6 Ag-binding site served as model Ag. The goal was to select mutant antibody sequences that altered the L6 reactivity with the anti-Id in subtle ways, i.e., to eliminate the binding to one anti-Id while preserving other reactivities or to identify mutants with increased binding. A high frequency of variant M13 phage clones exhibiting altered specificity for the anti-Id were identified. Codon-based mutagenesis in conjunction with the M13 antibody expression and screening system should provide an efficient and general approach for redirecting the specificity and potentially improving the affinity of antibodies in vitro.     

Reprogramming the infection mechanism of a filamentous phage

When they infect Escherichia coli cells, the filamentous phages IF1 and fd first interact with a pilus and then target TolA as their common receptor. They use the domains N2 and N1 of their gene-3-proteins (G3P) for these interactions but differ in the mechanism of infection. In G3P of phage IF1, N1 and N2 are independent modules that are permanently binding-active. G3P of phage fd is usually in a closed state in which N1 and N2 are tightly associated. The TolA binding site is thus inaccessible and the phage incompetent for infection. Partial unfolding and prolyl isomerization must occur to abolish the domain interactions and expose the TolA binding site. This complex mechanism of phage fd could be changed to the simple infection mechanism of phage IF1 by reprogramming its G3P following physicochemical rules of protein stability. The redesigned phage fd was robust and as infectious as wild-type phage fd.     

Copper staining: a five-minute protein stain for sodium dodecyl sulfate-polyacrylamide gels

We present a new method for visualizing proteins electrophoresed in sodium dodecyl sulfate-polyacrylamide gels. After electrophoresis, gels are incubated in CuCl2 to produce a negative image of colorless protein bands against a semiopaque background. Gels are stained completely within 5 min, do not require destaining, and can be stored indefinitely without loss of the image. Because proteins are not permanently fixed within the gel, they can be quantitatively eluted after chelation of Cu with EDTA. The sensitivity of the CuCl2 stain falls between that of Coomassie blue and silver. We anticipate that CuCl2 will be useful in the rapid analysis of proteins by polyacrylamide gel electrophoresis and in the preparation of purified polypeptides by elution from gel slices.     

The symmetries of filamentous phage particles

The helical symmetries of two classes of filamentous bacteriophage particles are distinctly different. The symmetry of the class I particles is²C₅S_~2.0 (a 5-fold rotation axis combined with an approximately 2-fold screw axis). The symmetry of the class II particles is C₁S_5.4 (a one-start helix with 27 subunits equally spaced along five turns). The same basic α-helical interlocking arrangement of the largely α-helical coat protein subunits can be accommodated by the symmetry of the two classes of phage particles. The conservation of this structural pattern reflects intrinsic packing properties of α-helices. The difference between the symmetries of the class I and class II particles suggests that different assembly processes may have evolved to form these structures with very similar protein packing architectures.     

The role of thioredoxin in filamentous phage assembly. Construction, isolation, and characterization of mutant thioredoxins

Filamentous phage assembly in vivo shows an absolute requirement for thioredoxin and a partial requirement for thioredoxin reductase. Mutants in which one or both of the active site cysteine residues of thioredoxin were changed to alanine or serine were constructed and shown to support filamentous phage assembly. Some of the mutants were almost as effective as wild-type thioredoxin, while others supported phage assembly only when high levels of the mutant protein were present in the infected cell. The mutant proteins were all inactive in an assay which couples oxidation of NADPH to reduction of 5,5'-dithiobis-2-nitrobenzoic acid) via thioredoxin reductase and thioredoxin. These active site mutants make phage assembly completely independent of thioredoxin reductase, which suggests that the phage needs, and the active site mutants provide, the proteins in the reduced conformation. Other mutants were isolated on the basis of their failure to support filamentous phage growth. These specified mutant thioredoxin proteins with varying levels of redox activity in vivo and in vitro. The locations of these mutations suggest that the surface of thioredoxin thought to interact with thioredoxin reductase also interacts with the filamentous phage assembly machinery. An in vivo assay for thioredoxin redox function, based on the ability of cells to utilize methionine sulfoxide, was developed. Met- cells containing mutant thioredoxins that are inactive in vitro do not form colonies on plates containing methionine sulfoxide as the sole methionine source.