Biosynthesis and characterization of deuterated polyhydroxyoctanoate

The synthesis of a polyhydroxyalkanoate with medium chain length alkyl substituents by Pseudomonas oleovoranswas investigated using protonated and deuterated forms of octanoic acid in a minimal salts medium. Cultivation with deuterated octanoic acid resulted in a reduced rate of polymer accumulation compared to that with its protonated counterpart (107 and 207 mg of polymer L(-1) of medium h(-1) of cultivation, respectively). Nuclear magnetic resonance and gas chromatography coupled mass spectrometry of the derivatized polymer was used to establish the extent and distribution of deuterium in the biopolymer. A partially deuterated heteropolymer with 3-hydroxyoctanoic acid as the main constituent was produced. Deuteration is an important tool for contrast variation studies using neutron scattering, but predicates that the deuterated polymer is otherwise comparable in its physiochemical and material properties to its protonated counterpart. In studies reported here, the deuterated biopolymer exhibited an additional diffraction maximum at 7.55 A and slight differences in its melting point (60 and 55 degrees C) and glass transition temperature (-39 and -36 degrees C) when compared to its protonated equivalent. While significant differences between the protonated and deuterated biopolymers were determined, our results support the use of this deuterated polyhydroxyalkanoate in its application in investigations using analytical neutron scattering techniques.     

Production and use of deuterated polyhydroxyoctanoate in structural studies of PHO inclusions

This work reports on the biosynthesis of polyhydroxyalkanoates with medium chain length alkyl substituents in the side chain by Pseudomonas oleovorans using hydrogenated and deuterated substrates. These investigations aimed to obtain polyhydroxyalkanoates with varying degrees of deuterium substitution, and establish whether they are suitable analogues for structural investigation. In order to understand the formation and structure of inclusions in their native state, whole inclusions were isolated from microbial cells and were analysed using Small Angle Neutron Scattering. A contrast variation study was conducted on hydrogenated and deuterated inclusions of polyhydroxyoctanoate, as well as inclusions resulting from co-feeding or sequentially feeding different precursors. The data indicated a core/shell structure resulting from feeding hydrogenated followed by perdeuterated PHO precursor, and demonstrated the utility of this analysis for characterising chemically similar systems.     

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.     

Periodic disorder along ramie cellulose microfibrils

Small angle neutron scattering studies have been carried out on cellulose fibers from ramie and Populus maximowicii (cotton wood). Labile hydrogen atoms were replaced by deuterium atoms, in water-accessible disordered regions of the fibers, to increase the neutron scattering contrast between the disordered and crystalline regions. A meridional Bragg reflection, corresponding to a longitudinal periodicity of 150 nm, was observed when scattering collected from hydrogenated and deuterated dry ramie fibers was subtracted. No Bragg reflection was observed with the cotton wood fibers, probably because of lower orientation of the microfibrils in the cell wall. The ramie fibers were then subjected to electron microscopy, acid hydrolysis, gel permeation chromatography, and viscosity studies. The leveling off degree of polymerization (LODP) of the hydrolyzed samples matched exactly the periodicity observed in the diffraction studies. The weight loss related to the LODP was only about 1.5%, and thus, the microfibrils can be considered to have 4-5 disordered residues every 300 residues.     

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.     

Mechanism of force generation studied by neutron scattering

Neutron scattering has been used to compare the structure of myosin S1 that is free in solution to that when it is bound to F-actin. To achieve this, deuterated actin was obtained from D. discoideum that had been fed deuterated E. coli. This deuterated actin was rendered “invisible” to neutrons when dissolved in 94% D₂O. The neutron scattering patterns obtained from S1 bound to deuterated actin were identical to those of free S1 except for oscillations due to S1's bound to the same actin filament. At low S1 to actin stoichiometrics, these oscillations diminish and the patterns become indistinguishable. The apparent radius of gyration of S1 bound to actin is identical to that of free S1 when the stoichiometry is low. Thus, no changes in the structure of S1 were observed to a resolution of 2.5 nm. Computer modelling studies were used to evaluate the compatibility of models for the mechanism of force generation with the neutron data. These studies show that for powerstrokes greater than 5.0 nm, the data are consistent with more than 80% of the crossbridge maintaining a rigid conformation during force generation.     

Recent neutron scattering studies of muscle contraction and its control

We have presented two applications of the method of neutron scattering utilizing selective deuteration of actin. In these experiments the actin was rendered effectively invisible to neutrons by matching the scattering-length densities of deuterated actin and the solvent. The scattering of neutrons by myosin S1 and by Tm bound to this actin was studied.     

On the long-range attraction between proteins due to nonadsorbing polysaccharide

The effect of a nonadsorbing polysaccharide (dextran) on the structure factor of a solution of lysozyme was studied using small-angle neutron scattering (SANS) experiments. By choosing the appropriate water/deuterium ratio as solvent, we made the scattering signal from dextran invisible for the SANS measurements. Dextran induces a weak long-range attraction between the lysozyme molecules. This attraction is described using a depletion interaction potential from theory for two spheres in an ideal polymer solution. Incorporation of the theory in a mean-spherical approximation shows that the wave vector below which the structure factor increases depends on the polymer size. The theoretical prediction is in fair agreement with the measured structure factor of lysozyme, as affected by nonadsorbing dextran.     

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 study of rhodopsin in detergent micelles by small-angle neutron scattering

Monodisperse solutions of bovine rhodopsin monomers, devoid of lipid, associated with a linear polyoxyethylene alcohol detergent have been prepared. The composition and homogeneity of these complexes have been determined by hydrodynamic characterisation. Each rhodopsin molecule is associated with about 110 monomers of the detergent. These rhodopsin-detergent complexes have been studied by small-angle neutron scattering. Partial or total deuteration of the detergent, as well as variation of the ²H₂O/H₂O ratio in the solvent, were used to eliminate the detergent—solvent contrast at various protein—solvent contrasts. The size and shape of the detergent micelle and of the rhodopsin-detergent complexes were shown to be independent of solvent or detergent deuteration. Mixture of selectively deuterated detergent molecules allowed us to obtain an homogeneous scattering density for the detergent part of the micelles and therefore to eliminate totally its contribution to the scattering when it is contrast matched. Neutron scattering from rhodopsin alone was then measured even in highly deuterated solvents, with low incoherent background, as for a water-soluble protein. Supplementary neutron scattering measurements on rhodopsin-dodecyl dimethylamine oxide micelles confirmed essentially the results reported by Yeager (1975). Analysis of the neutron scattering data indicates that most of the hydrophobic residues of rhodopsin form a compact region which has zero hydration, this probably being the part which is embedded in the disc membrane, and that the unhydrated rhodopsin molecule is asymmetrically arranged with respect to the membrane. Comparison with the results of a small-angle X-ray scattering study (Sardet et al., 1976) implies that the peripheral regions on both sides of the membrane are highly hydrated. Several schematic models are discussed.     

Neutron scattering shows a droplet of oleic acid at the center of the BAMLET complex

The anti‐cancer complex, Bovine Alpha‐lactalbumin Made LEthal to Tumors (BAMLET), has intriguing broad‐spectrum anti‐cancer activity. Although aspects of BAMLET's anti‐cancer mechanism are still not known, it is understood that it involves the oleic acid or oleate component of BAMLET being preferentially released into cancer cell membranes leading to increased membrane permeability and lysis. The structure of the protein component of BAMLET has previously been elucidated by small angle X‐ray scattering (SAXS) to be partially unfolded and dramatically enlarged. However, the structure of the oleic acid component of BAMLET and its disposition with respect to the protein component was not revealed as oleic acid has the same X‐ray scattering length density (SLD) as water. Employing the difference in the neutron SLDs of hydrogen and deuterium, we carried out solvent contrast variation small angle neutron scattering (SANS) experiments of hydrogenated BAMLET in deuterated water buffers, to reveal the size, shape, and disposition of the oleic acid component of BAMLET. Our resulting analysis and models generated from SANS and SAXS data indicate that oleic acid forms a spherical droplet of oil incompletely encapsulated by the partially unfolded protein component. This model provides insight into the anti‐cancer mechanism of this cache of lipid. The model also reveals a protein component “tail” not associated with the oleic acid component that is able to interact with the tail of other BAMLET molecules, providing a plausible explanation of how BAMLET readily forms aggregates. Proteins 2017; 85:1371–1378. © 2017 Wiley Periodicals, Inc.     

Frozen spin targets in ribosomal structure research.

Polarized neutron scattering strongly depends on nuclear spin polarisation, particularly on proton spin polarisation. A single proton in a deuterated environment then is as efficient as 10 electrons in X-ray anomalous diffraction. Neutron scattering from the nuclear spin label is controlled by the polarisation of neutron spins and nuclear spins. Pure deuteron spin labels and proton spin labels are created by NMR saturation. We report on results obtained from the large subunit of E. coli ribosomes which have been obtained at the research reactor of GKSS using the polarized target facility developed by CERN. The nuclear spins were oriented with respect to an external field by dynamic nuclear polarisation. Proton spin polarisations of more than 80% were obtained in ribosomes at temperatures below 0.5 K. At T = 130 mK the relaxation time of the polarized target is one month (frozen spin target). Polarized small-angle neutron scattering of the in situ structure of rRNA and the total ribosomal protein (TP) has been determined from the frozen spin targets of the large ribosomal subunit, which has been deuterated in the TP and rRNA respectively. The results agree with those from neutron scattering in H2O/D2O mixtures obtained at room temperature. This is a necessary prerequisite for the planned determination of the in situ structure of individual ribosomal proteins and especially of that of ribosome bound mRNA and tRNAs.     

Methionyl-tRNA synthetase from E. coli: direct evidence for exchange of protomers in the dimeric enzyme by using deuteration and small-angle neutron scattering

Direct demonstration of the reversible dissociation of native dimeric methionyl-tRNA synthetase from E. coli has been obtained using small angle neutron scattering and deuterated enzyme. Structural parameters of the fully deuterated dimer are very similar to the hydrogenated one. Analysis of the variations of the intensity and of the radius of gyration of a stoichiometric mixture of the two types of dimer (hydrogenated and deuterated), as a function of D2O content in the solvent, enabled us to characterize an hybrid dimer, having both hydrogenated and deuterated protomers. By separating the contribution of each protomer to the scattering, the radius of gyration of the protomer in situ and the distance between the centers of mass of each protomer in the dimer are determined.     

The reaction center profile structure derived from neutron diffraction

Both reaction center protein from the photosynthetic bacteria Rhodopseudomonas sphaeroides and egg phosphatidylcholine can be deuterium labelled; the reaction center protein can be incorporated into the phosphatidylcholine bilayers forming a homogeneous population of unilamellar vesicles. The lipid profile and the reaction center profile within these reconstituted membrane profiles were directly determined to 32 Å resolution using lamellar neutron diffraction from oriented membrane multilayers containing either deuterated or protonated reaction centers, and either deuterated or protonated phosphatidylcholine. The 32 Å resolution reaction center profile shows that the protein spans the membranes, and has an asymmetric mass distribution along the perpendicular to the membrane plane. These results were combined with previously described X-ray diffraction results in order to extend the resolution of the derived reaction center profile to 9 Å.     

Production and characterization of recombinant perdeuterated cholesterol oxidase

Cholesterol oxidase (CO) is a FAD (flavin adenine dinucleotide) containing enzyme that catalyzes the oxidization and isomerization of cholesterol. Studies directed toward elucidating the catalytic mechanism of CO will provide an important general understanding of Flavin-assisted redox catalysis. Hydrogen atoms play an important role in enzyme catalysis; however, they are not readily visualized in protein X-ray diffraction structures. Neutron crystallography is an ideal method for directly visualizing hydrogen positions at moderate resolutions because hydrogen and deuterium have comparable neutron scattering lengths to other heavy atoms present in proteins. The negative coherent and large incoherent scattering lengths of hydrogen atoms in neutron diffraction experiments can be circumvented by replacing hydrogen atoms with its isotope, deuterium. The perdeuterated form of CO was successfully expressed from minimal medium, purified, and crystallized. X-ray crystallographic structures of the enzyme in the perdeuterated and hydrogenated states confirm that there are no apparent structural differences between the two enzyme forms. Kinetic assays demonstrate that perdeuterated and hydrogenated enzymes are functionally identical. Together, structural and functional studies indicate that the perdeuterated protein is suitable for structural studies by neutron crystallography directed at understanding the role of hydrogen atoms in enzyme catalysis.     

Mass spectrometric measurement of protein amide hydrogen exchange rates of apo- and holo-myoglobin.

Measurement of backbone amide hydrogen exchange rates can provide detailed information concerning protein structure, dynamics, and interactions. Although nuclear magnetic resonance is typically used to provide these data, its use is restricted to lower molecular weight proteins that are soluble at millimolar concentrations. Not subject to these limitations is a mass spectrometric approach for measuring deuterium incorporation into proteins that are subsequently proteolyzed by pepsin; the resulting peptide masses are measured using a flowing-fast atom bombardment ionization source (Zhang Z, Smith DL, 1993, Protein Sci 2:522-531). In the current study, amide deuterium incorporation for intact apo- and holo-myoglobin was measured using liquid chromatography coupled directly to an electrospray ionization (LC/MS) source. Electrospray ionization provided a more complete coverage of the protein sequence and permitted the measurement of deuterium incorporation into intact proteins. Tandem mass spectrometry was used to rapidly identify the peptic peptides. It was found that within 30 s, the amides in apo-myoglobin were 47% deuterated, whereas holo-myoglobin was 12% deuterated. Peptic digestion and LC/MS demonstrated that regions represented by peptic peptides encompassing positions 1-7, 12-29, and 110-134 were not significantly altered by removal of the heme. Likewise, destabilized regions were identified within positions 33-106 and 138-153.     

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.     

Use of deuterium labelled glucose in evaluating the pathway of hepatic glycogen synthesis

Deuterium labelled glucose has been used to study the pathway of hepatic glycogen synthesis during the fasted-refed transition in rats. Deuterium enrichment of liver glycogen was determined using nuclear magnetic resonance as well as mass spectroscopy. Sixty minutes after oral administration of deuterated glucose to fasted rats, the portal vein blood was fully enriched with deuterated glucose. Despite this, less than half of the glucose molecules incorporated into liver glycogen contained deuterium. The loss of deuterium label from glucose is consistent with hepatic glycogen synthesis by an indirect pathway requiring prior metabolism of glucose. The use of deuterium labelled glucose may prove to be a useful probe to study hepatic glycogen metabolism. Its use may also find application in the study of liver glycogen metabolism in humans by a noninvasive means.     

C subunits binding to the protein kinase A RI alpha dimer induce a large conformational change

We present structural data on the RI alpha isoform of the cAMP-dependent protein kinase A that reveal, for the first time, a large scale conformational change within the RI alpha homodimer upon catalytic subunit binding. This result infers that the inhibition of catalytic subunit activity is not the result of a simple docking process but rather is a multi-step process involving local conformational changes both in the cAMP-binding domains as well as in the linker region of the regulatory subunit that impact the global structure of the regulatory homodimer. The results were obtained using small-angle neutron scattering with contrast variation and deuterium labeling. From these experiments we derived information on the shapes and dispositions of the catalytic subunits and regulatory homodimer within a holoenzyme reconstituted with a deuterated regulatory subunit. The scattering data also show that, despite extensive sequence homology between the isoforms, the overall structure of the type I alpha holoenzyme is significantly more compact than the type II alpha isoform. We present a model of the type I alpha holoenzyme, built using available high-resolution structures of the component subunits and domains, which best fits the neutron-scattering data. In this model, the type I alpha holoenzyme forms a flattened V shape with the RI alpha dimerization domain at the point of the V and the cAMP-binding domains of the RI alpha subunits with their bound catalytic subunits at the ends.     

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.