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.     

Is there a role for oligosaccharides in seed longevity? An assessment of intracellular glass stability

We examined whether oligosaccharides extend seed longevity by increasing the intracellular glass stability. For that purpose, we used a spin probe technique to measure the molecular mobility and glass transition temperature of the cytoplasm of impatiens (Impatiens walleriana) and bell pepper (Capsicum annuum) seeds that were osmo-primed to change oligosaccharide content and longevity. Using saturation transfer electron paramagnetic resonance spectroscopy, we found that the rotational correlation time of the polar spin probe 3-carboxy-proxyl in the cytoplasm decreased, together with longevity, as a function of increasing seed water content, suggesting that longevity may indeed be regulated by cytoplasmic mobility. Osmo-priming of the seeds resulted in considerable decreases in longevity and oligosaccharide content, while the sucrose content increased. No difference in the glass transition temperature was found between control and primed impatiens seeds at the same temperature and water content. Similarly, there was no difference in the rotational motion of the spin probe in the cytoplasm between control and primed impatiens and bell pepper seeds. We therefore conclude that oligosaccharides in seeds do not affect the stability of the intracellular glassy state, and that the reduced longevity after priming is not the result of increased molecular mobility in the cytoplasm.     

Membrane-bound conformation of M13 major coat protein: a structure validation through FRET-derived constraints

M13 major coat protein, a 50-amino-acid-long protein, was incorporated into DOPC/DOPG (80/20 molar ratio) unilamellar vesicles. Over 60% of all amino acid residues was replaced with cysteine residues, and the single cysteine mutants were labeled with the fluorescent label I-AEDANS. The coat protein has a single tryptophan residue that is used as a donor in fluorescence (or F?rster) resonance energy transfer (FRET) experiments, using AEDANS-labeled cysteines as acceptors. Based on FRET-derived constraints, a straight alpha-helix is proposed as the membrane-bound conformation of the coat protein. Different models were tested to represent the molecular conformations of the donor and acceptor moieties. The best model was used to make a quantitative comparison of the FRET data to the structures of M13 coat protein and related coat proteins in the Protein Data Bank. This shows that the membrane-bound conformation of the coat protein is similar to the structure of the coat protein in the bacteriophage that was obtained from x-ray diffraction. Coat protein embedded in stacked, oriented bilayers and in micelles turns out to be strongly affected by the environmental stress of these membrane-mimicking environments. Our findings emphasize the need to study membrane proteins in a suitable environment, such as in fully hydrated unilamellar vesicles. Although larger proteins than M13 major coat protein may be able to handle environmental stress in a different way, any membrane protein with water exposed parts in the C or N termini and hydrophilic loop regions should be treated with care.     

Quantification of Protein-Lipid Selectivity using FRET: Application to the M13 Major Coat Protein

Quantification of lipid selectivity by membrane proteins has been previously addressed mainly from electron spin resonance studies. We present here a new methodology for quantification of protein-lipid selectivity based on fluorescence resonance energy transfer. A mutant of M13 major coat protein was labeled with 7-diethylamino-3((4'iodoacetyl)amino)phenyl-4-methylcoumarin to be used as the donor in energy transfer studies. Phospholipids labeled with N-(7-nitro-2-1,3-benzoxadiazol-4-yl) were selected as the acceptors. The dependence of protein-lipid selectivity on both hydrophobic mismatch and headgroup family was determined. M13 major coat protein exhibited larger selectivity toward phospholipids which allow for a better hydrophobic matching. Increased selectivity was also observed for anionic phospholipids and the relative association constants agreed with the ones already presented in the literature and obtained through electron spin resonance studies. This result led us to conclude that fluorescence resonance energy transfer is a promising methodology in protein-lipid selectivity studies.     

Membrane assembly of M13 major coat protein: evidence for a structural adaptation in the hinge region and a tilted transmembrane domain

New insights into the low-resolution structure of the hinge region and the transmembrane domain of the membrane-bound major coat protein of the bacteriophage M13 are deduced from a single cysteine-scanning approach using fluorescence spectroscopy. New mutant coat proteins are labeled and reconstituted into phospholipid bilayers with varying headgroup compositions (PC, PE, and PG) and thicknesses (14:1PC, 18:1PC, and 22:1PC). 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. It is found that the protein is almost entirely embedded in the membrane, whereas the phospholipid headgroup composition of the membrane hardly affects the overall embedment of the protein in the membrane. From the assessment of a hydrophobic and hydrophilic face of the transmembrane helix, it is concluded that the helix is tilted with respect to the membrane normal. As compared to the thicker 18:1PC and 22:1PC membranes, reconstitution of the protein in the thin 14:1PC membranes results in a loss of helical structure and in the formation of a stretched conformation of the hinge region. It is suggested that the hinge region acts as a flexible spring between the N-terminal amphipathic arm and transmembrane hydrophobic helix. On average, the membrane-bound state of the coat protein can be seen as a gently curved and tilted, "banana-shaped" molecule, which is strongly anchored in the membrane-water interface at the C-terminus. From our experiments, we propose a rather small conformational adaptation of the major coat protein as the most likely reversible mechanism for responding to environmental changes during the bacteriophage disassembly and assembly process.     

2D exchange 31P NMR spectroscopy of bacteriophage M13 and tobacco mosaic virus.

Two-dimensional (2D) exchange 31P nuclear magnetic resonance spectroscopy is used to study the slow overall motion of the rod-shaped viruses M13 and tobacco mosaic virus in concentrated gels. Even for short mixing times, observed diagonal spectra differ remarkably from projection spectra and one-dimensional spectra. Our model readily explains this to be a consequence of the T2e anisotropy caused by slow overall rotation of the viruses about their length axis. 2D exchange spectra recorded for 30% (w/w) tobacco mosaic virus with mixing times < 1 s do not show any off-diagonal broadening, indicating that its overall motion occurs in the sub-Hz frequency range. In contrast, the exchange spectra obtained for 30% M13 show significant off-diagonal intensity for mixing times of 0.01 s and higher. A log-gaussian distribution around 25 Hz of overall diffusion coefficients mainly spread between 1 and 10(3) Hz faithfully reproduces the 2D exchange spectra of 30% M13 recorded at various mixing times in a consistent way. A small but notable change in diagonal spectra at increasing mixing time is not well accounted for by our model and is probably caused by 31P spin diffusion.     

Analysis of 31P MAS NMR spectra and transversal relaxation of bacteriophage M13 and tobacco mosaic virus.

Phosphorus magic angle spinning nuclear magnetic resonance (NMR) spectra and transversal relaxation of M13 and TMV are analyzed by use of a model, which includes both local backbone motions of the encapsulated nucleic acid molecules and overall rotational diffusion of the rod-shaped virions about their length axis. Backbone motions influence the sideband intensities by causing a fast restricted reorientation of the phosphodiesters. To evaluate their influence on the observed sideband patterns, we extend the model that we used previously to analyze nonspinning 31P NMR lineshapes (Magusin, P.C.M.M., and M. A. Hemminga. 1993a. Biophys. J. 64:1861-1868) to magic angle spinning NMR experiments. Backbone motions also influence the conformation of the phosphodiesters, causing conformational averaging of the isotropic chemical shift, which offers a possible explanation for the various linewidths of the centerband and the sidebands observed for M13 gels under various conditions. The change of the experimental lineshape of M13 as a function of temperature and hydration is interpreted in terms of fast restricted fluctuation of the dihedral angles between the POC and the OCH planes on both sides of the 31P nucleus in the nucleic acid backbone. Backbone motions also seem to be the main cause of transversal relaxation measured at spinning rates of 4 kHz or higher. At spinning rates less than 2 kHz, transversal relaxation is significantly faster. This effect is assigned to slow, overall rotation of the rod-shaped M13 phage about its length axis. Equations are derived to simulate the observed dependence of T2e on the spinning rate.     

Conformation of a pentacosapeptide representing the RNA-binding N-terminus of cowpea chlorotic mottle virus coat protein in the presence of oligophosphates: a two-dimensional proton nuclear magnetic resonance and distance geometry study.

Conformational studies were performed on a synthetic pentacosapeptide representing the RNA-binding N-terminal region of the coat protein of cowpea chlorotic mottle virus. Two-dimensional proton NMR experiments were performed on the highly positively charged peptide containing six arginines and three lysines in the presence of an excess of monophosphates, tetra(poly)phosphates, or octadeca(poly)phosphates mimicking the phosphates of the RNA. The results show that the peptide alternates between various extended and helical structures in the presence of monophosphate and that this equilibrium shifts toward the helical structures (with the helical region situated between residues 10 and 20) in the presence of oligophosphates. Distance geometry calculations using distance constraints derived from a NOESY spectrum of the peptide in the presence of tetra(poly)phosphate resulted in eight structures belonging to two structure families. The first family consists of five structures with an alpha-helixlike conformation in the middle of the peptide, and the second family consists of three structures with a more open conformation. The propensity to form an alpha-helical conformation in the N-terminal part of this viral coat protein upon binding of phosphate groups to the positively charged side chains is suggested to play an essential role in RNA binding.