Analysis of 31P nuclear magnetic resonance lineshapes and transversal relaxation of bacteriophage M13 and tobacco mosaic virus.

The experimentally observed 31P lineshapes and transversal relaxation of 15% (wt/wt) M13, 30% M13, and 30% tobacco mosaic virus (TMV) are compared with lineshapes and relaxation curves that are simulated for various types of rotational diffusion using the models discussed previously (Magusin, P. C. M. M., and M. A. Hemminga. 1993. Biophys. J. 64:1851-1860). It is found that isotropic diffusion cannot explain the observed lineshape effects. A rigid rod diffusion model is only successful in describing the experimental data obtained for 15% M13. For 30% M13 the experimental lineshape and relaxation curve cannot be interpreted consistently and the TMV lineshape cannot even be simulated alone, indicating that the rigid rod diffusion model does not generally apply. A combined diffusion model with fast isolated motions of the encapsulated nucleic acid dominating the lineshape and a slow overall rotation of the virion as a whole, which mainly is reflected in the transversal relaxation, is able to provide a consistent picture for the 15 and 30% M13 samples, but not for TMV. Strongly improved lineshape fits for TMV are obtained assuming that there are three binding sites with different mobilities. The presence of three binding sites is consistent with previous models of TMV. The best lineshapes are simulated for a combination of one mobile and two static sites. Although less markedly, the assumption that two fractions of DNA with different mobilities exist within M13 also improves the simulated lineshapes. The possible existence of two 31P fractions in M13 sheds new light on the nonintegral ratio 2.4:1 between the number of nucleotides and protein coat subunits in the phage: 83% of the viral DNA is less mobile, suggesting that the binding of the DNA molecule to the protein coat actually occurs at the integral ratio of two nucleotides per protein subunit.     

The in situ aggregational and conformational state of the major coat protein of bacteriophage M13 in phospholipid bilayers mimicking the inner membrane of host Escherichia coli

The major coat protein of bacteriophage M13 has been reconstituted into phospholipids with a composition comparable to that found in the host (Escherichia coli) inner membrane. Reconstitution experiments have revealed conditions in which the alpha-oligomeric state is favored over the beta-polymeric state. Discrimination between the two states of the membrane-bound coat protein (alpha-oligomeric and beta-polymeric states) has been achieved using high-performance size-exclusion chromatography and circular dichroism. Interprotein electrostatic interactions, probably induced by head-tail binding, are initiated and facilitating the aggregation-related conformational change process, in which alpha-oligomeric coat protein is converted into beta-polymeric coat protein. A model for this beta-polymerization process of the coat protein is presented. The alpha-helical protein has been studied by the in situ Trp fluorescence quantum yield. This shows that the average distances between coat proteins decrease upon lowering the L/P ratio. In situ cross-linking reactions of the coat protein at high L/P ratios reveal a monomeric state, thus excluding specific aggregation of the coat protein. A monomeric state of detergent-solubilized coat protein is also observed using SDS-PAGE and SDS-HPSEC. On the basis of these results, the smallest in situ aggregational entity of the coat protein is proposed to be a monomer. This finding is discussed in relation to the functional state of the M13 coat protein in the membrane-bound assembly and disassembly processes during infection.     

Conformational studies on a peptide fragment representing the RNA-binding N-terminus of a viral coat protein using circular dichroism and NMR spectroscopy

Conformational studies were performed on a synthetic pentacosapeptide representing the RNA-binding N-terminal region of the coat protein of cowpea chlorotic mottle virus. The effects of ionic strength, addition of (oligo)phosphates and temperature on the conformation of this highly positively charged peptide containing six arginines and three lysines were studied. CD experiments show that the peptide has 15-18% alpha-helical conformation and about 80% random-coil conformation in the absence of inorganic salt at 25 degrees C, and 20-21% alpha-helical conformation under the same conditions at 10 degrees C. Addition of inorganic salts results in an increase of alpha-helix content, up to 42% in the presence of oligophosphate with an average chain length of 18 phosphates, which was used as an RNA analog. NMR experiments show that the alpha-helix formation starts in the region between Thr9 and Gln12, and is extended in the direction of the C terminus. Relaxation measurements show that binding to oligophosphates of increasing length results in reduced internal mobilities of the positively charged side chains of the arginyl and lysyl residues and of the side chain of Thr9 in the alpha-helical region. The alpha-helix formation 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.     

The performance of the leaf mining microlepidopteran Bucculatrix maritima (Stt.) on the salt marsh halophyte,Aster tripolium (L.), exposed to different salinity conditions

Summary The performance of phytophagous insects is influenced by the nutritional quality of the food plant, which may vary with environmental conditions. Hardly any information exists on food-plant mediated effects of variable soil salinity on the performance of phytophagous insects. Conspicuous differences in salinity levels, however, are found in soils of intertidal wetlands such as salt marshes and mangroves. The growth of larvae of Bucculatrix maritima, a leaf miner of the salt marsh halophyte Aster tripolium, was studied on the host plant along the salinity gradient of the Westerschelde estuary (S.W. Netherlands). In addition, its performance on A. tripolium grown on low or high salinity culture medium was investigated experimentally. Although salinity conditions significantly influenced the chemistry of the host plants, insect performance seemed almost unaffected, although near the mouth of the estuary high environmental salinities may have caused some inhibition of larval growth. The results contrast with our previous studies on the stem-borer Agapanthia villosoviridescens, which showed that growth and development was conspicuously influenced by the changing characteristics of Aster tripolium along the estuarine salinity gradient. The location-dependent qualities of halophytes in an estuary thus appear to have species-specific effects on insect performance. We hypothesize that this phenomenon contributes to the existence of non-identical distribution patterns of phytophagous insects associated with the same halophyte in an estuary.     

Decomposition in salt marsh ecosystems of the S.W. Netherlands: the effects of biotic and abiotic factors

Decomposition rates, determined with the litterbag technique in salt marshes of the S.W. Netherlands during the past decade are compared; the biotic and abiotic factors influencing these rates are identified and discussed.Tissue composition is the main variable affecting decay rates of halophytes, particularly variations in lignin content between plant parts and between species.Experiments in which the loss of the tensile strength of cotton strips was used as an index of cellulolytic decay, show that there is a conspicuous variation in decay rates on different sites in a salt marsh. Nonetheless, the locally varying environmental conditions within salt marshes of the S.W. Netherlands have less impact on the variation in decomposition rates of halophyte litter than the chemical make-up of the plant material.Larger fauna elements (> 300 m) may increase decomposition rates, but this effect is only limited and depends on location and litter type. The role of small fauna elements such as nematodes, which occur abundantly in association with halophyte litter, remains largely unknown.     

Magic angle spinning carbon-13 NMR of tobacco mosaic virus. An application of the high-resolution solid-state NMR spectroscopy to very large biological systems.

Magic angle spinning 13C NMR was used to study tobacco mosaic virus (TMV) in solution. Well-resolved 13C NMR spectra were obtained, in which several carbon resonances of amino acids of the TMV coat protein subunits that are not observable by conventional high-resolution NMR spectroscopy can be designed. RNA resonance were absent, however, in the magic angle spinning 13C NMR spectra. Since three different binding sites are available for each nucleotide of the RNA, this is probably due to a line broadening caused by distributions of isotropic chemical shift values. In 13C-enriched TM 13C-13C dipolar interactions also gave rise to line broadening. By suitable pulse techniques that discriminate carbon resonances on the basis of their T1 and T1 rho values, it was possible to select particular groups of carbon nuclei with characteristic motional properties. Magic angle spinning 13C NMR spectra obtained with these pulse techniques are extremely well resolved.     

FRET study of membrane proteins: determination of the tilt and orientation of the N-terminal domain of M13 major coat protein

A formalism for membrane protein structure determination was developed. This method is based on steady-state FRET data and information about the position of the fluorescence maxima on site-directed fluorescent labeled proteins in combination with global data analysis utilizing simulation-based fitting. The methodology was applied to determine the structural properties of the N-terminal domain of the major coat protein from bacteriophage M13 reconstituted into unilamellar DOPC/DOPG (4:1 mol/mol) vesicles. For our purpose, the cysteine mutants A7C, A9C, N12C, S13C, Q15C, A16C, S17C, and A18C in the N-terminal domain of this protein were produced and specifically labeled with the fluorescence probe AEDANS. The energy transfer data from the natural Trp-26 to AEDANS were analyzed assuming a two-helix protein model. Furthermore, the polarity Stokes shift of the AEDANS fluorescence maxima is taken into account. As a result the orientation and tilt of the N-terminal protein domain with respect to the bilayer interface were obtained, showing for the first time, to our knowledge, an overall alpha-helical protein conformation from amino acid residues 12-46, close to the protein conformation in the intact phage.     

The effects of moisture and temperature on the ageing kinetics of pollen: interpretation based on cytoplasmic mobility

This study shows that characterization of the molecular mobility in the cytoplasm of pollen provides a new understanding of the effects of moisture and temperature on ageing rates. Using EPR spectroscopy, we determined the rotational motion of the polar spin probe, 3-carboxy-proxyl, in the cytoplasm of Typha latifolia pollen, under different temperature and moisture content conditions. Increasing the temperature resulted in faster rotational motion, analogous to faster ageing rates. With decreasing moisture content, rotational motion first decreased until a minimum was reached, after which rotational motion slightly increased again. The moisture content at which this minimal rotational motion was observed increased with decreasing temperature, comparable to the pattern of ageing rate. A significant linear relationship was found between ageing rates and rotational motion in the cytoplasm, suggesting that these parameters are causally linked. Upon melting of the intracellular glass, a twofold increase in activation energy of rotational motion and ageing rate was observed. In contrast, melting of the sucrose glass resulted in an increase in rotational motion of five orders of magnitude. The difference in rotational motion upon melting glasses of pollen or sucrose suggests that other molecules beside sugars play a role in intracellular glass formation in pollen.     

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.