Conformation of the Drosophila motor protein non-claret disjunctional in solution from X-ray and neutron scattering

The quaternary structures of monomeric and dimeric Drosophila non-claret disjunctional (ncd) constructs were investigated using synchrotron x-ray and neutron solution scattering, and their low resolution shapes were restored ab initio from the scattering data. The experimental curves were further compared with those computed from crystallographic models of one monomeric and three available dimeric ncd structures in the microtubule-independent ADP-bound state. These comparisons indicate that accounting for the missing parts in the crystal structures for all these constructs is indispensable to obtain reasonable fits to the scattering patterns. A ncd construct (MC6) lacking the coiled-coil region is monomeric in solution, but the calculated scattering from the crystallographic monomer yields a poor fit to the data. A tentative configuration of the missing C-terminal residues in the form of an antiparallel beta-sheet was found that significantly improves the fit. The atomic model of a short dimeric ncd construct (MC5) without 2-fold symmetry is found to fit the data better than the symmetric models. Addition of the C-terminal residues to both head domains gives an excellent fit to the x-ray and neutron experimental data, although the orientation of the beta-sheet differs from that of the monomer. The solution structure of the long ncd construct (MC1) including complete N-terminal coiled-coil and motor domains is modeled by adding a straight coiled-coil section to the model of MC5.     

Molecular adaptation: the malate dehydrogenase from the extreme halophilic bacterium Salinibacter ruber behaves like a non-halophilic protein

Malate dehydrogenase from the extreme halophilic bacterium, Salinibacter ruber (Sr MalDH) was purified and characterised as a tetramer by sedimentation velocity measurements, showing the enzyme belongs to the LDH-like group of MalDHs. In contrast to most other halophilic enzymes, which unfold when incubated at low salt concentration, Sr MalDH is completely stable in absence of salt. Its amino acid composition does not display the strong acidic character specific of halophilic proteins. The enzyme displays a strong KCl-concentration dependent variation in K(m) for oxaloacetate, but not for the NADH co-factor. Its activity is reduced by high salt concentration, but remains sufficient for the enzyme to sustain catalysis at approximately 30% of its maximal rates in 3 M KCl. The properties of the protein were compared with those from other LDH-like MalDHs of bacterial and archaeal origins, showing that Sr MalDH in fact behaves like a non-halophilic enzyme.     

Generation of CD1 tetramers as a tool to monitor glycolipid-specific T cells

CD1 molecules are beta(2)m-associated HLA class-I-like glycoproteins which have the unique ability to present glycolipid and phospholipid antigens to specific T lymphocytes. To study the biology of CD1 and its role in human disease we developed novel techniques for generation of recombinant CD1/lipid complexes by in vitro refolding. Fluorescent tetrameric complexes made from soluble recombinant CD1d/alpha-galactosylceramide complexes allowed highly sensitive and specific ex vivo and in vitro detection and functional characterization of novel human T-lymphocyte populations. Furthermore, protein crystals were obtained from soluble recombinant CD1b/beta(2)m-proteins loaded either with phosphatidylinositol or ganglioside GM2, which led to the first atomic structure determination of a CD1/lipid complex. The analysis of these crystal structures clarified how CD1b molecules can bind lipid ligands of different size, and revealed a broader spectrum of potential CD1b ligands than previously predicted.     

Protein dynamics studied by neutron scattering

This review of protein dynamics studied by neutron scattering focuses on data collected in the last 10 years. After an introduction to thermal neutron scattering and instrumental aspects, theoretical models that have been used to interpret the data are presented and discussed. Experiments are described according to sample type, protein powders, solutions and membranes. Neutron-scattering results are compared to those obtained from other techniques. The biological relevance of the experimental results is discussed. The major conclusion of the last decade concerns the strong dependence of internal dynamics on the macromolecular environment.     

Tetrahedral aminopeptidase: a novel large protease complex from archaea

A dodecameric protease complex with a tetrahedral shape (TET) was isolated from Haloarcula marismortui, a salt-loving archaeon. The 42 kDa monomers in the complex are homologous to metal-binding, bacterial aminopeptidases. TET has a broad aminopeptidase activity and can process peptides of up to 30-35 amino acids in length. TET has a central cavity that is accessible through four narrow channels (<17 A wide) and through four wider channels (21 A wide). This architecture is different from that of all the proteolytic complexes described to date that are made up by rings or barrels with a single central channel and only two openings.     

Novel cationic vesicle platform derived from vernonia oil for efficient delivery of DNA through plant cuticle membranes

Novel cationic amphiphilic compounds were prepared from vernonia oil, a natural epoxidized triglyceride, and studied with respect to vesicle formation, encapsulation of biomaterials such as DNA, and their physical stability and transport through isolated plant cuticle membranes. The amphiphiles studied were a single-headed compound III (a quaternary ammonium head group with two alkyl chains) and a triple-headed compound IV, which is essentially three molecules of compound III bound together through a glycerol moiety. Vesicles of the two amphiphiles, prepared by sonication in water and solutions of uranyl acetate or the herbicide 2,4-D (2,4-dichloropenoxy acetic acid), were examined by TEM, SEM, AFM, and confocal laser systems and had a spherical shape which encapsulated the solutes with diameters between 40 and 110 nm. Vesicles from amphiphile IV could be made large enough to encapsulate a condensed 5.2kb DNA plasmid (pJD328). Vesicles of amphiphile IV were also shown to pass intact across isolated plant cuticle membranes and the rate of delivery of encapsulated radio-labeled 2,4-D through isolated plant cuticle membranes obtained with these vesicles was clearly greater in comparison to liposomes prepared from dipalmitopyl phosphatidylcholine (DPPC) and the control, nonencapsulated 2,4-D. Vesicles from amphiphiles III and IV were found to be more stable than those of liposomes from DPPC. The data indicate the potential of vesicles prepared from the novel amphiphile IV to be a relatively efficient nano-scale delivery system to transport DNA and other bioactive agents through plant biological barriers. This scientific approach may open the way for further development of efficient in vivo plant transformation systems.     

Liquid-like water confined in stacks of biological membranes at 200 k and its relation to protein dynamics

Confined water is of considerable current interest owing to its biophysical importance and relevance to cryopreservation. It can be studied in its amorphous or supercooled state in the "no-man's land", i.e., in the temperature range between 150 and 235 K, in which bulk water is always crystalline. Amorphous deuterium oxide (D(2)O) was obtained in the intermembrane spaces of a stack of purple membranes from Halobacterium salinarum by flash cooling to 77 K. Neutron diffraction showed that upon heating to 200 K the intermembrane water space decreased sharply with an associated strengthening of ice diffraction, indicating that water beyond the first membrane hydration layer flowed out of the intermembrane space to form crystalline ice. It was concluded that the confined water undergoes a glass transition at or below 200 K to adopt an ultraviscous liquid state from which it crystallizes to form ice as soon as it finds itself in an unconfined, bulk-water environment. Our results provide model-free evidence for translational diffusion of confined water in the no-man's land. Potential effects of the confined-water glass transition on nanosecond membrane dynamics were investigated by incoherent elastic neutron scattering experiments. These revealed no differences between flash-cooled and slow-cooled samples (in the latter, the intermembrane space at temperatures <250 K is occupied only by the first membrane hydration layers), with dynamical transitions at 150 and 260 K, but not at 200 K, suggesting that nanosecond membrane dynamics are not sensitive to the state of the water beyond the first hydration shell at cryotemperatures.     

The influence of solvent composition on global dynamics of human butyrylcholinesterase powders: a neutron-scattering study

A major result of incoherent elastic neutron-scattering experiments on protein powders is the strong dependence of the intramolecular dynamics on the sample environment. We performed a series of incoherent elastic neutron-scattering experiments on lyophilized human butyrylcholinesterase (HuBChE) powders under different conditions (solvent composition and hydration degree) in the temperature range from 20 to 285 K to elucidate the effect of the environment on the enzyme atomic mean-square displacements. Comparing D(2)O- with H(2)O-hydrated samples, we were able to investigate protein as well as hydration water molecular dynamics. HuBChE lyophilized from three distinct buffers showed completely different atomic mean-square displacements at temperatures above approximately 200 K: a salt-free sample and a sample containing Tris-HCl showed identical small-amplitude motions. A third sample, containing sodium phosphate, displayed highly reduced mean-square displacements at ambient temperature with respect to the other two samples. Below 200 K, all samples displayed similar mean-square displacements. We draw the conclusion that the reduction of intramolecular protein mean-square displacements on an Angstrom-nanosecond scale by the solvent depends not only on the presence of salt ions but also on their type.     

Dynamics-stability relationships in apo- and holomyoglobin: a combined neutron scattering and molecular dynamics simulations study

The removal of the heme group from myoglobin (Mb) results in a destabilization of the protein structure. The dynamic basis of the destabilization was followed by comparative measurements on holo- (holo-Mb) and apomyoglobin (apo-Mb). Mean-squared displacements (MSD) and protein resilience on the picosecond-to-nanosecond timescale were measured by elastic incoherent neutron scattering. Differences in thermodynamic parameters, MSD, and resilience were observed for both proteins. The resilience of holo-Mb was significantly lower than that of apo-Mb, indicating entropic stabilization by a higher degree of conformational sampling in the heme-bound folded protein. Molecular dynamics simulations provided site-specific information. Averaged over the whole structure, the molecular dynamics simulations yielded similar MSD and resilience values for the two proteins. The mobility of residues around the heme group in holo-Mb showed a smaller MSD and higher resilience compared to the same residue group in apo-Mb. It is of interest that in holo-Mb, higher MSD values are observed for the residues outside the heme pocket, indicating an entropic contribution to protein stabilization by heme binding, which is in agreement with experimental results.