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PubMed1995

Moving through the membrane with filamentous phages.

Filamentous phages are small, highly evolved parasites that can reproduce and disseminate without killing their host. During assembly, virion proteins are transferred from the host membrane to the single-stranded DNA phase genome and simultaneously secreted from the cell. Filamentous phage assembly shares certain features with bacterial processes responsible for the assembly of cell-surface structures and for extracellular protein secretion.

Coliphages↗
PubMed2003

Vibrio cholerae O139 Bengal: odyssey of a fortuitous variant.

Vibrio cholerae O139, the new serogroup associated with epidemic cholera, came into being in the second half of the year 1992 in an explosive fashion and was responsible for several outbreaks in India and other neighbouring countries. This was an unprecedented event in the history of cholera and the genesis of the O139 serogroup was, at that time, thought to be the beginning of the next or the eighth pandemic of cholera. However, with the passage of time, the O1 serogroup of the El Tor biotype again reappeared and displaced the O139 serogroup on the Indian subcontinent, and there was a feeling among cholera workers that the appearance of this new serogroup may have been a one-time event. The resurgence of the O139 serogroup in September 1996 in Calcutta and the coexistence of both the O1 and O139 serogroups in much of the cholera endemic areas in India and elsewhere, suggested that the O139 serogroup has come to stay and is a permanent entity to contend with in the coming years. During the past 10 years, intensive work on all aspects of the O139 serogroup was carried out by cholera researchers around the world. The salient findings on this serogroup over the past 10 years pertinent to its prevalence, clinico-epidemiological features, virulence-associated genes, rapid screening and identification, molecular epidemiology, and vaccine developments have been highlighted.

Antigens, Bacterial↗
PubMed2003

Filamentous phages linked to virulence of Vibrio cholerae.

The pathogenicity of Vibrio cholerae depends upon its production of two key virulence factors: the toxin co-regulated pilus (TCP), a colonization factor, and cholera toxin, an exotoxin. Genes encoding both virulence factors were introduced into V. cholerae by horizontal gene transfer. The toxin genes are contained within the genome of CTXphi, an integrated filamentous phage identified in 1996. In the past few years, it has been shown that CTXphi relies on novel processes for phage DNA integration, replication and secretion. In addition, expression of CTXphi genes--including the toxin genes--and transmission of CTXphi were recently found to be promoted by the antirepressor RstC, which is encoded within RS1, a newly described satellite phage of CTXphi. The genetic island that encodes TCP has also been described as a filamentous phage; however, these sequences are unlike the genome of any previously characterized filamentous phage.

Cholera↗
PubMed2006

[Large recombinant protein displayed on filamentous phage surface and its interaction with small molecule].

Recombinant proteins were expressed as fusions with the phagemid system of pHEN-KM13 and the characteristics and activities of the fusion proteins displayed on the surface of filamentous phagintain the were studiedon abilty. The altered titer of rescued phages from the phagemid system after trypsin treatment indicated the relative quantity of the phages displaying fusion proteins. The rescue phages displayed foreign proteins could keep the bacterial infection ability, while the bald phage without foreign protein displayed on its surface was sensitive to trypsin treatment and lost the bacterial infection ability. To determine the upper limit for filamentous phage display, four recombinant proteins, glutathione-S-transferase and glutathione-S-transferase fused with three various size peptide linkers, were fused to N terminus of capsid protein gp3 and rescued by helper phage KM13. The rescued phages which displayed fused protein with the size of 40kD or less maintain the infection ability. To assay the activity of the phage displayed protein, the known small molecule probe was used in the interaction study with protein incorporated on phage surface. Results showed that the glutathione-S-transferase on phage surface still bound to glutathione specifically. It indicated that the glutathione-S-transferase displayed on phage surface was correctly folded and functionally active. The results demonstrated that it was feasible to use small molecule probes to interact with the protein displayed on phage surface. In turn, the method described here also demonstrated that phage display system could be utilized to investigate the interactions between protein and small molecules.

Glutathione↗
PubMed1995

Comparison of Pf1 and Fd gene 5 proteins and their single-stranded DNA complexes by NMR spectroscopy and differential scanning calorimetry.

The Pf1 gene 5 protein forms a large helical nucleoprotein complex (Mr = 3.1 x 10(7)) with single-stranded viral DNA, from which a 32 amino acid sequence rich in alanine, proline, and glutamine residues can be removed from the C-terminus by limited proteolysis. Sharp resonances in the 1H NMR spectrum of the Pf1 nucleoprotein complex indicate that the C-terminal region of the protein subunits enjoys remarkable conformational flexibility in the complex. In contrast, the globular N-terminal domain of the protein subunits is rigidly held and does not contribute to the spectrum. The Fd gene 5 protein lacks this C-terminal flexible domain, and no distinct resonances can be observed in the 1H NMR spectrum when this protein is complexed to single-stranded viral DNA. Differential scanning calorimetry shows that the thermal stability of both the Pf1 and Fd gene 5 protein is increased by 8 degrees C in the complex with DNA, and the transition is highly cooperative. Removal of the C-terminal domain of the Pf1 gene 5 protein subunits has no appreciable effect either on the Tm of the DNA-protein complex or on the cooperative nature of the thermal transition. It is suggested that the C-terminal domain of the Pf1 gene 5 protein acts as a dynamic clamp which kinetically stabilizes the nucleoprotein complex.

Amino Acid Sequence↗
PubMed1995

Raman spectroscopy of the filamentous virus Ff (fd, fl, M13): structural interpretation for coat protein aromatics.

Site-specific isotope substitutions in the coat protein (pVIII) of the filamentous bacterial virus Ff (fd, fl, M13) have been employed to advance vibrational band assignments and facilitate structural interpretation of the Raman spectrum. We report spectra of phage fd assembled in vivo from pVIII subunits incorporating either deuteriophenylalanine (Fd5), deuteriotryptophan (Wd5), or deuteriotyrosine (Yd4) residues with labeled ring sites. The deuterated aromatics were introduced into fd individually and in combination. On the basis of observed isotope shifts, definitive assignments have been developed for all prominent Raman bands diagnostic of the pVIII aromatic residues (F11, F42, F45, W26, Y21, Y24). The present study constitutes the first direct experimental determination of Raman fingerprints of tyrosine and phenylalanine side chains within hydrophobic alpha-helical domains and yields unexpected results. Importantly, neither Y21 nor Y24 of pVIII exhibits the "canonical" Fermi doublet expected in the 820-860 cm-1 interval of the Raman spectrum. Instead, each tyrosine exhibits a single band near 853 cm-1. Since the application of denaturing conditions is sufficient to generate in fd an apparent Fermi doublet, it is concluded that the anomalous singlet is intrinsic to tyrosine environments in the native virion assembly. In addition, the Raman results clearly demonstrate an interdependence of the environments of aromatic side chains in virion subunits. We show that the results on fd isotopomers are also confirmed by Raman spectroscopy of Ff virions incorporating the tyrosine mutations Y21M, Y24M, and Y21F/Y24S. The Raman marker bands identified for pVIII aromatics modify and extend Raman correlations proposed previously for proteins. The unusual environments detected for aromatic residues in the mature Ff assembly are discussed in relation to recently proposed models for filamentous virion architecture.

Amino Acid Sequence↗
PubMed1995

FT-IR spectroscopy of the major coat protein of M13 and Pf1 in the phage and reconstituted into phospholipid systems.

FT-IR spectroscopy has been applied to study the secondary structure of the major coat protein of Pf1 and M13 as present in the phage and reconstituted in DOPG and mixed DOPC/DOPG (4/1) bilayers. Infrared absorbance spectra of the samples were examined in dehydrated films and in suspensions of D2O and H2O. The secondary structure of the coat protein is investigated by second-derivative analysis, Fourier self-deconvolution, and curve fitting of the infrared bands in the amide I region (1600-1700 cm-1). It is found that, in dehydrated films of Pf1 and M13 phage, the amide I region contains three bands located at about 1633, 1657, and 1683 cm-1, that are assigned to hydrogen-bonded turn, alpha-helix/random coil, and non-hydrogen-bonded turn, respectively. From a comparison of the infrared spectra in dehydrated film with those in aqueous suspension, the percentages of secondary structure were found with an accuracy of about +/- 5%. For the coat protein of Pf1 phage, the FT-IR quantification gives 69% alpha-helix conformation, 19% turn structure, and 12% random coil structure. For Pf1 coat protein in the membrane-embedded state, the amount of alpha-helix is 57%, whereas 42% is in a turn structure and 1% in a random coil structure. The same assignment strategy was used for the analysis of the data obtained for M13 coat protein reconstitution into phospholipid systems. For M13 coat protein in the phage, this gives 75% alpha-helix conformation, 21% turn structure, and 4% random coil structure.(ABSTRACT TRUNCATED AT 250 WORDS)

Bacteriophage M13↗
PubMed1995

Relationship between in vivo activity and in vitro measures of function and stability of a protein.

The in vivo activities of mutant proteins are readily measured and can potentially be used to estimate changes in in vitro properties such as stability or function, but this connection has not been rigorously established. Gene V protein is a small protein produced by bacteriophage f1 that binds to single-stranded DNA and to RNA and for which fitness can be assayed both in vivo and in vitro. We have assembled a large number of temperature-sensitive mutants of the gene V protein of bacteriophage f1 and measured their ability to support phage growth and replication in vivo. We have also purified many of these mutant gene V proteins and measured their stabilities and ssDNA binding affinities in vitro. Mutations at surface residues frequently yielded temperature-sensitive mutants, but remarkably, no overall correlation between in vivo activity and in vitro measures of either stability or function was found for this group. Mutations at buried residues often lead to the temperature-sensitive phenotype. At buried sites temperature sensitivity was strongly correlated with in vitro stability changes, but not with in vitro ssDNA binding affinity. The implication of these observations for protein engineering efforts is that phenotypes conferred by amino acid substitutions at buried sites can be used to identify mutants whose stabilities fall into ranges of interest, while phenotypes of mutants with surface substitutions may be much less readily interpreted, even in the case of a single-stranded-DNA-binding protein.

Binding Sites↗
PubMed1995

Accessibility and dynamics of Cys residues in Bacteriophage IKe and M13 major coat protein mutants.

The filamentous bacteriophage major coat protein occurs as a membrane-spanning assembly intermediate prior to incorporation into the lipid-free virion. To gain insight into how this small, multifunctional protein is able to be stably incorporated into both of these distinct environments, the reactive sulfhydryl group of IKe and M13 coat protein Cys mutants was exploited to probe the mobility and environment of this residue at several loci within the hydrophobic domain of these proteins. IKe mutants P30C, G39C, and G39C-V36A and M13 mutant Y24C-V31A, each previously obtained from randomized mutagenesis, were characterized in the intact virion, the intermediate spheroidal S-form, and in membrane-mimetic sodium dodecyl sulfate (SDS) micelles. The accessibility of the Cys sulfhydryl in the virion was examined by reaction with [14C]iodoacetamide (14C-IAN) and other alkylating agents. The IKe mutants G39C and G39C-V36A were found to be the most reactive with 14C-IAN and thus the most accessible, although this accessibility was subject to strict steric constraints since only the smallest sulfhydryl-specific alkylating agents were able to modify the Cys39 locus. The spin probe proxyliodoacetamide (PIAN) was used to characterize Cys side chain mobility by electron paramagnetic resonance (EPR) spectroscopy. The M13 mutant Y24C-V31A Cys side chain in the phage was observed to be the most mobile, with slightly less mobility for IKe mutant P30C and considerably less for G39C mutants. The SDS micelle-bound forms of the Cys mutants all exhibited enhanced side chain mobility compared to the virion form, with the extent of mobility dependent upon the specific location of the Cys residue. EPR and fluorescence quenching results show that the Cys side chain in the Y24C-V31A S-form is largely immobilized and inaccessible in comparison to the virion and micelle-solubilized forms. The overall results are interpreted in terms of the structural changes accompanying disassembly and insertion of the coat protein into the Escherichia coli inner membrane.

Alkylation↗
PubMed1993

Structural changes accompanying chloroform-induced contraction of the filamentous phage fd.

Contact with a chloroform/water interface at 2 degrees C induces contraction of fd filamentous phage into rodlike I-forms; this contraction is accompanied by a decrease in the magnitude of circular dichroism spectral intensity near 222 nm and an increase near 210 nm. Comparisons with circular dichroism spectra of 100% helical poly-L-lysine and N-bromosuccinimide-oxidized fd phage indicate that the spectral change accompanying the fd to I-forms transition is due primarily to a change in the contributions from the single tryptophan (W26) of the major coat protein, with probably no significant change in the alpha-helix content. Further contraction of the rodlike I-forms to spherical S-forms at 25 degrees C is accompanied by a substantial general decrease in the magnitude of the ellipticity throughout the 230-210-nm region, which is indicative of a decrease in the alpha-helix content of the major coat protein. The similarity of the circular dichroism spectrum of S-forms with that of coat protein in detergents suggests that the S-form coat protein resembles the coat protein in lipid bilayers. The intrinsic fluorescence of W26 is quenched without red-shift (but perhaps a barely detectable blue-shift) following fd contraction to I-forms and S-forms. The accessibility of W26 to aqueous quenchers does not change significantly upon contraction. However, interaction with hydrophobic quenchers is dramatically altered in the contracted forms in a manner suggesting that the environment surrounding the tryptophan changes from native-protein-like in the fd filament to molten globule-like in the I-form rods and S-form spheroids. As discussed herein, certain features of these data support previous suggestions that chloroform-induced filamentous phage contraction may provide information about phage penetration and assembly in vivo.

Capsid↗
PubMed1993

Biophysical studies of the Pf1 coat protein in the filamentous phage, in detergent micelles, and in a membrane environment.

During the assembly of the Pf1 phage, the membrane-bound coat proteins convert into subunits of the filamentous phage. Fourier-transform infrared (FT-IR) transmission spectroscopy has been applied to a study of the secondary structure of these coat proteins when present (a) in the phage, (b) in detergent micelles, and (c) in a phospholipid membrane aqueous system. Suspensions of the Pf1 phage in H2O and 2H2O show an amide I band at 1652 cm-1, indicative of a high content of the alpha-helical structure present. Oriented films of the Pf1 phage studied by polarized FT-IR transmission spectroscopy indicate that the alpha-helical structures as well as the tyrosine residues (band at 1515 cm-1) are both aligned along the axis of the phage. When the Pf1 coat protein is present in sodium dodecyl sulfate detergent micelles and in lipid membrane systems, the FT-IR spectra show an amide I band at 1657-1658 cm-1, also indicative of a predominantly alpha-helical secondary structure. H/2H amide proton exchange studies show that when present in a phospholipid membrane system some 50-60% of the Pf1 protein exchanges rapidly, while the rest undergoes slow exchange. This is consistent with a proportion of the protein being exposed to the solvent and the other being embedded in the lipid bilayer. The presence of a band at 1630-1640 cm-1 is indicative of the presence of random structures.(ABSTRACT TRUNCATED AT 250 WORDS)

Calorimetry, Differential Scanning↗
PubMed1994

Pf3 coat protein forms voltage-gated ion channels in planar lipid bilayers.

The coat protein of bacteriophage Pf3 forms discrete and stable ion channels of uniform size in planar bilayers of asolectin. Its primary sequence suggests a channel formed by a bundle of transmembrane helices. Since the apparent transmembrane region only consists of strongly hydrophobic residues, it represents a new class of channel-forming proteins. The channel activity is strongly voltage-dependent. The single-channel conductance of 60 pS (at 100 mV) in 0.2 M NaCl is slightly voltage-dependent, indicating conformational changes of the pore upon variation of the transmembrane electric field. The channel is unselective which suggests that the pore is of aqueous character. For the observed conductance, a channel diameter of 3.6 A is consistent with a tetrameric alpha-helix bundle, as calculated from a barrel-stave model. A pronounced dependence of the gating kinetics with increasing voltage arises from two opposing effects: an increase in the number of open channel structures, and a simultaneous, more than 3-fold decrease in the channel lifetime. Thus, a maximum activity is reached around 100 mV, a range which corresponds well with physiological membrane potentials. The channels activate only upon application of a positive voltage on the side of the membrane to which the protein had been added. The slow relaxation of the mean current upon application of sudden voltage jumps indicates a strong activation barrier in the channel gating process, which may result from the membrane translocation of the charged residues of the peptide ends. A channel-mediated import mechanism is suggested for the bacterial infection by phage DNA.

Amino Acid Sequence↗
PubMed1994

Novel tyrosine markers in Raman spectra of wild-type and mutant (Y21M and Y24M) Ff virions indicate unusual environments for coat protein phenoxyls.

The tyrosine side chain generates a pair of distinctive Raman bands--a Fermi doublet near 850 and 830 cm-1--with relative intensities diagnostic of hydrogen bonding states of the phenolic acceptor and donor atoms [Siamwiza et al. (1975) Biochemistry 14, 4870-4876]. This structural correlation has been tested extensively and is used widely as an indicator of tyrosine interactions in globular proteins and their assemblies. However, in Ff filamentous viruses (fd, f1, M13) the apparent Fermi doublet intensity ratio (I853/I826 approximately 4.0) is much greater than the maximum predicted or observed in other proteins. To understand this anomaly, we have reevaluated the basis for the Fermi doublet assignment in Ff. We report Raman spectra of site-specific mutants of Ff in which either one (Y21M and Y24M) or both (Y21F/Y24S) tyrosines of the coat protein subunit (pVIII) have been mutated. These Raman data, together with those obtained from Ff virions carrying residue-specific tyrosyl (Y-d4) and phenylalanyl (F-d5) deuterations in pVIII, demonstrate conclusively that the 853 and 826 cm-1 bands of Ff do not constitute a typical tyrosine Fermi doublet: The observed 826 cm-1 Raman band of Ff is due not to tyrosine but to phenylalanine residues of pVIII. The 853 cm-1 Raman band thus constitutes the first known example of a "tyrosine singlet" in the Raman spectrum of a protein. The implications of this finding for Ff virion structure and its relevance to tyrosine markers in other proteins are discussed.

Amino Acid Sequence↗
PubMed1994

Ultraviolet absorbance and circular dichroism of Pf1 virus: nucleotide/subunit ratio of unity, hyperchromic tyrosines and DNA bases, and high helicity in the subunits.

Data have been obtained for the Pf1 virion that establish its stoichiometry and conformational features of its DNA and its protein. The absorbance spectrum of the dissociated virus under alkaline denaturing conditions is fit exactly by spectra for DNA and protein at a mole ratio of one nucleotide per protein subunit. This result, together with three previous values by independent methods, establishes that the nucleotide/subunit ratio (n/s) of Pf1 is unity. The absorbance spectrum of DNA in the intact native virus is assigned as the spectrum for heat denatured Pf1 DNA, with epsilon (P) = 8400 M-1 cm-1 at 259 nm. The absorbance spectrum assigned to protein (two tyrosines) in the intact virus has = 2500 M-1 cm-1 per tyrosine at lambda max of 281.5 nm; this is the most red-shifted and hyperchromic tyrosine spectrum known. The CD spectrum of the intact virus from 250 to 320 nm has no apparent DNA contribution, but has a strong contribution from the red-shifted tyrosine(s). The CD spectrum from 185 to 250 nm has the shape of alpha-helical CD reference spectra, but is perceptibly blue-shifted, with a crossover from negative to positive ellipticity at 199.7 nm, and it has very high amplitudes (e.g. [theta 207.5nm] = -44,000 deg cm2 dmol-1). This spectrum indicates completely helical protein in the virus, with a predominance of alpha-helix and perhaps some 3(10)-helix. The unit n/s ratio, the high absorbance and negligible near-UV CD for the DNA bases, and the high amplitudes for the helical protein are critical input data for the determination of Pf1 virus structure.

Circular Dichroism↗
PubMed2001

Ff gene 5 protein has a high binding affinity for single-stranded phosphorothioate DNA.

The gene 5 protein (g5p) of Ff bacteriophages is a well-studied model ssDNA-binding protein that binds cooperatively to the Ff ssDNA genome and single-stranded polynucleotides. Its affinity, K omega (the intrinsic binding constant times a cooperativity factor), can differ by several orders of magnitude for ssDNAs of different nearest-neighbor base compositions [Mou, T. C., Gray, C. W., and Gray, D. M. (1999) Biophys. J. 76, 1537-1551]. We found that the DNA backbone can also dramatically affect the binding affinity. The K omega for binding phosphorothioate-modified S-d(A)(36) was >300-fold higher than for binding unmodified P-d(A)(36) at 0.2 M NaCl. CD titrations showed that g5p bound phosphorothioate-modified oligomers with the same stoichiometry as unmodified oligomers. The CD spectrum of S-d(A)(36) underwent the same qualitative change upon protein binding as did the spectrum of unmodified DNA, and the phosphorothioate-modified DNA appeared to bind in the normal g5p binding site. Oligomers of d(A)(36) with different proportions of phosphorothioate nucleotides had binding affinities and CD perturbations intermediate to those of the fully modified and unmodified sequences. The influence of phosphorothioation on binding affinity was nearly proportional to the extent of the modification, with a small nearest-neighbor dependence. These and other results using d(ACC)(12) oligomers and mutant proteins indicated that the increased binding affinity of g5p for phosphorothioate DNA was not a polyelectrolyte effect and probably was not an effect due to the altered nucleic acid structure, but was more likely a general effect of the properties of the sulfur in the context of the phosphorothioate group.

Circular Dichroism↗
PubMed2001

Tyrosine Raman signatures of the filamentous virus Ff are diagnostic of non-hydrogen-bonded phenoxyls: demonstration by Raman and infrared spectroscopy of p-cresol vapor.

p-Cresol is a simple molecular model for the para phenolic side chain of tyrosine. Previously, Siamwiza and co-workers [(1975) Biochemistry 14, 4870-4876] investigated p-cresol solutions to identify Raman spectroscopic signatures for different hydrogen-bonding states of the tyrosine phenoxyl group in proteins. They found that the phenolic moiety exhibits an intense Raman doublet in the spectral interval 820-860 cm(-1) and that the doublet intensity ratio (I2/I1, where I2 and I1 are Raman peak intensities of the higher- and lower-wavenumber members of the doublet) is diagnostic of specific donor and acceptor roles of the phenoxyl OH group. The range of the doublet intensity ratio in proteins (0.30 < I2/I1 < 2.5) was shown to be governed by Fermi coupling between the phenolic ring-stretching fundamental nu1 and the first overtone of the phenolic ring-deformation mode nu(16a), such that when the tyrosine phenoxyl proton is a strong hydrogen-bond donor, I2/I1 = 0.30, and when the tyrosine phenoxyl oxygen is a strong hydrogen-bond acceptor, I2/I1 = 2.5. Here, we interpret the Raman and infrared spectra of p-cresol vapor and extend the previous correlation to the non-hydrogen-bonded state of the tyrosine phenoxyl group. In the absence of hydrogen bonding, the Raman intensity of the higher-wavenumber component of the canonical Fermi doublet is greatly enhanced such that I2/I1 = 6.7. Thus, for the non-hydrogen-bonded phenoxyl, the lower-wavenumber member of the Fermi doublet loses most of its Raman intensity. This finding provides a basis for understanding the anomalous Raman singlet signature (approximately 854 cm(-1)) observed for tyrosine in coat protein subunits of filamentous viruses Ff and Pf1 [Overman, S. A., et al. (1994) Biochemistry 33, 1037-1042; Wen, Z. Q., et al. (1999) Biochemistry 38, 3148-3156]. The implications of the present results for Raman analysis of tyrosine hydrogen-bonding states in other proteins are considered.

Capsid↗
PubMed1994

The adsorption protein of filamentous phage fd: assignment of its disulfide bridges and identification of the domain incorporated in the coat.

The mature adsorption protein (g3p) of filamentous phage fd consists of 406 amino acids. It contains eight cysteine residues in total. To determine the disulfide bond pattern, purified g3p was proteolytically digested, and the resulting peptides were separated using RP-HPLC. N-terminal sequencing and mass spectrometry of cysteine-containing fragments showed that each cysteine is involved in an intramolecular disulfide bond. The cystine sites are Cys7-Cys36, Cys46-Cys53, Cys188-Cys201, and Cys354-Cys371. In the native conformation of g3p, none of the disulfide bridges is accessible to alkylating agents after treatment with DTT. The cystine sites seem therefore to be located in the interior of the folded molecule or are shielded by components of the phage envelope. The part of g3p which is incorporated in the phage coat and hence is inaccessible to proteolytic cleavage was identified after treatment of phage particles with subtilisin: Immunoblots performed with antibody directed against g3p revealed essentially one g3p fragment with an apparent molecular weight of approximately 17,000 which resisted the proteolytic attack. The amino terminus of this peptide was determined to be glycine 254. This amino acid position correlates with the carboxy-terminal end of the second glycine-rich region (amino acids 218-256) in the primary structure of g3p. The notion that the extended carboxy-terminal g3p fragment is indeed entirely buried within the phage envelope is further supported by the fact that only polyclonal antibodies raised against purified g3p are able to react with this peptide, whereas those against phage coat-associated g3p are not.

Alkylating Agents↗