Physical properties of the dimyristoylphosphatidylcholine vesicle and of complexes formed by its interaction with apolipoprotein C-III.

The structure of a single bilayer vesicle of dimyristoylphosphatidylcholine has been characterized by sedimentation, densimetry, and light-scattering measurements. The molecular weight, partial specific volume, Stokes radius, and degree by hydration were found to be 2.68 X 10(6), 0.972 cm3/g, 125 A, and 0.86 g/g, respectively. From these quantities, a spherically symmetrical model has been derived that features a phospholipid bilayer 35.5 A thick and a hydration shell 9.3 A thick. This particle was shown to bind apolipoprotein C-III (apoC-III) up to 0.08 g/g without loss of its original vesicular structure. At protein-lipid ratios in excess of 0.08 g/g, sedimentation, gel chromatography, and light-scattering measurement indicated a dramatic decrease in Stokes radius and molecular weight. The sedimentation data showed these parameters to become constant at protein-lipid ratios in excess of 0.25 g/g. In this region, the Stokes radius and molecular weight were found to be approximately 80 A and 442 000, respectively. Within the constraints of these values and other data, several models for this complex are discussed.     

Structure of Colorado potato beetle lipophorin: differential scanning calorimetric and small-angle X-ray scattering studies

The structure of lipophorin, isolated from hemolymph of the Colorado potato beetle, was investigated by differential scanning calorimetry (DSC) and small-angle X-ray scattering. The DSC heating curves of intact lipophorin showed endothermic peaks that were similar to peaks obtained with the hydrocarbon fraction isolated from this lipophorin. The observed peaks correlated with the transition of the hydrocarbons from an ordered into a more disordered state. Changes in structure of the lipophorin particles with increasing temperature were also observed by small-angle X-ray scattering studies. The structural organization of lipophorin was further elucidated by simulation analysis, using a three-layered symmetrical sphere as a model. These studies revealed that lipophorin from the Colorado potato beetle is a sphere with a maximum diameter of 175 A. The sphere is composed of three radially symmetrical layers of different electron densities. The outer layer (37.5-39.5 A in thickness) is composed of phospholipid, apolipophorin I, and part of apolipophorin II. The middle layer (5-10 A) contains diacylglycerol, the rest of apolipophorin II, and probably beta-carotene. The core of the particle (40-45 A) only contains hydrocarbons. This structure differs from another model, previously proposed for cockroach and locust lipophorins [Katagiri, C., Sato, M., & Tanaka N. (1987) J. Biol. Chem. 262, 15857-15861], in the small size of the middle layer. The volume of the middle layer correlated well with the low diacylglycerol content of this lipophorin.     

X-ray and neutron small-angle scattering analysis of the complex formed by the Met receptor and the Listeria monocytogenes invasion protein InlB

The Listeria monocytogenes surface protein InlB binds to the extracellular domain of the human receptor tyrosine kinase Met, the product of the c-met proto-oncogene. InlB binding activates the Met receptor, leading to uptake of Listeria into normally nonphagocytic host cells. The N-terminal half of InlB (InlB₃₂₁) is sufficient for Met binding and activation. The complex between this Met-binding domain of InlB and various constructs of the Met ectodomain was characterized by size exclusion chromatography and dynamic light scattering, and structural models were built using small-angle X-ray scattering and small-angle neutron scattering. Although most receptor tyrosine kinase ligands induce receptor dimerization, InlB₃₂₁ consistently binds the Met ectodomain with a 1:1 stoichiometry. A construct comprising the Sema and PSI domains of Met, although sufficient to bind the physiological Met ligand hepatocyte growth factor/scatter factor, does not form a complex with InlB₃₂₁ in solution, highlighting the importance of Met Ig domains for InlB binding. Small-angle X-ray scattering and small-angle neutron scattering measurements of ligand and receptor, both free and in complex, reveal an elongated shape for the receptor. The four Ig domains form a bent, rather than a fully extended, conformation, and InlB₃₂₁ binds to Sema and the first Ig domain of Met, in agreement with the recent crystal structure of a smaller Met fragment in complex with InlB₃₂₁. These results call into question whether receptor dimerization is the basic underlying event in InlB₃₂₁-mediated Met activation and demonstrate differences in the mechanisms by which the physiological ligand hepatocyte growth factor/scatter factor and InlB₃₂₁ bind and activate the Met receptor.     

Structure of human low-density lipoprotein subfractions determined by X-ray small-angle scattering

The structure of low-density lipoprotein (LDL) particles from three different density ranges (LDL-1: d = 1.006−1.031 g/ml; LDL-3: d = 1.034−1.037 g/ml; LDL-6: d = 1.044−1.063 g/ml) was determined by X-ray small-angle scattering. By using a theoretical particle model, which accounted for the polydispersity of the samples, we were able to obtain fits of the scattering intensity that were inside the noise interval of the measured intensity. The assumption of deviations from radial symmetry is not supported by our data. This implies a spread-out conformation of the apolipoprotein B (apoB) molecule, which appears to be localized in the outer surface shell. A globular structure is not consistent with our data. Furthermore, different models exist concerning the structure of the cholesterol ester core below the phase transition temperature. The electron density data suggest an arrangmeent in which the steroid moieties are localized at average radii of 3.2 and 6.4 nm. Model calculations show that packing problems can only be avoided if approximately half of the acyl chains of each shell are pointing towards the center of the particle, the other half towards the surface. This arrangement of the acyl chains has never been proposed before. The LDL particles of different density classes differ mainly with respect to the size of the core but also with respect to the width of the surface shells. Model calculations show that the size of different LDL particles can be accurately predicted from the compositional data.     

X-ray and neutron small-angle scattering studies of the complex between protein S1 and the 30-S ribosomal subunit.

X-ray neutron solution scattering experiments have been done to investigate the influence of the binding of ribosomal protein S1 on the conformation of the 30-S ribosomal subunit of Escherichia coli. The following conclusions were made. 1. The alterations (if any) in conformation of the non-S1 parts of the 30-S subunit induced by S1 binding are too small to be detected (less than 0.1 nm change in radius of gyration). 2. The center of gravity of protein S1 bound to the 30-S subunit is quite far from the center of gravity of the particle (approximately 7.5 nm).     

A review of multi-domain and flexible molecular chaperones studies by small-angle X-ray scattering

Intrinsic flexibility is closely related to protein function, and a plethora of important regulatory proteins have been found to be flexible, multi-domain or even intrinsically disordered. On the one hand, understanding such systems depends on how these proteins behave in solution. On the other, small-angle X-ray scattering (SAXS) is a technique that fulfills the requirements to study protein structure and dynamics relatively quickly with few experimental limitations. Molecular chaperones from Hsp70 and Hsp90 families are multi-domain proteins containing flexible and/or disordered regions that play central roles in cellular proteostasis. Here, we review the structure and function of these proteins by SAXS. Our general approach includes the use of SAXS data to determine size and shape parameters, as well as protein shape reconstruction and their validation by using accessory biophysical tools. Some remarkable examples are presented that exemplify the potential of the SAXS technique. Protein structure can be determined in solution even at limiting protein concentrations (for example, human mortalin, a mitochondrial Hsp70 chaperone). The protein organization, flexibility and function (for example, the J-protein co-chaperones), oligomeric status, domain organization, and flexibility (for the Hsp90 chaperone and the Hip and Hep1 co-chaperones) may also be determined. Lastly, the shape, structural conservation, and protein dynamics (for the Hsp90 chaperone and both p23 and Aha1 co-chaperones) may be studied by SAXS. We believe this review will enhance the application of the SAXS technique to the study of the molecular chaperones.     

Structure of human low-density lipoprotein subfractions, determined by X-ray small-angle scattering

The structure of low-density lipoprotein (LDL) particles from three different density ranges (LDL-1: d = 1.006-1.031 g/ml; LDL-3: d = 1.034-1.037 g/ml; LDL-6: d = 1.044-1.063 g/ml) was determined by X-ray small-angle scattering. By using a theoretical particle model, which accounted for the polydispersity of the samples, we were able to obtain fits of the scattering intensity that were inside the noise interval of the measured intensity. The assumption of deviations from radial symmetry is not supported by our data. This implies a spread-out conformation of the apolipoprotein B (apoB) molecule, which appears to be localized in the outer surface shell. A globular structure is not consistent with our data. Furthermore, different models exist concerning the structure of the cholesterol ester core below the phase transition temperature. The electron density data suggest an arrangement in which the steroid moieties are localized at average radii of 3.2 and 6.4 nm. Model calculations show that packing problems can only be avoided if approximately half of the acyl chains of each shell are pointing towards the center of the particle, the other half towards the surface. This arrangement of the acyl chains has never been proposed before. The LDL particles of different density classes differ mainly with respect to the size of the core but also with respect to the width of the surface shells. Model calculations show that the size of different LDL particles can be accurately predicted from the compositional data.     

Temperature dependence of the ripple structure in dimyristoylphosphatidylcholine studied by synchrotron X-ray small-angle diffraction

The ripple structure of 1,2-dimyristoyl-L-phosphatidylcholine (DMPC) multibilayer containing excess water (60 wt%) was studied by synchrotron X-ray small-angle diffraction. The (0,1) spacing which corresponds to the ripple repeat distance depends on temperature: At 13 degrees C the (0,1) spacing is 14.15 nm, the spacing decreases at higher temperatures and reaches 12.1 nm at 23.5 degrees C, just below the main transition temperature. The spacing is in good agreement between heating process and cooling process except for the supercooling region. The result suggests that the rearrangement of the ripple structure takes place during temperature change successively. The Landau-de Gennes free energy equation explains well the temperature dependence of the ripple repeat distance.     

Structure and plasticity of the peptidyl-prolyl isomerase Par27 of Bordetella pertussis revealed by X-ray diffraction and small-angle X-ray scattering

Par27 from Bordetella pertussis belongs to a newly discovered class of dimeric peptidyl-prolyl isomerase (PPIase)/chaperones from the parvulin family. It is a tripartite protein with a central PPIase domain surrounded by N- and C-terminal sub-domains (NTD and CTD). Here, the Par27 structure was characterized by X-ray crystallography, small-angle X-ray scattering and template-based modeling. In the crystal lattice, Par27 consists of alternating well ordered and poorly ordered domains. The PPIase domains gave rise to diffuse scattering and could not be solved, whereas a 2.2 Å resolution crystal structure was obtained for the NTD and CTD, revealing a cradle-shaped dimeric platform. Despite a lack of sequence similarity with corresponding sub-domains, the topology of the peptide chain in the NTD/CTD core is similar to that of other monomeric PPIase/chaperones such as SurA and trigger factor from Escherichia coli. In Par27, dimerization occurs by sub-domain swapping. Because of the strong amino acid sequence similarity to other parvulin domains, a model for the Par27 PPIase domain was built by template-based modeling and validated against small-angle X-ray scattering (SAXS) data. A model of the full-length dimeric Par27 structure was built by rigid-body modeling and filtering against SAXS data using the partial crystal structure of the NTD/CTD core and the template-based PPIase model. The flexibility of protein was accounted for by representing the structure as an ensemble of different conformations that collectively reproduce the scattering data. The refined models exhibit a cradle-like shape reminiscent of other PPIase/chaperones, and the variability in the orientation of the PPIase domains relative to the NTD/CTD core platform observed in the different models suggests inter-domain flexibility that could be important for the biological activity of this protein.     

The solution structure of the Sac7d/DNA complex: a small-angle X-ray scattering study.

Small-angle X-ray scattering has been used to study the structure of the multimeric complexes that form between double-stranded DNA and the archaeal chromatin protein Sac7d from Sulfolobus acidocaldarius. Scattering data from complexes of Sac7d with a defined 32-mer oligonucleotide, with poly[d(GC)], and with E. coli DNA indicate that the protein binds along the surface of an extended DNA structure. Molecular models of fully saturated Sac7d/DNA complexes were constructed using constraints from crystal structure and solution binding data. Conformational space was searched systematically by varying the parameters of the models within the constrained set to find the best fits between the X-ray scattering data and simulated scattering curves. The best fits were obtained for models composed of repeating segments of B-DNA with sharp kinks at contiguous protein binding sites. The results are consistent with extrapolation of the X-ray crystal structure of a 1:1 Sac7d/octanucleotide complex [Robinson, H., et al. (1998) Nature 392, 202-205] to polymeric DNA. The DNA conformation in our multimeric Sac7d/DNA model has the base pairs tilted by about 35 degrees and displaced 3 A from the helix axis. There is a large roll between two base pairs at the protein-induced kink site, resulting in an overall bending angle of about 70 degrees for Sac7d binding. Regularly repeating bends in the fully saturated complex result in a zigzag structure with negligible compaction of DNA. The Sac7d molecules in the model form a unique structure with two left-handed helical ribbons winding around the outside of the right-handed duplex DNA.     

Structure of a mouse immunoglobulin G that lacks the entire CH1 domain: protein sequencing and small-angle X-ray scattering studies

The structure of a short-chain IgG2a antibody, which is a member of the family of mouse anti-dansyl switch variant antibodies with identical variable regions but different heavy-chain constant regions [Dangl, J.L., Parks, D. R., Oi, V. T., & Herzenberg, L. A. (1982) Cytometry 2, 395-401], is reported. Amino acid sequencing analyses have demonstrated that in the short-chain IgG2a antibody the entire CH1 domain is deleted whereas the hinge region remains intact. Small-angle X-ray scattering data were collected for the short-chain IgG2a antibody and compared with those for the switch variant IgG1, IgG2a, and IgG2b antibodies with the normal heavy chain. It has been concluded that deletion of the CH1 domain results in a large structural change and the short-chain IgG2a antibody possesses an elongated molecular shape with a much smaller hinge angle as compared with the normal IgG2a antibody that is a Y-shaped molecule.     

Structure of Escherichia coli uracil DNA glycosylase and its complexes with nonhydrolyzable substrate analogues in solution observed by synchrotron small-angle X-ray scattering

The structure of native and modified uracil DNA glycosylase from E. coli in solution was studied by synchrotron small-angle X-ray scattering. The modified enzyme (6His-uracyl DNA glycosylase) differs from the native one by the presence of an additional N-terminal 11-meric sequence amino acid residues including a block of six His residues. It was found that the conformations of these enzymes in solution at moderate ionic strength (60 mM NaCI) substantially differ in spite of minimal differences in the amino acid sequences and functional activity. The structure of native uracil DNA glycosylase in solution is close to that in crystal, showing a tendency for association. The interaction of this enzyme with nonhydrolyzable analogues of DNA ligands causes a partial dissociation of associates and a compactization of protein structure. At the same time, 6His-uracyl DNA glycosylase has a compact structure essentially different from the crystal one. A decrease in the ionic strength of solution results in a partial disruption of compact structure of the modified protein, without changes in its functional activity.     

Structure of the cyanobacterial Magnesium Chelatase H subunit determined by single particle reconstruction and small-angle X-ray scattering

The biosynthesis of chlorophyll, an essential cofactor for photosynthesis, requires the ATP-dependent insertion of Mg²⁺ into protoporphyrin IX catalyzed by the multisubunit enzyme magnesium chelatase. This enzyme complex consists of the I subunit, an ATPase that forms a complex with the D subunit, and an H subunit that binds both the protoporphyrin substrate and the magnesium protoporphyrin product. In this study we used electron microscopy and small-angle x-ray scattering to investigate the structure of the magnesium chelatase H subunit, ChlH, from the thermophilic cyanobacterium Thermosynechococcus elongatus. Single particle reconstruction of negatively stained apo-ChlH and Chl-porphyrin proteins was used to reconstitute three-dimensional structures to a resolution of ∼30 Å. ChlH is a large, 148-kDa protein of 1326 residues, forming a cage-like assembly comprising the majority of the structure, attached to a globular N-terminal domain of ∼16 kDa by a narrow linker region. This N-terminal domain is adjacent to a 5 nm-diameter opening in the structure that allows access to a cavity. Small-angle x-ray scattering analysis of ChlH, performed on soluble, catalytically active ChlH, verifies the presence of two domains and their relative sizes. Our results provide a basis for the multiple regulatory and catalytic functions of ChlH of oxygenic photosynthetic organisms and for a chaperoning function that sequesters the enzyme-bound magnesium protoporphyrin product prior to its delivery to the next enzyme in the chlorophyll biosynthetic pathway, magnesium protoporphyrin methyltransferase.     

Solution structure of the chicken skeletal muscle troponin complex via small-angle neutron and X-ray scattering

Troponin is a Ca2+-sensitive switch that regulates the contraction of vertebrate striated muscle by participating in a series of conformational events within the actin-based thin filament. Troponin is a heterotrimeric complex consisting of a Ca2+-binding subunit (TnC), an inhibitory subunit (TnI), and a tropomyosin-binding subunit (TnT). Ternary troponin complexes have been produced by assembling recombinant chicken skeletal muscle TnC, TnI and the C-terminal portion of TnT known as TnT2. A full set of small-angle neutron scattering data has been collected from TnC-TnI-TnT2 ternary complexes, in which all possible combinations of the subunits have been deuterated, in both the +Ca2+ and -Ca2+ states. Small-angle X-ray scattering data were also collected from the same troponin TnC-TnI-TnT2 complex. Guinier analysis shows that the complex is monomeric in solution and that there is a large change in the radius of gyration of TnI when it goes from the +Ca2+ to the -Ca2+ state. Starting with a model based on the human cardiac troponin crystal structure, a rigid-body Monte Carlo optimization procedure was used to yield models of chicken skeletal muscle troponin, in solution, in the presence and in the absence of regulatory calcium. The optimization was carried out simultaneously against all of the scattering data sets. The optimized models show significant differences when compared to the cardiac troponin crystal structure in the +Ca2+ state and provide a structural model for the switch between +Ca2+ and -Ca2+ states. A key feature is that TnC adopts a dumbbell conformation in both the +Ca2+ and -Ca2+ states. More importantly, the data for the -Ca2+ state suggest a long extension of the troponin IT arm, consisting mainly of TnI. Thus, the troponin complex undergoes a large structural change triggered by Ca2+ binding.     

Shape and size of bovine rhodopsin: A small-angle X-ray scattering study of a rhodopsin-detergent complex

Rhodopsin is extracted from rod outer segments of retinas with dodecyldimethylamine oxide (DDAO), a non-ionie detergent. The rhodopsin-DDAO complex is characterized by binding experiments, gel filtration, sedimentation, densimetry; its homogeneity, chemical composition, weight and partial specific volume are determined. The complex turns out to be a reasonably monodisperse association of one rhodopsin and 156 DDAO molecules. The rhodopsin-DDAO complex and the detergent micelles are studied by small-angle X-ray scattering techniques using a water/sucrose solvent of variable density. The experiments are performed on an absolute scale; mainly the value and curvature of the scattering curves at zero angle are exploited. The structure of the complex and of the micelles is shown to be independent of sucrose. Under these conditions the final result of the X-ray scattering study of each type of particle is the numerical value of a set of five parameters: molecular weight, volume and radius of gyration of the volume occupied by the particles, average electron density and second moment of the electron density fluctuations inside the particles. It is also shown that in the complex the centres of gravity of rhodopsin and of the detergent moiety are very near to each other. The analysis of these parameters leads to the determination of the size and shape of the detergent micelles and to an estimate of the size and shape of the volumes occupied by protein and by detergent in the complex. We find rhodopsin to be a very elongated molecule (maximum diameter ~95 Å) which spans a flat detergent micelle. These results suggest that in the rod outer segment discs the rhodopsin molecules span the membranes, that the rhodopsin molecules of the two opposite membranes of each disc come near to each other and that a high fraction of the intra-disc space is occupied by rhodopsin.     

Structural resolution of the complex between a fungal polygalacturonase and a plant polygalacturonase-inhibiting protein by small-angle X-ray scattering

We report here the low-resolution structure of the complex formed by the endo-polygalacturonase from Fusarium phyllophilum and one of the polygalacturonase-inhibiting protein from Phaseolus vulgaris after chemical cross-linking as determined by small-angle x-ray scattering analysis. The inhibitor engages its concave surface of the leucine-rich repeat domain with the enzyme. Both sides of the enzyme active site cleft interact with the inhibitor, accounting for the competitive mechanism of inhibition observed. The structure is in agreement with previous site-directed mutagenesis data and has been further validated with structure-guided mutations and subsequent assay of the inhibitory activity. The structure of the complex may help the design of inhibitors with improved or new recognition capabilities to be used for crop protection.     

Determination of the fraction composition of blood lipoproteins by the small-angle X-ray scattering technique.

A new method for analyzing the fraction composition of blood lipoproteins (LP) was developed based on the small-angle X-ray scattering (SAXS) technique. The method allows quantitative determination of the contents of basic LP fractions (high-density LP, low-density LP, very low-density LP and their subfractions) in the blood plasma or serum. The results of LP analysis by the new method were compared with electron microscopy, ultracentrifugation and gel electrophoresis data. The results obtained by SAXS correlated with those obtained by traditional methods. The new method for the determination of the LP fraction composition in the blood is rapid (1-1.5 h), uses only one reagent (e.g., sucrose) and features a high accuracy and resolution up to LP subfractions. A total of 0.05 ml of the blood plasma or serum is required for an assay. The assays can be carried out in purified preparations or in the blood plasma or serum. The method developed can be used in clinical practice for diagnostics and in scientific research.