In vivo deuteration strategies for neutron scattering analysis of bacterial polyhydroxyoctanoate

The cultivation of microorganisms on deuterated substrates has allowed us to control deuterium incorporation into biopolymer systems which is important for characterisation using neutron scattering techniques. Bacterial polyhydroxyoctanoate (PHO) is a polyester formed within inclusions inside bacterial cells and was deuterated in vivo under various conditions to characterise the formation of these inclusions by neutron scattering. Manipulation of deuterated media during microbial growth and PHO production phases resulted in polymer with partial or complete substitution of hydrogen by deuterium, as shown by gas chromatography. Sequential feeding of hydrogenated and deuterated forms of the same precursor was used to demonstrate that neutron scattering analysis could be used to differentiate between chemically similar phases in these polymer inclusions.     

The location of retinal in the purple membrane profile by neutron diffraction.

The trans-membrane location of retinal in the purple membrane of Halobacterium halobium, has been determined by low-angle neutron scattering studies on aqueous dispersions of the membranes. The membrane was bleached and regenerated with deuterated and with hydrogen-containing retinal. The modified retinal was obtained by extraction from bacteria grown in a totally deuterated medium. The determination of the retinal position is based on the differences in neutron scattering between a purple membrane sample with normal, protonated retinal and another sample with deuterated retinal. A distinct scattering density difference between the two preparations was observed. A direct structure determination was used with the retinal localized from a Fourier difference density profile. We conclude that the β-ionone ring portion of the retinal is situated centrally in the membrane.     

NMR crystallography: the effect of deuteration on high resolution 13C solid state NMR spectra of a 7-TM protein

The effect of deuteration on the 13C linewidths of U-13C, 15N 2D crystalline bacteriorhodopsin (bR) from Halobacterium salinarium, a 248-amino acid protein with seven-transmembrane (7TM) spanning regions, has been studied in purple membranes as a prelude to potential structural studies. Spectral doubling of resonances was observed for receptor expressed in 2H medium (for both 50:50% 1H:2H, and a more highly deuterated form) with the resonances being of similar intensities and separated by <0.3 ppm in the methyl spectral regions in which they were readily distinguished. Line-widths of the methyl side chains were not significantly altered when the protein was expressed in highly deuterated medium compared to growth in fully protonated medium (spectral line widths were about 0.5 ppm on average for receptor expressed both in the fully protonated and highly deuterated media from the C delta, C gamma 1, and C gamma 2 Ile 13C signals observed in the direct, 21-39 ppm, and indirect, 9-17 ppm, dimensions). The measured 13C NMR line-widths observed for both protonated and deuterated form of the receptor are sufficiently narrow, indicating that this crystalline protein morphology is suitable for structural studies. 1) decoupling comparison of the protonated and deuterated bR imply that deuteration may be advantageous for samples in which low power 1H decoupling is required.     

Selective deuteration of tryptophan and methionine residues in maltose binding protein: a model system for neutron scattering

We describe methods that have been developed within the ILL-EMBL Deuteration Laboratory for the production of maltose binding protein (MBP) that has been selectively labelled either with deuterated tryptophan or deuterated methionine (single labelling), or both (double labelling). MBP is used as an important model system for biophysical studies, and selective labelling can be helpful in the analysis of small-angle neutron scattering (SANS) data, neutron reflection (NR) data, and high-resolution neutron diffraction data. The selective labelling was carried out in E. coli high-cell density cultures using auxotrophic mutants in minimal medium containing the required deuterated precursors. Five types of sample were prepared and studied: (1) unmodified hydrogenated MBP (H-MBP), (2) perdeuterated MBP (D-MBP), (3) singly labelled MBP with the tryptophan residues deuterated (D-trp MBP), (4) singly labelled MBP with methionine residues deuterated (D-met MBP) and (5) doubly labelled MBP with both tryptophan and methionine residues deuterated (D-trp/met MBP). Labelled samples were characterised by size exclusion chromatography, gel electrophoresis, light scattering and mass spectroscopy. Preliminary small-angle neutron scattering (SANS) experiments have also been carried out and show measurable differences between the SANS data recorded for the various labelled analogues. More detailed SANS experiments using these labelled MBP analogues are planned; the degree to which such data could enhance structure determination by SANS is discussed.     

Accumulation of a Polyhydroxyalkanoate Containing Primarily 3-Hydroxydecanoate from Simple Carbohydrate Substrates by Pseudomonas sp. Strain NCIMB 40135

A number of Pseudomonas species have been identified which accumulate a polyhydroxyalkanoate containing mainly 3-hydroxydecanoate monomers from sodium gluconate as the sole carbon source. One of these, Pseudomonas sp. strain NCIMB 40135, was further investigated and shown to accumulate such a polyhydroxyalkanoate from a wide range of carbon sources (C₂ to C₆); however, when supplied with octanoic acid it produced a polyhydroxyalkanoate containing mainly 3-hydroxyoctanoate monomers. Polymer synthesis occurred in batch culture after cessation of growth due to exhaustion of nitrogen. In continuous culture under nitrogen limitation up to 16.9% (wt/wt) polyhydroxyalkanoate was synthesized from glucose as the carbon source. The monomer units are mainly of the R-(−) configuration. Nuclear magnetic resonance studies confirmed the composition of the polymer. Differential scanning calorimetry suggested that the solvent-extracted polymer contained a significant proportion of crystalline material. The weight-average molecular weight of the polymer from glucose-grown cells was 143,000.     

NMR crystallography: The effect of deuteration on high resolution C solid state NMR spectra of a 7-TM protein

The effect of deuteration on the ¹³C linewidths of U-¹³C, ¹⁵N 2D crystalline bacteriorhodopsin (bR) from Halobacterium salinarium, a 248-amino acid protein with seven-transmembrane (7TM) spanning regions, has been studied in purple membranes as a prelude to potential structural studies. Spectral doubling of resonances was observed for receptor expressed in ²H medium (for both 50:50% 1H:2H, and a more highly deuterated form) with the resonances being of similar intensities and separated by < 0.3 ppm in the methyl spectral regions in which they were readily distinguished. Line-widths of the methyl side chains were not significantly altered when the protein was expressed in highly deuterated medium compared to growth in fully protonated medium (spectral line widths were about 0.5 ppm on average for receptor expressed both in the fully protonated and highly deuterated media from the Cδ, Cγ₁, and Cγ₂ Ile ¹³C signals observed in the direct, 21-39 ppm, and indirect, 9-17 ppm, dimensions). The measured ¹³C NMR line-widths observed for both protonated and deuterated form of the receptor are sufficiently narrow, indicating that this crystalline protein morphology is suitable for structural studies. ¹H decoupling comparison of the protonated and deuterated bR imply that deuteration may be advantageous for samples in which low power ¹H decoupling is required.     

Inter-protein distances within the large subunit from Escherichia coli ribosomes.

Selected pairs of protonated ribosomal proteins were reconstituted into deuterated 50S subunits from Escherichia coli ribosomes. The rRNA of the deuterated ribosomal matrix was derived from cells grown in 76% D2O, the deuterated protein moiety from cells grown in 84% D2O. This procedure warrants that the coherent neutron scattering of deuterated proteins and rRNA is nearly the same and equals that of a D2O solution of approximately 90%. The neutron scattering is recorded in a reconstitution buffer containing approximately 90% D2O. The result is a significant improvement of the coherent signal:noise ratio over traditional methods; due to this dilute solutions can be used, thus preventing unfavorable inter-particle effects. From the diffraction pattern the distance between the mass centers of gravity of the two protonated proteins can be deduced. In this way, 50 distances between proteins within the large subunit have been determined which provide a basis for future models of the large ribosomal subunit describing the spatial distribution of the ribosomal proteins. A model containing seven ribosomal proteins is presented.     

Structural dynamics of translating ribosomes.

We describe three groups of small angle neutron scattering (SANS) experiments with translating ribosomes: 1) regular protonated (normal abundance hydrogen) particles; 2) two isotopic hybrid particles which are reconstituted from one protonated and the other deuterated subunit; 3) four isotypic hybrid particles differing from each other by the extent of protein and RNA deuteration. Using the SANS contrast variation method the radii of gyration of protein and RNA components in both ribosomal subunits as well as the intersubunit distance in the pre- and post-translocation states were determined. The results obtained suggest the following model of the ribosome as a dynamic machine. The ribosome oscillates between two major conformers differing in geometrical dimensions. The 'active' (pulsating) part of the ribosome is the 30S subunit. We believe that the movement of its 'head' relative to the passive 50S subunit is the main mechanical act of translocation. The radius of gyration of the 30S subunit and the intersubunit distance change upon the movement. This is corroborated by neutron scattering data.     

Low-resolution structure of the tetrameric phenylalanyl-tRNA synthetase from Escherichia coli. A neutron small-angle scattering study of hybrids composed of protonated and deuterated protomers

Escherichia coli phenylalanyl-tRNA synthetase is a tetrameric protein composed of two types of protomers. In order to resolve the subunit organization, neutron small-angle scattering experiments have been performed in different contrasts with all types of isotope hybrids that could be obtained by reconstituting the alpha 2 beta 2 enzyme from the protonated and deuterated forms of the alpha and beta subunits. Experiments have been also made with the isolated alpha promoter. A model for the alpha 2 beta 2 tetramer is deduced where the two alpha promoters are elongated ellipsoids (45 x 45 x 160 A3) lying side by side with an angle of about 40 degrees between their long axes and where the two beta subunits are also elongated ellipsoids (31 x 31 x 130 A3) with an angle of 30 degrees between their axes. This model was obtained by assuming that the two pairs of subunits are in contact in an orthogonal manner and by taking advantage of the measured distance between the centers of mass of the alpha 2 and beta 2 pairs (d = 23 +/- 2 A).     

Neutron small angle scattering of matched proteoliposomes with incorporated F0F1 ATPase complex from Rhodospirillum rubrum FR1. An approach to the structure of membrane proteins in their natural environment

Purified F0F1 ATPase from Rhodospirillum rubrum FR1 has been incorporated into lipid vesicles from the partially deuterated phospholipid dimyristoylglycerophosphocholine (DMPC-D54). These proteoliposomes were able to carry out energy transducing reactions. The incorporation of the membrane protein was controlled by freeze fracture electron microscopy. A method for structural research of the membrane protein in its natural environment has been developed by means of neutron small angle scattering. Using the contrast variation technique, the lipid part of the proteoliposomes was matched by adding an appropriate amount of D2O to the solvent. Thus the neutron scattering profile of F0F1 ATPase incorporated into vesicles was separated from the neutron scattering of the liposome. F0F1 ATPase incorporated in a lipid bilayer, as well as the free enzyme, yields a radius of gyration of Rg = 6.0 +/- 0.1 nm which leads to an overall diameter of 15.5 nm. This result suggests that the monomeric form of F0F1 ATPase is incorporated in DMPC-D54 membranes at 20 degrees C.     

Quasielastic neutron scattering measurements of fast local translational diffusion of lipid molecules in phospholipid bilayers

Quasielastic incoherent neutron scattering has been used to investigate the rate of local translational diffusion of lipid molecules in phospholipid bilayers of dipalmitoyl-phosphatidylcholine. The measured translational diffusion constants (4 x 10(-10) m2 s-1 at 63 degrees C and 1.4 x 10(-11) m2 s-1 at 30 degrees C) are considerably faster than those deduced using other less direct methods, but are in agreement with those measured in soap-water lyotropic liquid crystals, and with calculated values. This disagreement is attributed to differences in the time and distance scales characterising the various measurements. Quasielastic neutron scattering experiments observe fast motions over molecular distances, whereas other methods tend to measure a rate of diffusion which is averaged over macroscopic distances, and may thus contain contributions from long distance slow diffusive motions such as diffusion between the bilayers.     

Small-angle neutron scattering characterization of polyhydroxyalkanoates and their BioPEGylated hybrids in solution

Small-angle neutron scattering was used to probe the molecular conformation of various polyhydroxyalkanoates (PHAs) and their bioPEGylated counterparts (PHA- b-PEG). Analysis of neutron scattering profiles of these polymers dissolved in deuterated chloroform at various concentrations from dilute (approximately 0.1% w/v) to semidilute (approximately 7% w/v) showed the two distinct regimes and established overlap concentrations around 4-9 mg mL(-1). Scattering profiles were similar for all polymers investigated; power laws of approximately Q(-1.66) at high Q demonstrated that chloroform behaves as a good solvent for PHAs and suggests that under conditions synonymous with processing the solvated chains were swollen rather than in Gaussian conformation as previously reported. A gradual change to Guinier knees was followed by slopes of Q(-3) suggesting the presence of supramolecular structures at larger length scales. These observations in both the dilute and semidilute concentrations have not been previously reported. Zimm analysis of the data provided gyration radii and absolute molecular weights consistent with trends established using light scattering but showed some variation in their second virial coefficients. While natural-synthetic hybrids of PHA- b-PEG can self-assemble into microporous films, they showed no noticeable differences in chain conformation when in solution, the fabricating medium. This suggests that some form of entropic inducement is required.     

Small-angle neutron scattering study of lateral phase separation in dimyristoylphosphatidylcholine-cholesterol mixed membranes

The small-angle neutron scattering (SANS) technique developed previously is used to study the lateral phase separation in dimyristoylphosphatidylcholine (DMPC)-cholesterol mixed vesicles in the L alpha (35 degrees C) and L beta' (7 degrees C) phase of DMPC. To increase the sensitivity of the previous method, we apply the so-called inverse contrast variation technique where contrast matching is performed at a constant H2O/D2O ratio by varying the ratio of DMPC with deuterated and protonated hydrocarbon chains. Phase boundaries can be determined to an accuracy of +/- 0.5 mol %. In parallel experiments phase separation in the L beta' phase was also studied by freeze-fracture electron microscopy. For DMPC in the L alpha phase complete miscibility is clearly established up to cholesterol molar fractions of xc = 0.14. Strong evidence is provided that this is also the case up to xc approximately equal to 0.45. Cholesterol is no longer soluble above this limit and precipitates as small crystallites. For the L beta' phase (7 degrees C) phase boundaries are clearly established at xc1 = 0.08 and xc2 = 0.24, and very strong evidence is provided for two additional boundaries at xc3 = 0.435 and xc4 approximately equal to 1.0. At 0 less than or equal to xc less than or equal to xc1 the mixture forms a tilted solid solution in both the L beta' and P beta' phase while at xc1 less than or equal to xc less than or equal to xc2 this phase coexists with a nontilted mixture containing 24 mol % cholesterol.(ABSTRACT TRUNCATED AT 250 WORDS)     

A study of factors influencing hydration of sodium hyaluronate from compressibility and high-precision densimetric measurements.

A study of the factors influencing the hydration of the biopolymer hyaluronic acid was made by compressibility and density measurements. The factors investigated were the hydration changes on glycosidic bond formation, and also the influence of counterion type, solution ionic strength and temperature. The results indicate that, with this biopolymer, the hydration of the glucuronate residue is significantly more than that of the N-acetylglucosamine residue, and further that the biopolymer is less hydrated than the sum of its component monosaccharide residues. Change of the counterion salt form of this polyelectrolyte from univalent to bivalent counterion type (Na+ to Ca2+) leads to a small though significant increase in the total hydration sheath surrounding the polymer. An increase in the background ionic strength of the solvent leads to a quantifiable lowering of the hydration of the polymer at physiological ionic strength compared with its value in salt-free aqueous solution. A decrease in hydration with increase in temperature in the range 20-50 degrees C is the opposite of previous reports, and was observed when the polymer was dissolved both in pure water and in 0.15 M-NaCl.     

Polyhydroxyalkanoate (PHA) production using waste vegetable oil by Pseudomonas sp. strain DR2

To produce polyhydroxyalkanoate (PHA) from inexpensive substrates by bacteria, vegetable-oil-degrading bacteria were isolated from a rice field using enrichment cultivation. The isolated Pseudomonas sp. strain DR2 showed clear orange or red spots of accumulated PHA granules when grown on phosphate and nitrogen limited medium containing vegetable oil as the sole carbon source and stained with Nile blue A. Up to 37.34% (w/w) of intracellular PHA was produced from corn oil, which consisted of three major 3-hydroxyalkanoates; octanoic (C8:0, 37.75% of the total 3-hydroxyalkanoate content of PHA), decanoic (C10:0, 36.74%), and dodecanoic (C12:0, 11.36%). Pseudomonas sp. strain DR2 accumulated up to 23.52% (w/w) of PHAMCL from waste vegetable oil. The proportion of 3- hydroxyalkanoate of the waste vegetable-oil-derived PHA [hexanoic (5.86%), octanoic (45.67%), decanoic (34.88%), tetradecanoic (8.35%), and hexadecanoic (5.24%)] showed a composition ratio different from that of the corn-oil-derived PHA. Strain DR2 used three major fatty acids in the same ratio, and linoleic acid was the major source of PHA production. Interestingly, the production of PHA in Pseudomonas sp. strain DR2 could not occur in either acetate- or butyrate-amended media. Pseudomonas sp. strain DR2 accumulated a greater amount of PHA than other well-studied strains (Chromobacterium violaceum and Ralstonia eutropha H16) when grown on vegetable oil. The data showed that Pseudomonas sp. strain DR2 was capable of producing PHA from waste vegetable oil.     

Synthesis,Characterization and Applications of a Perdeuterated Amphipol

Amphipols are short amphipathic polymers that can substitute for detergents at the hydrophobic surface of membrane proteins (MPs), keeping them soluble in the absence of detergents while stabilizing them. The most widely used amphipol, known as A8-35, is comprised of a polyacrylic acid (PAA) main chain grafted with octylamine and isopropylamine. Among its many applications, A8-35 has proven particularly useful for solution-state NMR studies of MPs, for which it can be desirable to eliminate signals originating from the protons of the surfactant. In the present work, we describe the synthesis and properties of perdeuterated A8-35 (perDAPol). Perdeuterated PAA was obtained by radical polymerization of deuterated acrylic acid. It was subsequently grafted with deuterated amines, yielding perDAPol. The number-average molar mass of hydrogenated and perDAPol, ~4 and ~5 kDa, respectively, was deduced from that of their PAA precursors, determined by size exclusion chromatography in tetrahydrofuran following permethylation. Electrospray ionization–ion mobility spectrometry–mass spectrometry measurements show the molar mass and distribution of the two APols to be very similar. Upon neutron scattering, the contrast match point of perDAPol is found to be ~120 % D₂O. In ¹H-¹H nuclear overhauser effect NMR spectra, its contribution is reduced to ~6 % of that of hydrogenated A8-35, making it suitable for extended uses in NMR spectroscopy. PerDAPol ought to also be of use for inelastic neutron scattering studies of the dynamics of APol-trapped MPs, as well as small-angle neutron scattering and analytical ultracentrifugation.     

Comparison of the structure of myosin subfragment 1 bound to actin and free in solution. A neutron scattering study using actin made "invisible" by deuteration

The structure of subfragment 1 (S1) bound to F-actin has been compared to the structure of free S1 using neutron scattering. The F-actin was rendered "invisible" to neutrons by selective deuteration and solvent contrast matching. Highly deuterated actin was purified from the slime mold Dictyostelium discoideum, which was fed deuterated Escherichia coli. The properties of this actin were found to be similar to those of protonated actin. The neutron-scattering pattern of S1 bound to this "invisible" actin was compared to that of free S1. At near-physiological ionic strength, a strong interference effect was observed, which arose from pairs of S1 molecules cross-linking actin filaments. However, at low ionic strength the only differences that could be observed were attributed to interference effects between neutrons scattered from S1s bound randomly to equivalent sites on an actin filament. These effects became negligible as the fraction of actin sites occupied by S1 approached zero. Thus, we conclude that the scattering by S1 attached to F-actin is identical with that of free S1, to a resolution of about 2.5 nm. The difference in apparent radii of gyration is less than 0.05 nm. Modeling calculations have been carried out to determine the sensitivity of neutron scattering to possible S1 deformations. The calculations showed that deformations of the structure of S1 that are large enough ultimately to produce a powerstroke of 5 nm or greater are only consistent with the data if they involve at most about 20% of the S1 mass. These results restrict the class of plausible models describing force generation in muscle contraction.     

Biosynthesis and characterization of polyhydroxyalkanoates in the polysaccharide-degrading marine bacterium <Emphasis Type="Italic">Saccharophagus degradans</Emphasis> ATCC 43961

The marine bacterium Saccharophagus degradans was investigated for the synthesis of polyhydroxyalkanoates (PHAs), using glucose as the sole source of carbon in a two-step batch culture. In the first step the microorganism grew under nutrient balanced conditions; in the second step the cells were cultivated under limitation of nitrogen source. The biopolymer accumulated in S. degradans cells was detected by Nile red staining and FT-IR analysis. From GC-MS analysis, it was found that this strain produced a homopolymer of 3-hydroxybutyric acid. The cellular polymer concentration, its molecular mass, glass transition temperature, melting point and heat of fusion were 17.2+/-2.7% of dry cell weight, 54.2+/-0.6 kDa, 37.4+/-6.0 degrees C, 165.6+/-5.5 degrees C and 59.6+/-2.2 J g(-1), respectively. This work is the first report determining the capacity of S. degradans to synthesize PHAs.     

Small-angle neutron scattering from the reconstituted TF1 of H(+)-ATPase from thermophilic bacterium PS3 with deuterated subunits

Subunits alpha, beta and gamma of adenosine triphosphatase (H(+)-ATPase) from the thermophilic bacterium PS3 (TF1) have been over-expressed in Escherichia coli. alpha and beta subunits deuterated to the level of 90% were obtained by culturing E. coli in 2H2O medium. Both the subunits and the reconstituted alpha beta gamma complex, TF1, which contain the deuterated components in various combinations, were studied in solution by small-angle neutron scattering. The individual shapes of the subunits and their organization in the alpha beta gamma-TF1 complex were examined using the techniques of selective deuteration and contrast variation. The alpha and beta subunits are well approximated as ellipsoids of revolution having minor semi-axes of 20.4(+/- 0.4) and 20.0(+/- 0.2) A, and major semi-axes of 53.0(+/- 1.4) and 55.8(+/- 0.9) A, respectively. In the TF1 complex, three beta subunits are aligned to form an equilateral triangle, with their major axes tilted by 35 degrees with respect to the 3-fold axis of the complex. The beta-beta distance is about 53 A. Three alpha subunits are similarly arranged, positioned between the beta subunits, and with their direction of tilt opposite to that of the beta subunits. The centers of the alpha and beta subunits lie in the same plane, forming a hexagon. Adjacent subunits overlap in this model, suggesting that they are not simple ellipsoids of revolution.     

The denaturation transition of DNA in mixed solvents

The helix-to-coil denaturation transition in DNA has been investigated in mixed solvents at high concentration using ultraviolet light absorption spectroscopy and small-angle neutron scattering. Two solvents have been used: water and ethylene glycol. The "melting" transition temperature was found to be 94 degrees C for 4% mass fraction DNA/d-water and 38 degrees C for 4% mass fraction DNA/d-ethylene glycol. The DNA melting transition temperature was found to vary linearly with the solvent fraction in the mixed solvents case. Deuterated solvents (d-water and d-ethylene glycol) were used to enhance the small-angle neutron scattering signal and 0.1M NaCl (or 0.0058 g/g mass fraction) salt concentration was added to screen charge interactions in all cases. DNA structural information was obtained by small-angle neutron scattering, including a correlation length characteristic of the inter-distance between the hydrogen-containing (desoxyribose sugar-amine base) groups. This correlation length was found to increase from 8.5 to 12.3 A across the melting transition. Ethylene glycol and water mixed solvents were found to mix randomly in the solvation region in the helix phase, but nonideal solvent mixing was found in the melted coil phase. In the coil phase, solvent mixtures are more effective solvating agents than either of the individual solvents. Once melted, DNA coils behave like swollen water-soluble synthetic polymer chains.