Solution structure of monomeric and trimeric photosystem I of <Emphasis Type="Italic">Thermosynechococcus elongatus</Emphasis> investigated by small-angle X-ray scattering

The structure of monomeric and trimeric photosystem I (PS I) of Thermosynechococcus elongatus BP1 (T. elongatus) was investigated by small-angle X-ray scattering (SAXS). The scattering data reveal that the protein–detergent complexes possess radii of gyration of 58 and 78 Å in the cases of monomeric and trimeric PS I, respectively. The results also show that the samples are monodisperse, virtually free of aggregation, and contain empty detergent micelles. The shape of the protein–detergent complexes can be well approximated by elliptical cylinders with a height of 78 Å. Monomeric PS I in buffer solution exhibits minor and major radii of the elliptical cylinder of about 50 and 85 Å, respectively. In the case of trimeric PS I, both radii are equal to about 110 Å. The latter model can be shown to accommodate three elliptical cylinders equal to those describing monomeric PS I. A structure reconstitution also reveals that the protein–detergent complexes are larger than their respective crystal structures. The reconstituted structures are larger by about 20 Å mainly in the region of the hydrophobic surfaces of the monomeric and trimeric PS I complexes. This seeming contradiction can be resolved by the addition of a detergent belt constituted by a monolayer of dodecyl-β-D-maltoside molecules. Assuming a closest possible packing, a number of roughly 1024 and 1472 detergent molecules can be determined for monomeric and trimeric PS I, respectively. Taking the monolayer of detergent molecules into account, the solution structure can be almost perfectly modeled by the crystal structures of monomeric and trimeric PS I.     

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.     

Small-angle X-ray studies of the human immunoglobulin molecule Kol.

The conformation of the human immunoglobulin molecul Kol [IgG I, kappa2 gamma2, Gm(f)+] was studied by small-angle X-ray scattering in 0.15 M NaCl solution. The radius of gyration was found to be 5.84 +/- 0.04 nm, the volume 329 +/- 15 nm3 and the molecular weight 150 000 +/- 10 000. Information on the overall shape was obtained by comparing the experimental scattering curve with the calculated curves for various models. The models were obtained by arranging the models found for the Fab and Fc fragments of the same immunoglobulin molecule in a different manner. A model which fits all the date and the form of the experimental scattering curve is presented.     

Photochemical Electron Transport in Photosynthetic Reaction Centers from Rhodopseudomonas spheroides: I. Kinetics of the Oxidation and Reduction of P-870 As Affected by External Factors

Photosynthetic reaction centers from Rhodopseudomonas spheroides were prepared with the detergent lauryl dimethylamine oxide (LDAO). In contrast to reaction centers made with Triton X-100, these contained no cytochromes and little or no ubiquinone (UQ). The reduction of P-870, after its photochemical oxidation, was studied in these materials with the following results. In reaction centers made with Triton X-100, slow kinetic components (seconds to minutes) could be attributed to secondary electron acceptors or traps. In reaction centers made with LDAO the kinetics were predominantly fast (half-times, 100 msec or less); slower components could be introduced by adding UQ. Added UQ appeared to become bound to reaction centers made with LDAO, but the binding might have meant only that both components were trapped within detergent micelles. Ferricyanide could retard the reduction of oxidized P-870, apparently by capturing electrons from the reducing side of the photochemical system. Under conditions in which the participation of secondary electron acceptors seemed to have been eliminated, the recovery of P-870 was mainly by a first-order process with a half-time of about 60 msec at room temperature and 20-30 msec at about -80°C and below. The transition with decreasing temperature suggested the presence of a mixed population, exhibiting both the 60 and 20 msec components, but variations in the absorption spectra with temperature did not suggest the presence of a mixed population. Absorption difference spectra in the ultraviolet were compatible with the idea that UQ added to reaction centers became reduced in the light.     

Small-angle X-ray scattering study of ribosomal proteins S3, S4 S7 and s20.

Ribosomal proteins S3, S4, S7, S20 from Escherichia coli have been studied by small-angle X-ray scattering techniques. The molecular weights found by X-ray scattering agree with other determinations. The large values of the radii of gyration indicate anisometric particles. A more detailed morphological analysis is hindered by low solubility. An interpretation of the experimental results is given in terms of compact objects of simple shape. Yet the possibility is envisaged that these proteins may be less rigid and compact than other proteins.     

Small-angle X-ray scattering studies of Escherichia colil-asparaginase

Small angle X-ray scattering studies on Escherichia colil-asparaginase solutions show that the enzyme has a radius of gyration of 34.0 Å ± 0.5 Å at pH 7. The radius of gyration of the dissociated monomer is 16.0 Å ± 1.0 Å; it has the general shape of a prolate ellipsoid with an axial ratio of 1.4. A tetramer of four such ellipsoids arranged with 222 symmetry gives good agreement between measured and calculated radii of gyration if the distance between subunit centers is 43 Å. The tetramer dissociates on dilution below 1% and at pH values below 3.0. Acid-induced denaturation at pH 2.0 is irreversible in contrast to the reversible guanidine-HCl-induced denaturation.     

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.     

Solution structures of DNA-bound gyrase

The DNA gyrase negative supercoiling mechanism involves the assembly of a large gyrase/DNA complex and conformational rearrangements coupled to ATP hydrolysis. To establish the complex arrangement that directs the reaction towards negative supercoiling, bacterial gyrase complexes bound to 137- or 217-bp DNA fragments representing the starting conformational state of the catalytic cycle were characterized by sedimentation velocity and small-angle X-ray scattering (SAXS) experiments. The experiments revealed elongated complexes with hydrodynamic radii of 70–80 Å. Molecular envelopes calculated from these SAXS data show 2-fold symmetric molecules with the C-terminal domain (CTD) of the A subunit and the ATPase domain of the B subunit at opposite ends of the complexes. The proposed gyrase model, with the DNA binding along the sides of the molecule and wrapping around the CTDs located near the exit gate of the protein, adds new information on the mechanism of DNA negative supercoiling.     

Bilayer structure of dihexadecyl phosphate

The X-ray scattering data of dihexadecyl phosphate (DHP) were analyzed by the distance distribution function directly obtained from the scattered intensity by Fourier transformation with no prior assumption about size and shape. The detergent in aqueous solutions is a random distribution of lamellae with a thickness of 58 Å and consistent with a hollow vesicular structure of an outer diameter of 340 Å with some overall size heterogeneity. The electron density profile perpendicular to the lamellar plane indicates a bilayer as the underlying structural principle.     

Probing the Conformation of FhaC with Small-Angle Neutron Scattering and Molecular Modeling

Probing the solution structure of membrane proteins represents a formidable challenge, particularly when using small-angle scattering. Detergent molecules often present residual scattering contributions even at their match point in small-angle neutron scattering (SANS) measurements. Here, we studied the conformation of FhaC, the outer-membrane, β-barrel transporter of the Bordetella pertussis filamentous hemagglutinin adhesin. SANS measurements were performed on homogeneous solutions of FhaC solubilized in n-octyl-d17-βD-glucoside and on a variant devoid of the α helix H1, which critically obstructs the FhaC pore, in two solvent conditions corresponding to the match points of the protein and the detergent, respectively. Protein-bound detergent amounted to 142 ± 10 mol/mol as determined by analytical ultracentrifugation. By using molecular modeling and starting from three distinct conformations of FhaC and its variant embedded in lipid bilayers, we generated ensembles of protein-detergent arrangement models with 120–160 detergent molecules. The scattered curves were back-calculated for each model and compared with experimental data. Good fits were obtained for relatively compact, connected detergent belts, which occasionally displayed small detergent-free patches on the outer surface of the β barrel. The combination of SANS and modeling clearly enabled us to infer the solution structure of FhaC, with H1 inside the pore as in the crystal structure. We believe that our strategy of combining explicit atomic detergent modeling with SANS measurements has significant potential for structural studies of other detergent-solubilized membrane proteins.     

Use of nile red as a fluorescent probe for the study of the hydrophobic properties of protein-sodium dodecyl sulfate complexes in solution.

Our results show that the noncovalent dye 9-diethylamino-5H-benzo[alpha]phenoxazine-5-one (Nile red) can be used as a fluorescent probe to study the hydrophobic properties of proteins associated with the anionic detergent sodium dodecyl sulfate (SDS). Nile red can interact with both SDS micelles and protein-SDS complexes. The enhancement of Nile red fluorescence observed with diverse types of proteins occurs at SDS concentrations lower than the critical micelle concentration of this detergent. This is also observed using the covalent fluorophore rhodamine B isothiocyanate. Additional results obtained in studies in solution show that the fluorescence intensity and the spectral characteristics of Nile red associated with different proteins complexed with SDS are very similar. These spectroscopic similarities are probably related to the equivalent synchrotron X-ray scattering results found for various protein-SDS complexes in solution. The scattering results suggest that SDS induces the formation of complexes in which the basic structural properties are independent of the different initial structures of native proteins. We speculate that Nile red is bound to regions with equivalent hydrophobic characteristics located in the uniform structures produced by the association of SDS with proteins.     

Semi-Rigid Solution Structures of Heparin by Constrained X-ray Scattering Modelling: New Insight into Heparin-Protein Complexes

The anionic polysaccharides heparin and heparan sulphate play essential roles in the regulation of many physiological processes. Heparin is often used as an analogue for heparan sulphate. Despite knowledge of an NMR solution structure and 19 crystal structures of heparin-protein complexes for short heparin fragments, no structures for larger heparin fragments have been reported up to now. Here, we show that solution structures for six purified heparin fragments dp6-dp36 (where dp stands for degree of polymerisation) can be determined by a combination of analytical ultracentrifugation, synchrotron X-ray scattering, and constrained modelling. Analytical ultracentrifugation velocity data for dp6-dp36 showed sedimentation coefficients that increased linearly from 1.09 S to 1.84 S with size. X-ray scattering of dp6-dp36 gave radii of gyration R_G that ranged from 1.33 nm to 3.12 nm and maximum lengths that ranged from 3.0 nm to 12.3 nm. The higher resolution of X-ray scattering revealed an increased bending of heparin with increased size. Constrained molecular modelling of 5000 randomised heparin conformers resulted in 9-15 best-fit structures for each of dp18, dp24, dp30, and dp36 that indicated flexibility and the presence of short linear segments in mildly bent structures. Comparisons of these solution structures with crystal structures of heparin-protein complexes revealed similar ranges of phi (φ) and psi (ψ) angles between iduronate and glucosamine rings. We conclude that heparin in solution has a semi-rigid and extended conformation that is preformed for its optimal binding to protein targets without major conformational changes.     

Determination of the topological shape of integral membrane protein light-harvesting complex LH2 from photosynthetic bacteria in the detergent solution by small-angle X-ray scattering

The topological shape of the integral membrane protein light-harvesting complex LH2 from photosynthetic bacteria Rhodobacter spheroides 2.4.1 in detergent solution has been determined from synchrotron small-angle X-ray scattering data using direct curve-fitting by the ellipsoid, ab initio shape determination methods of simulated annealing algorithm and multipole expansion, respectively. The results indicate that the LH2 protein in aqueous solution is encapsulated by a monolayered detergent shell. The detergent-stabilized structure has the shape of an oblate plate, with a thickness of 40 A, a long axis of 110 A, and a short axis of 85 A. After correction for the detergent shell, the shape of the LH2 core is also an oblate plate with a height of 40 A, a long axis of 80 A, and a short axis of 55 A. In contrast to the cylindrical crystal structure with a height of 40 A and a diameter of 68 A, the molecular shape of the LH2 complex in detergent solution clearly deviates from the ringlike crystal structure, with an eccentricity found to be 0.59-consistent with the result of single molecular spectroscopy study of the isolated single LH2 molecules.     

Protein S1 from Escherichia coli ribosomes: an improved isolation procedure and shape determination by small-angle X-ray scattering.

Ribosomal protein S1 from Escherichia coli was studied in solution by small-angle X-ray scattering and the following parameters were obtained. The radius of gyration R = 8.0 +/- 0.2 nm; largest diameter D = 28 nm; molecular weight = (8--9) x 10(4). The data also yielded (with the assumption of a rigid particle with almost constant electron density) two radii of gyration of cross-section Rq1 = 2.5 +/- 0.1 nm and Rq2 = 1.05 +/- 0.05 nm and molecular volume = 140 nm3. The experimental scattering curve of S1 was compared with the theoretical scattering curves for several rigid triaxial homogeneous bodies and the closest fit was given by that of a flat elliptical cylinder with the dimensions of 4.5 nm and 0.88 nm for the two semiaxes and 26.5 nm for height. The results from the present X-ray scattering studies and those from limited proteolytic digestion of protein S1 [J. Mol. Biol. 127, 41--54, (1979)] support the notion that the structure of protein S1 is organized into two distinct subdomains within its elongated overall shape. Protein S1 was purified for this study by an efficient procedure which yielded 12 mg S1/g ribosomes. The isolated protein was fully active in functional tests both before and after X-ray irradiation.     

Properties of reaction centers of Rhodopseudomonas sphaeroides in dried gelatin films.: Linear dichroism and low temperature spectra

Methods of preparing dried gelatin films containing purified reaction centers of Rhodopseudomonas sphaeroides are described. The spectral properties of reaction centers in solution are essentially maintained in dried gelatin films. These films are uniform and have excellent optical properties, showing little particulate scattering at temperatures down to about 4K. Film contraction on cooling to 90K is less than 1% in linear dimension. Linear dichroism spectra are reported for films at room and low temperature. Reaction centers show a moderate amount of linear dichroism in unstretched gelatin films; the magnitude of the linear dichroism becomes much greater when the films are stretched. In stretched films, linear dichroic ratios ($\text{A}{}_{\mathrm{\parallel }}\text{A}{}_{\mathrm{\perp }}$; absorbance measured with electric vector parallel and perpendicular to stretching direction) between 1.7 and 2.2 were obtained for the 860 nm absorption band of the bacteriochlorophyll component that undergoes primary photooxidation. The relative polarizations of light-induced absorption changes of reaction centers in stretched films are similar to those reported by Vermeglio and Clayton ((1976) Biochim. Biophys. Acta 449, 500–515) and support their hypothesis that absorbance decreases, maximal near 860 and 810 nm, and an increase near 790 nm are associated with the respective disappearance and appearance of discrete bands characteristic of the reduced and oxidized bacteriochlorophyll dimer. This interpretation is also supported by the polarization of the absolute absorption spectrum near 810 and 860 nm. An absorption band near 540 nm, ascribed to the Q_x transitions of two molecules of bacteriopheophytin in the reaction center, is split at low temperatures into two bands having similar polarizations. This splitting is probably not due to exciton coupling of the two molecules, since excition theory predicts different polarizations.     

Analysis of models of irregular shape by solution X-ray scattering: the case of the 50S ribosomal subunit from E. coli

Structural models of biological macromolecules can be tested by comparing calculated and experimental solution scattering curves. We have developed an approach for computing scattering shape functions at medium resolution from models proposed on the basis of other techniques such as electron microscopy. We present the results obtained with the 50S ribosomal subunit from Escherichia coli; two models are considered, one proposed by Lake (1976), the other one by Tischendorf et al. (1975). Although the two models are similar in many respects, their scattering shape functions are significantly different. The comparison with the experimental scattering curve allows us to check the scale of the models and, after scaling, to quantitate the agreement between the observed and the calculated curves. Finally, it can provide a starting point for the structural interpretation of the X-ray data.     

The Internal Organization of Mycobacterial Partition Assembly: Does the DNA Wrap a Protein Core?

Before cell division in many bacteria, the ParBs spread on a large segment of DNA encompassing the origin-proximal parS site(s) to form the partition assembly that participates in chromosome segregation. Little is known about the structural organization of chromosomal partition assembly. We report solution X-ray and neutron scattering data characterizing the size parameters and internal organization of a nucleoprotein assembly formed by the mycobacterial chromosomal ParB and a 120-meric DNA containing a parS-encompassing region from the mycobacterial genome. The cross-sectional radii of gyration and linear mass density describing the rod-like ParB-DNA assembly were determined from solution scattering. A “DNA outside, protein inside” mode of partition assembly organization consistent with the neutron scattering hydrogen/deuterium contrast variation data is discussed. In this organization, the high scattering DNA is positioned towards the outer region of the partition assembly. The new results presented here provide a basis for understanding how ParBs organize the parS-proximal chromosome, thus setting the stage for further interactions with the DNA condensins, the origin tethering factors and the ParA.     

Physicochemical characterization of dodecylphosphocholine/palmitoyllysophosphatidic acid/myelin basic protein complexes.

The stoichiometry of dodecylphosphocholine/palmitoyllysophosphatidic acid/myelin basic protein complexes and the location of the protein in the micelles have been investigated by electron paramagnetic resonance, ultracentrifugation, small-angle X-ray scattering, 31P, 13C, and 1H nuclear magnetic resonance spectroscopy, and electron microscopy. Ultracentrifugation measurements indicated that well-defined complexes are formed by association of one protein molecule with approximately 133 detergent molecules. The spin-labels 5-, 12-, and 16-doxylstearate have been incorporated into detergent/protein aggregates. Electron paramagnetic resonance spectral parameters and 13C and 1H nuclear magnetic resonance relaxation times showed that the addition of myelin basic protein does not affect the environment and location of the labels or the organization of the micelles. Previous results suggesting that the protein lies primarily near the surface of the micelles have been confirmed by comparing 13C spectra of the detergents with and without protein with spectra of detergent/protein aggregates containing the spin labels. Electron micrographs of the complexes taken by using the freeze-fracture technique revealed the presence of particles with an estimated radius about three times the radius of the micelles measured by small-angle X-ray scattering. The structural integrity of the complexes appears to be based on intramolecular protein interactions as well as protein-detergent interactions.     

Dynamic light scattering: a practical guide and applications in biomedical sciences

Dynamic light scattering (DLS), also known as photon correlation spectroscopy (PCS), is a very powerful tool for studying the diffusion behaviour of macromolecules in solution. The diffusion coefficient, and hence the hydrodynamic radii calculated from it, depends on the size and shape of macromolecules. In this review, we provide evidence of the usefulness of DLS to study the homogeneity of proteins, nucleic acids, and complexes of protein–protein or protein–nucleic acid preparations, as well as to study protein–small molecule interactions. Further, we provide examples of DLS’s application both as a complementary method to analytical ultracentrifugation studies and as a screening tool to validate solution scattering models using determined hydrodynamic radii.