Coat Protein Of Bacteriophage M13 Research Articles

Engineered M13-Derived Bacteriophages Capable of Gold Nanoparticle Synthesis and Nanogold Manipulations

For years, gold nanoparticles (AuNPs) have been widely used in medicine and industry. Although various experimental procedures have been reported for their preparation and manipulation, none of them is optimal for all purposes. In this work, we engineered the N-terminus of the pIII minor coat protein of bacteriophage (phage) M13 to expose a novel HLYLNTASTHLG peptide that effectively and specifically binds gold. In addition to binding gold, this engineered phage could synthesize spherical AuNPs of 20 nm and other sizes depending on the reaction conditions, aggregate them, and precipitate gold from a colloid, as revealed by transmission electron microscopy (TEM), atomic force microscopy (AFM), and scanning electron microscopy (SEM), as well as ultraviolet–visible (UV–vis) and Fourier-transform infrared (FTIR) spectroscopic methods. We demonstrated that the engineered phage exposing a foreign peptide selected from a phage-displayed library may serve as a sustainable molecular factory for both the synthesis of the peptide and the subsequent overnight preparation of AuNPs from gold ions at room temperature and neutral pH in the absence of strong reducing agents, such as commonly used NaBH4. Taken together, the results suggest the potential applicability of the engineered phage and the new, in vitro-identified gold-binding peptide in diverse biomimetic manipulations.

Polarity and Charge as Determinants for Translocase Requirement for Membrane Protein Insertion

Membrane protein biogenesis in bacteria is a well-studied but poorly understood area in the field of membrane proteins. In bacteria, the Sec translocon and the YidC insertase promote the membrane insertion process. Both proteins are essential, and universally conserved, and together insert about 95% of the proteins in E.coli. Membrane insertion of SecYEG-dependent proteins likely occurs at the lateral gate of SecY, the channel-forming subunit of the bacterial Sec translocon. On the other hand, YidC catalyzes insertion by possessing a hydrophilic groove, which can accommodate the hydrophilic region of the substrate that inserts across the membrane. Recently, Soman et al (2014) have reported that increasing the charge of the translocated region of procoat can route substrates from the YidC pathway into the Sec pathway. Here, we have further tested this polarity/charge hypothesis by showing that the major coat protein of bacteriophage M13 can become increasingly YidC/Sec dependent by making the periplasmic loop highly polar in the absence of charged residues. We also show that adding hydrophobic amino acids to highly polar or charged loop can decrease the Sec-dependence of the otherwise strictly Sec-dependent substrates. Additionally we demonstrate that increasing the driving force of insertion by adding four leucyl residues to the transmembrane segment leads to translocation of a highly charged region that was not inserted by native TM segment. Finally, we show that the length of procoat loop is a determinant for Sec dependent insertion. The combined results support the polarity/charge hypothesis and is consistent with the notion that membrane insertion occurs at the interface of SecYEG and YidC.

Asymmetric dipping of bacteriophage M13 coat protein with increasing lipid bilayer thickness

Knowledge about the vertical movement of a protein with respect to the lipid bilayer plane is important to understand protein functionality in the biological membrane. In this work, the vertical displacement of bacteriophage M13 major coat protein in a lipid bilayer is used as a model system to study the molecular details of its anchoring mechanism in a homologue series of lipids with the same polar head group but different hydrophobic chain length. The major coat proteins were reconstituted into 14:1PC, 16:1PC, 18:1PC, 20:1PC, and 22:1PC bilayers, and the fluorescence spectra were measured of the intrinsic tryptophan at position 26 and BADAN attached to an introduced cysteine at position 46, located at the opposite ends of the transmembrane helix. The fluorescence maximum of tryptophan shifted for 700 cm -1 on going from 14:1PC to 22:1PC, the corresponding shift of the fluorescence maximum of BADAN at position 46 was approximately 10 times less (∼ 70 cm -1). Quenching of fluorescence with the spin label CAT 1 indicates that the tryptophan is becoming progressively inaccessible for the quencher with increasing bilayer thickness, whereas quenching of BADAN attached to the T46C mutant remained approximately unchanged. This supports the idea that the BADAN probe at position 46 remains at the same depth in the bilayer irrespective of its thickness and clearly indicates an asymmetrical nature of the protein dipping in the lipid bilayer. The anchoring strength at the C-terminal domain of the protein (provided by two phenylalanine residues together with four lysine residues) was estimated to be roughly 5 times larger than the anchoring strength of the N-terminal domain.

The position of the Gly-xxx-Gly motif in transmembrane segments modulates dimer affinityThis paper is one of a selection of papers published in this Special Issue, entitled CSBMCB — Membrane Proteins in Health and Disease.

Although the intrinsic low solubility of membrane proteins presents challenges to their high-resolution structure determination, insight into the amino acid sequence features and forces that stabilize their folds has been provided through study of sequence-dependent helix-helix interactions between single transmembrane (TM) helices. While the stability of helix-helix partnerships mediated by the Gly-xxx-Gly (GG4) motif is known to be generally modulated by distal interfacial residues, it has not been established whether the position of this motif, with respect to the ends of a given TM segment, affects dimer affinity. Here we examine the relationship between motif position and affinity in the homodimers of 2 single-spanning membrane protein TM sequences: glycophorin A (GpA) and bacteriophage M13 coat protein (MCP). Using the TOXCAT assay for dimer affinity on a series of GpA and MCP TM segments that have been modified with either 4 Leu residues at each end or with 8 Leu residues at the N-terminal end, we show that in each protein, centrally located GG4 motifs are capable of stronger helix-helix interactions than those proximal to TM helix ends, even when surrounding interfacial residues are maintained. The relative importance of GG4 motifs in stabilizing helix-helix interactions therefore must be considered not only in its specific residue context but also in terms of the location of the interactive surface relative to the N and C termini of alpha-helical TM segments.

Rapid Preparation of Stable Isotope Labeled Peptides that Bind to Target Proteins by a Phage Library System

We have developed a system for directly isolating foreign peptides displayed on the N-terminus of the major coat protein of bacteriophage M13. The phage particle in this system is formed as a mixture of wild type and modified coat proteins. The N-terminal segment of the modified coat protein was mutated for chemical cleavage, in order to obtain the displayed peptide from the major coat protein. Using 13C, 15N- labeled medium, we introduced stable isotopes, 13C and/or 15N, into the coat proteins. The NMR spectra for the cleaved peptides from the phage particles could be recorded within a few days after the selection of the phage clone.

Fluorescence resonance energy transfer shows a close helix-helix distance in the transmembrane M13 procoat protein.

During the membrane insertion process the major coat protein of bacteriophage M13 assumes a conformation in which two transmembrane helices corresponding to the leader sequence and the anchor region in the mature part of the protein coming into close contact with each other. Previous studies on the molecular mechanism of membrane insertion of M13 procoat protein have shown that this interaction between the two helices might drive the actual translocation process. We investigated the intramolecular distance between the two helices of the transmembrane procoat protein by measuring fluorescence resonance energy transfer (FRET) between the donor (Tyr) placed in one helix and the acceptor (Trp) placed in the other helix. Various mutant procoat proteins with differently positioned donor-acceptor pairs were generated, purified, and reconstituted into artificial lipid bilayers. The results obtained from the FRET measurements, combined with molecular modeling, show that the transmembrane helices are in close contact on the order of 1-1.5 nm. The present approach might be of general interest for determining the topology and the folding of membrane proteins.

Conformation and orientation of the gene 9 minor coat protein of bacteriophage M13 in phospholipid bilayers

The membrane-bound state of the gene 9 minor coat protein of bacteriophage M13 was studied in model membrane systems, which varied in lipid head group and lipid acyl chain composition. By using FTIR spectroscopy and subsequent band analysis a quantitative analysis of the secondary structure of the protein was obtained. The secondary structure of the gene 9 protein predominantly consists of α-helical (67%) and turn (33%) structures. The turn structure is likely to be located C-terminally where it has a function in recognizing the phage DNA during bacteriophage assembly. Attenuated total reflection FTIR spectroscopy was used to determine the orientation of gene 9 protein in the membrane, revealing that the α-helical domain is mainly transmembrane. The conformational and orientational measurements result in two models for the gene 9 protein in the membrane: a single transmembrane helix model and a two-helix model consisting of a 15 amino acid long transmembrane helix and a 10 amino acid long helix oriented parallel to the membrane plane. Potential structural consequences for both models are discussed.

Spontaneous insertion of gene 9 minor coat protein of bacteriophage M13 in model membranes

Gene 9 minor coat protein from bacteriophage M13 is known to be located in the inner membrane after phage infection of Escherichia coli. The way of insertion of this small protein (32 amino acids) into membranes is still unknown. Here we show that the protein is able to insert in monolayers. The limiting surface pressure of 35 mN/m for 1,2-dioleoyl- sn-glycero-3-phosphocholine and 1,2-dioleoyl- sn-glycero-3-phosphoglycerol lipid systems indicates that this spontaneous insertion can also occur in vivo. By carboxyfluorescein leakage experiments of vesicles it is demonstrated that protein monomers, or at least small aggregates, are more effective in releasing carboxyfluorescein than highly aggregated protein. The final orientation of the protein in the bilayer after insertion was addressed by proteinase K digestion, thereby making use of the unique C-terminal location of the antigenic binding site. After insertion the C-terminus is still available for the enzymatic digestion, while the N-terminus is not. This leads to the overall conclusion that the protein is able to insert spontaneously into membranes without the need of any machinery or transmembrane gradient, with the positively charged C-terminus remaining on the outside. The orientation after insertion of gene 9 protein is in agreement with the ‘positive inside rule’.

Localization and rearrangement modulation of the N-terminal arm of the membrane-bound major coat protein of bacteriophage M13

During infection the major coat protein of the filamentous bacteriophage M13 is in the cytoplasmic membrane of the host Escherichia coli. This study focuses on the configurational properties of the N-terminal part of the coat protein in the membrane-bound state. For this purpose X-Cys substitutions are generated at coat protein positions 3, 7, 9, 10, 11, 12, 13, 14, 15, 17, 19, 21, 22, 23 and 24, covering the N-terminal protein part. All coat protein mutants used are successfully produced in mg quantities by overexpression in E. coli. Mutant coat proteins are labeled and reconstituted into mixed bilayers of phospholipids. Information about the polarity of the local environment around the labeled sites is deduced from the wavelength of maximum emission using AEDANS attached to the SH groups of the cysteines as a fluorescent probe. Additional information is obtained by determining the accessibility of the fluorescence quenchers acrylamide and 5-doxyl stearic acid. By employing uniform coat protein surroundings provided by TFE and SDS, local effects of the backbone of the coat proteins or polarity of the residues could be excluded. Our data suggest that at a lipid to protein ratio around 100, the N-terminal arm of the protein gradually enters the membrane from residue 3 towards residue 19. The hinge region (residues 17–24), connecting the helical parts of the coat protein, is found to be more embedded in the membrane. Substitution of one or more of the membrane-anchoring amino acid residues lysine 8, phenylalanine 11 and leucine 14, results in a rearrangement of the N-terminal protein part into a more extended conformation. The N-terminal arm can also be forced in this conformation by allowing less space per coat protein at the membrane surface by decreasing the lipid to protein ratio. The influence of the phospholipid headgroup composition on the rearrangement of the N-terminal part of the protein is found to be negligible within the range thought to be relevant in vivo. From our experiments we conclude that membrane-anchoring and space-limiting effects are key factors for the structural rearrangement of the N-terminal protein part of the coat protein in the membrane.

Role of aromatic residues at the lipid-water interface in micelle-bound bacteriophage M13 major coat protein.

Analyses of transmembrane domains of proteins have revealed that aromatic residues tend to cluster at or near the lipid-water interface of the membrane. To assess protein-membrane interactions of such residues, a viable mutant library was generated of the major coat protein of bacteriophage M13 (a model single membrane-spanning protein) in which one or the other of its interfacial tyrosine residues (Tyr-21 and Tyr-24) is mutated. Using the interfacial tryptophan (Trp-26) as an intrinsic probe, blue shifts in fluorescence emission spectra and quenching constants indicated that mutants with a polar amino acid substitution (such as Y24D or Y24N) are less buried in a deoxycholate micelle environment than in the wild type protein. These polar mutants also exhibited alpha-helix to beta-structure transition temperatures in incremental-heating circular dichroism studies relatively lower than those of wild type and nonpolar mutants (such as Y21V, Y21I, and Y24A), indicating that specific side chains in the lipid-water interface influence local protein-micelle interactions. Mutant Y21F exhibited the highest transition temperature, suggesting that phenylalanine is ostensibly the most effective interfacial anchoring residue. Using phage viability as the assay in a combination of site-directed and saturation mutagenesis experiments, it was further observed that both Tyr residues could not simultaneously be "knocked out". The overall results support the notion that an interfacial Tyr is a primary recognition element for precise strand positioning in vivo, a function that apparently cannot be performed optimally by residues with simple aliphatic character.

Conformational and aggregational properties of the gene 9 minor coat protein of bacteriophage M13 in membrane-mimicking systems.

The membrane-bound state of the gene 9 minor coat protein of bacteriophage M13 was studied in various membrane-mimicking systems, including organic solvents, detergent micelles, and phospholipid bilayers. For this purpose we determined the conformational and aggregational properties of the chemically synthesized protein by CD, FTIR, and HPSEC. The protein appears to be in a monomeric or small oligomeric alpha-helical state in TFE but adopts a mixture of alpha-helical and random structure after subsequent incorporation into SDS or DOPG. When solubilized by sodium cholate, however, the protein undergoes a transition in time into large aggregates, which contain mainly beta-sheet conformation. The rate of this beta-polymerization process was decreased at lower temperature and higher concentrations of sodium cholate. This aggregation was reversed only upon addition of high concentrations of the strong detergent SDS. By reconstitution of the cholate-solubilized protein into DOPG, it was found that the state of the protein, whether initially alpha-helical monomeric/oligomeric or beta-sheet aggregate, did not change. On the basis of our results, we propose that the principal conformational state of membrane-bound gene 9 protein in vivo is alpha-helical.

Solution structure of the M13 major coat protein in detergent micelles: a basis for a model of phage assembly involving specific residues

The three-dimensional structure of the major coat protein of bacteriophage M13, solubilized in detergent micelles, has been determined using heteronuclear multidimensional NMR and restrained molecular dynamics. The protein consists of two α-helices, running from residues 8 to 16 and 25 to 45, respectively. These two helices are connected by a flexible and distorted helical hinge region. The structural properties of the coat protein make it resemble a flail, in which the hydrophobic helix (residues 25 to 45) is the handle and the other, amphipathic, helix the swingle. In this metaphor, the hinge region is the connecting piece of leather. The mobility of the residues in the hinge region is likely to enable a smooth transformation from the membrane-bound form, mimicked by the structure in detergent micelles, into the structure in the mature phage. A specific distribution of the residues over the surface of the two helices was observed in the presented high-resolution structure of the membrane-bound form of the major coat protein as well as in the structure in the mature phage. All data suggest that this arrangement of residues is important for the interactions of the protein with the membrane, for correct protein-DNA and protein-protein interactions in the phage and for a proper growth of the phage during the assembly process. By combining our findings with earlier NMR results on the major coat protein in detergent micelles, we were able to construct a model that addresses the role of specific residues in the assembly process.

Conventional and saturation-transfer EPR of spin-labeled mutant bacteriophage M13 coat protein in phospholipid bilayers

A mutant of bacteriophage M13 was prepared in which a cysteine residue was introduced at position 25 of the major coat protein. The mutant coat protein was spin-labeled with a nitroxide derivative of maleimide and incorporated at different lipid-to-protein (L/P) ratios in DOPC or DOPG. The rotational dynamics of the reconstituted mutant coat protein was studied using EPR and saturation transfer (ST) EPR techniques. The spectra are indicative for an anisotropic motion of the maleimide spin label with a high order parameter ( S=0.94). This is interpreted as a wobbling motion of the spin label with a correlation time of about 10 −6 to 10 −5 s within a cone, and a rotation of the spin label about its long molecular axis with a correlation time of about 10 −7 s. The wobbling motion is found to correspond generally to the overall rotational motion of a coat protein monomer about the normal to the bilayer. This motion is found to be sensitive to the temperature and L/P ratio. The high value of the order parameter implies that the spin label experiences a strong squeezing effect by its local environment, that reduces the amplitude of the wobbling motion. This squeezing effect is suggested to arise from a turn structure in the coat protein from Gly23 to Glu20.

Backbone dynamics of the major coat protein of bacteriophage M13 in detergent micelles by 15N nuclear magnetic resonance relaxation measurements using the model-free approach and reduced spectral density mapping.

The backbone dynamics of the major coat protein (gVIIIp) of the filamentous bacteriophage M13, solubilized in detergent micelles, have been studied using 15N nuclear magnetic resonance spectroscopy at three frequencies. Motional parameters and overall and internal correlation times were derived with the model-free approach. It was also checked whether these parameters had to be modified due to anisotropic motion of the protein/micelle complex. Reduced spectral density mapping was used to calculate the spectral densities at J(O), J(omegaN), and [J(omegaH)]. The spectral densities were interpreted by mapping a linear or scaled linear combination of two Lorentzians onto a J(O)-J(omega) plot. The major coat protein of bacteriophage M13 consists of two alpha-helices, one of which is hydrophobic and located within the micelle, while the other is amphipathic and located on the surface of the micelle. Our results indicate that the motion of the hydrophobic helix is restricted such that it corresponds to the overall tumbling of the protein/micelle complex. The interpretation of the relaxation data of the amphipathic helix by means of the model-free approach and the reduced spectral density mapping indicate that in addition to the overall motion all residues in this helix are subject to motion on the fast nanosecond and picosecond time scales. The motions of the vectors in the low nanosecond range are characterized by similar values of the spectral densities and correlation times and represent the motion of the amphipathic helix on and away from the surface of the micelle. The relaxation data of the residues in the hinge region connecting the helices show that there is an abrupt change from highly restricted to less restricted motion. Both the C-terminal and N-terminal residues are very mobile.

Improving the copy numbers of antibody fragments expressed on the major coat protein of bacteriophage M13

Background: Antibody fragments have been expressed on the major coat protein of filamentous phage using a gene VIII expression system, but with low copy numbers (averaging 0.2 Fab/phage). Objectives: As a general strategy to increase copy number, the phage vector was optimized by site directed mutagenesis. Study Design: One mutation was the introduction of a random six amino acid tether between the heavy chain and pseudo gene VIII to form a phage library. The other mutation was the removal of two cysteine residues which form a disulfide bond between the heavy and light chains. An assay was developed to measure Fab concentrations and used to calculate the average number of copies displayed on phage. Results: Phage libraries containing random tethers were panned, and clones containing a proline rich motif were extracted. Removing the interchain disulfide had a greater effect on copy number and soluble Fab concentrations in the periplasmic space of the bacterial cultures. Conclusion: A tenfold increase in the copy number was achieved using the optimized vector. Incorporation of these vector mutations may be a general strategy for optimizing Fab display on the major coat protein of bacteriophage M13.

Accessibility and environment probing using cysteine residues introduced along the putative transmembrane domain of the major coat protein of bacteriophage M13.

The major coat protein of the filamentous bacteriophage M13 is located in the inner membrane of host cell Escherichia coli prior to assembly into virions. To identify the transmembrane domain of the coat protein, we have introduced unique cysteine residues along the putative transmembrane domain at position 25, 31, 33, 36, 38, 46, 47, 49, or 50. The mutant major coat protein was solubilized by membrane-mimicking detergents or reconstituted into mixed bilayers of phospholipids. Information about the environmental polarity was deduced from the wavelength of maximum emission, using N-[[(iodoacetyl)-amino)ethyl]-1-sulfonaphthylamine (IAEDANS) attached to the SH groups of the cysteines as a fluorescent probe. Additional information was obtained by determining the accessibility of AEDANS for the fluorescence quencher molecules acrylamide and 5-doxylstearic acid, and the reactivity of the cysteine's sulfhydryl group toward 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB). Our data suggest transmembrane boundaries close to residue 25 and 46, with residue 25 inside the hydrophobic part of the membrane in very close proximity to the membrane-water interface and residue 46 located at the membrane-water interface. Domains of the mutant coat protein which are packed or coated by cholate molecules and various other detergents [except for sodium dodecyl sulfate (SDS)] are at least similarly packed by phospholipid molecules in bilayers. SDS is a good solubilizing detergent but badly mimics the typical nature of a membrane structure. The overall results are interpreted with respect to the established conformation of the coat protein and its membrane anchoring mechanism.

NMR Studies of the Major Coat Protein of Bacteriophage M13. Structural Information of gVIIIp in Dodecylphosphocholine Micelles

The membrane-bound form of the major coat protein (gVIIIp) of bacteriophage M13 has been studied using nuclear magnetic resonance spectroscopy. As membrane mimetics, we used dodecylphosphocholine (DodPCho) detergent micelles to solubilize the protein. We were able to nearly completely assign all resonances of the protein solubilized in DodPCho micelles by using both homonuclear and heteronuclear multidimensional experiments. Based on the patterns of the nuclear Overhauser enhancements and the chemical shifts of the resonances, we deduced the secondary structure of the protein. Additional structural information was obtained from amide proton exchange data and J-coupling constants. The protein consists of two α-helices which are connected by a hinge region around residue 21. From spin-label experiments, the location of the protein relative to the DodPCho micelles was determined. One, hydrophobic, helix spans the micelle, and another, amphipathic, helix, is located beneath the surface of the micelle. Comparison of the data of gVIIIp in DodPCho micelles with those of gVIIIp in sodium dodecyl sulfate (SDS) micelles [Van de Ven, F. J. M., van Os, J. W. M., Aelen, J. M. A., Wymenga, S. S., Remerowski, M. L., Konings, R. N. H. & Hilbers, C. W. (1993) Biochemistry 32, 8322–8328; Papavoine, C. H. M., Konings, R. N. H., Hilbers, C. W. & Van de Ven, F. J. M. (1994) Biochemistry 33, 12990–12997] reveals that the structures of the protein in the two detergent micelles are very similar. They differ only in the arrangement of the detergent molecules around the protein. For gVIIIp in SDS micelles, we found a micellar structure which is distorted near the C-terminus of the protein; whereas for DodPCho micelles, both distorted and regular elliptical micelles occur. This distortion is probably due to the interaction of the positively charged lysine side chains with the negatively charged head group of the detergent molecules.

NMR Studies of the Major Coat Protein of Bacteriophage M13

The membrane-bound form of the major coat protein (gVIIIp) of bacteriophage M13 has been studied using nuclear magnetic resonance spectroscopy. As membrane mimetics, we used dodecylphosphocholine (DodPCho) detergent micelles to solubilize the protein. We were able to nearly completely assign all resonances of the protein solubilized in DodPCho micelles by using both homonuclear and heteronuclear multidimensional experiments. Based on the patterns of the nuclear Overhauser enhancements and the chemical shifts of the resonances, we deduced the secondary structure of the protein. Additional structural information was obtained from amide proton exchange data and J-coupling constants. The protein consists of two alpha-helices which are connected by a hinge region around residue 21. From spin-label experiments, the location of the protein relative to the DodPCho micelles was determined. One, hydrophobic, helix spans the micelle, and another, amphipathic, helix, is located beneath the surface of the micelle. Comparison of the data of gVIIIp in DodPCho micelles with those of gVIIIp in sodium dodecyl sulfate (SDS) micelles [Van de Ven, F. J. M., van Os, J. W. M., Aelen, J. M. A., Wymenga, S. S., Remerowski, M. L., Konings, R. N. H. & Hilbers, C. W. (1993) Biochemistry 32, 8322-8328; Papavoine, C. H. M., Konings, R. N. H., Hilbers, C. W. & Van de Ven, F. J. M. (1994) Biochemistry 33, 12,990-12,997] reveals that the structures of the protein in the two detergent micelles are very similar. They differ only in the arrangement of the detergent molecules around the protein. For gVIIIp in SDS micelles, we found a micellar structure which is distorted near the C-terminus of the protein; whereas for DodPCho micelles, both distorted and regular elliptical micelles occur. This distortion is probably due to the interaction of the positively charged lysine side chains with the negatively charged head group of the detergent molecules.

Location of M13 coat protein in sodium dodecyl sulfate micelles as determined by NMR.

The major coat protein (gVIIIp) of bacteriophage M13 solubilized in sodium dodecyl sulfate (SDS) detergent micelles was used as a model system to study this protein in the lipid-bound form. In order to probe the position of gVIIIp relative to the SDS micelles, stearate was added, spin-labeled at the 5- or 16-position with a doxyl group containing a stable nitroxide radical. The average position of the spin-labels in the micelles was derived from the line broadening of the resonances in the 13C spectrum of SDS. Subsequently, we derived a model of the relative position of gVIIIp in the SDS micelle from the effect of the spin-labels on the gVIIIp resonances, monitored via 1H-15N HSQC and TOCSY experiments. The results are consistent with the structure of gVIIIp having two helical strands. One strand is a long hydrophobic helix that spans the micelle, and the other is a shorter amphipathic helix on the surface of the micelle. These results are in good agreement with the structure of gVIIIp in membranes proposed by McDonnell et al. on the basis of solid state NMR data [McDonnell, P. A., Shon, K., Kim, Y., & Opella, S. J. (1993) J. Mol. Biol. 233, 447-463]. This study indicates that high-resolution NMR on this membrane protein, solubilized in detergent micelles, is a very suitable technique for mimicking these proteins in their natural environment. Furthermore, the data indicate that the structure of the micelle near the C-terminus of the major coat protein is distorted.(ABSTRACT TRUNCATED AT 250 WORDS)

Val-->Ala mutations selectively alter helix-helix packing in the transmembrane segment of phage M13 coat protein.

Val-->Ala mutations within the effective transmembrane segment of a model single-spanning membrane protein, the 50-residue major coat (gene VIII) protein of bacteriophage M13, are shown to have sequence-dependent impacts on stabilization of membrane-embedded helical dimeric structures. Randomized mutagenesis performed on the coat protein hydrophobic segment 21-39 (YIGYAWAMV-VVIVGATIGI) produced a library of viable mutants which included those in which each of the four valine residues was replaced by an alanine residue. Significant variations found among these Val-->Ala mutants in the relative populations and thermal stabilities of monomeric and dimeric helical species observed on SDS/PAGE, and in the range of their alpha-helix-->beta-sheet transition temperatures confirmed that intramembranous valine residues are not simply universal contributors to membrane anchoring. Additional analyses of (i) nonmutatable sites in the mutant protein library, (ii) the properties of the double mutant V29A-V31A obtained by recycling mutant V31A DNA through mutagenesis procedures, and (iii) energy-minimized helical dimer structures of wild-type and mutant V31A transmembrane regions indicated that the transmembrane hydrophobic core helix of the M13 coat protein can be partitioned into alternating pairs of potential protein-interactive residues (V30, V31; G34, A35; G38, I39) and membrane-interactive residues (M28, V29; I32, V33; T36, I37). The overall results consitute an experimental approach to categorizing the distinctive contributions to structure of the residues comprising a protein-protein packing interface vs. those facing lipid and confirm the sequence-dependent capacity of specific residues within the transmembrane domain to modulate protein-protein interactions which underlie regulatory events in membrane proteins.