Cor J. A. M. Wolfs

Protein-lipid interactions of bacteriophage M13 major coat protein

During the past years, remarkable progress has been made in our understanding of the replication cycle of bacteriophage M13 and the molecular details that enable phage proteins to navigate in the complex environment of the host cell. With new developments in molecular membrane biology in combination with spectroscopic techniques, we are now in a position to ask how phages carry out this delicate process on a molecular level, and what sort of protein-lipid and protein-protein interactions are involved. In this review we will focus on the molecular details of the protein-protein and protein-lipid interactions of the major coat protein (gp8) that may play a role during the infection of Escherichia coli by bacteriophage M13.

Viruses: incredible nanomachines. New advances with filamentous phages

During recent decades, bacteriophages have been at the cutting edge of new developments in molecular biology, biophysics, and, more recently, bionanotechnology. In particular filamentous viruses, for example bacteriophage M13, have a virion architecture that enables precision building of ordered and defect-free two and three-dimensional structures on a nanometre scale. This could not have been possible without detailed knowledge of coat protein structure and dynamics during the virus reproduction cycle. The results of the spectroscopic studies conducted in our group compellingly demonstrate a critical role of membrane embedment of the protein both during infectious entry of the virus into the host cell and during assembly of the new virion in the host membrane. The protein is effectively embedded in the membrane by a strong C-terminal interfacial anchor, which together with a simple tilt mechanism and a subtle structural adjustment of the extreme end of its N terminus provides favourable thermodynamical association of the protein in the lipid bilayer. This basic physicochemical rule cannot be violated and any new bionanotechnology that will emerge from bacteriophage M13 should take this into account.

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.

Structural characterization of bacteriophage M13 solubilization by amphiphiles

The structural properties of bacteriophage M13 during disassembly were studied in different membrane model systems, composed of a homologue series of the detergents sodium octyl sulfate, sodium decyl sulfate, and sodium dodecyl sulfate. The structural changes during phage disruption were monitored by spin-labeled electron spin resonance (ESR) and circular dichroism spectroscopy. For the purpose of ESR spectroscopy the major coat protein mutants V31C and G38C were site-directed spin labeled in the intact phage particle. These mutants were selected because the mutated sites are located in the hydrophobic part of the protein, and provide good reporting locations for phage integrity. All amphiphiles studied were capable of phage disruption. However, no significant phage disruption was detected below the critical micelle concentration of the amphiphile used. Based on this finding and the linear dependence of phage disruption by amphiphiles on the phage concentration, it is suggested that the solubilization of the proteins of the phage coat by amphiphiles starts with an attachment to and penetration of amphiphile molecules into the phage particle. The amphiphile concentration in the phage increases in proportion to the amphiphile concentration in the aqueous phase. Incorporation of the amphiphile in the phage particle is accompanied with a change in local mobility of the spin-labeled part of the coat protein and its secondary structure. With increasing the amphiphile concentration in the phage particle, a concentration is reached where the concentration of the amphiphile in the aqueous phase is around its critical micelle concentration. A further increase in amphiphile concentration results in massive phage disruption. Phage disruption by amphiphiles appears to be dependent on the phage coat mutations. It is concluded that phage disruption is dependent on a hydrophobic effect, since phage solubilization could significantly be increased by keeping the hydrophilic part of the amphiphile constant, while increasing its hydrophobic part.

Analysis of time-resolved fluorescence anisotropy in lipid-protein systems

Fluorescent probes located in heterogeneous environments give rise to anomalous time-resolved fluorescence anisotropy. A simple analytical expression of anisotropy has been derived for the case of a small difference in local fluorescence lifetimes. The expression has the diagnostic advantage that the time dependence of the fluorescence anisotropy can be predicted from the differences in fluorescence lifetimes and residual anisotropies of the probes located in different sites. Using this model, the local fluorescence anisotropy parameters and the relative contributions of the lipid probe octadecyl rhodamine B in a lipid environment and in the vicinity of bacteriophage M13 coat protein reconstituted in phospholipid bilayers, composed of 80% 1,2-dimyristoyl-sn-glycero-3-phosphocholine and 20% 1,2-dimyristoyl-sn-glycero-3-phosphoglycerol have been determined experimentally. At 40°C, the correlation times for bound and free probes are 2.3 and 3.0 ns, respectively, while the corresponding order parameters are 0.85 and 0.62, respectively.

Deuterium nuclear magnetic resonance investigation of bacteriophage M13 coat protein in dimyristoylphosphatidylcholine liposomes using palmitic acid as a probe

The effect of incorporation of various amounts of M13 bacteriophage coat protein on the bilayer order and acyl chain motion in dimyristoylphosphatidylcholine (DMPC) liposomes has been investigated using deuterium NMR of specifically deuterated palmitic acid as a bilayer probe, phosphorus NMR and additional spin-label electron spin resonance (ESR). The secondary structure of the M13 coat protein in these bilayers was determined from circular dichroism spectra. Phosphorus NMR spectra of the mixed liposomes are characteristic for DMPC organized in bilayers, also after incorporation of various levels of M13 protein. Circular dichroism spectra of the coat protein indicate that the protein conformation is predominantly a beta-structure (more than 75%). Various incorporation levels of M13 coat protein do not affect the order of the deuterium-labelled positions along the acyl chain at the carbon-2, 9 and 16 positions. In contrast, the spin-spin relaxation times decrease at higher protein levels, especially at the carbon-16 position. The spin-label ESR spectra of the same system using 14-doxylstearic acid as a label show a second, motionally restricted component, that is not observed by deuterium NMR. The NMR and ESR results are consistent with a model in which the fatty acid molecules are in a fast two-site exchange (at a rate of approx. 10(7) Hz) between the sites in the bulk of the lipid bilayer and the motionally restricted sites on the coat protein.

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 alpha-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 alpha-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.

Lipid-protein interactions involved in bacteriophage M13 infection, Protein-lipid interactions.

Publisher Summary This chapter describes the α-helical form of M13 major coat protein and its properties in relation to the in vivo biological processes. M13 bacteriophage and the closely related phages, fl and fd, are Escherichia coli -specific filamentous phages belonging to the genus, Inovirus. The chapter discusses the molecular properties of the membrane-bound bacteriophage disassembly and assembly processes by studying the structural and functional behavior of the major coat protein of bacteriophage M13 and related phages, when incorporated into model membrane systems. Various spectroscopic techniques have been applied that provide detailed information about these reconstituted systems, whereas biochemical separation techniques allow purifying and characterizing these systems. The chapter discusses the role of M13 coat protein in the infection process, leading to conditions to reconstitute the protein in its native state into model membrane systems. In addition, the structure of the coat protein and protein–lipid interactions is discussed in the chapter.

Exploring the Structure of the 100 Amino-Acid Residue Long N-Terminus of the Plant Antenna Protein CP29

The structure of the unusually long (∼100 amino-acid residues) N-terminal domain of the light-harvesting protein CP29 of plants is not defined in the crystal structure of this membrane protein. We studied the N-terminus using two electron paramagnetic resonance (EPR) approaches: the rotational diffusion of spin labels at 55 residues with continuous-wave EPR, and three sets of distances with a pulsed EPR method. The N-terminus is relatively structured. Five regions that differ considerably in their dynamics are identified. Two regions have low rotational diffusion, one of which shows α-helical character suggesting contact with the protein surface. This immobile part is flanked by two highly dynamic, unstructured regions (loops) that cover residues 10-22 and 82-91. These loops may be important for the interaction with other light-harvesting proteins. The region around residue 4 also has low rotational diffusion, presumably because it attaches noncovalently to the protein. This section is close to a phosphorylation site (Thr-6) in related proteins, such as those encoded by the Lhcb4.2 gene. Phosphorylation might influence the interaction with other antenna complexes, thereby regulating the supramolecular organization in the thylakoid membrane.

Site-Directed Spin-Labeling Study of the Light-Harvesting Complex CP29

The topology of the long N-terminal domain (approximately 100 amino-acid residues) of the photosynthetic Lhc CP29 was studied using electron spin resonance. Wild-type protein containing a single cysteine at position 108 and nine single-cysteine mutants were produced, allowing to label different parts of the domain with a nitroxide spin label. In all cases, the apoproteins were either solubilized in detergent or they were reconstituted with their native pigments (holoproteins) in vitro. The spin-label electron spin resonance spectra were analyzed in terms of a multicomponent spectral simulation approach, based on hybrid evolutionary optimization and solution condensation. These results permit to trace the structural organization of the long N-terminal domain of CP29. Amino-acid residues 97 and 108 are located in the transmembrane pigment-containing protein body of the protein. Positions 65, 81, and 90 are located in a flexible loop that is proposed to extend out of the protein from the stromal surface. This loop also contains a phosphorylation site at Thr81, suggesting that the flexibility of this loop might play a role in the regulatory mechanisms of the light-harvesting process. Positions 4, 33, 40, and 56 are found to be located in a relatively rigid environment, close to the transmembrane protein body. On the other hand, position 15 is located in a flexible region, relatively far away from the transmembrane domain.