Structural stability and solution structure of chaperonin GroES heptamer studied by synchrotron small-angle X-ray scattering

The GroES protein from Escherichia coli is a well-known member of the molecular chaperones. GroES consists of seven identical 10 kDa subunits, and forms a dome-like oligomeric structure. In order to obtain information on the structural stability and unfolding-refolding mechanism of GroES protein, especially at protein concentrations (0.4-1.2 mM GroES monomer) that would mimic heat stress conditions in vivo, we have performed synchrotron small-angle X-ray scattering (SAXS) experiments. Surprisingly, in spite of the high protein concentration, reversibility in the unfolding-refolding reaction was confirmed by SAXS experiments structurally. Although the unfolding-refolding reaction showed an apparent single transition with a Cm of 1.1 M guanidium hydrochloride, a more detailed analysis of this transition demonstrated that the unfolding mechanism could be best explained by a sequential three-state model, which consists of native heptamer, dissociated monomer, and unfolded monomer. Together with our previous result that GroES unfolded completely via a partially folded monomer according to a three-state model at low protein concentration (5 microM monomer), the unfolding-refolding mechanism of GroES protein could be explained uniformly by the three-state model from low to high protein concentrations. Furthermore, to clarify an ambiguity of the native GroES structure in solution, especially mobile loop structures, we have estimated a solution structure of GroES using SAXS profiles obtained from experiments and simulation analysis. The result suggested that the native structure of GroES in solution was very similar to that seen in GroES-GroEL complex determined by crystallography.     

Temperature dependence of the structure of aggregates of tobacco mosaic virus protein at pH 7.2. Static synchrotron small-angle X-ray scattering

The small-angle X-ray scattering (SAXS) method using a synchrotron radiation source was applied to the study of the self-aggregation process of tobacco mosaic virus protein (TMVP) at a concentration of 5.0 or 12.0 mg ml-1 in 50 mM or 100 mM-phosphate buffer (ionic strengths approx. 0.1 and 0.2, respectively) at pH 7.2 in the temperature region of 4.8 to 25.0 degrees C. This paper presents the results of static measurements of SAXS. Sedimentation velocity experiments were performed simultaneously under the same conditions. These results are qualitatively parallel to those of the SAXS measurements, although the size of stacked disks derived from the SAXS measurements is larger than that derived from the sedimentation experiments, suggesting a change in the equilibrium conditions in the centrifugal field. Qualitative analysis of the SAXS data with model simulation calculations implies that the aggregation of TMVP consists of two steps: (1) the aggregation of A-protein comprising a few subunits to form double-layered disks; and (2) the random polymerization of double-layered disks by disk-stacking. Increase in temperature, ionic strength or protein concentration induced TMVP to polymerize to form a double-layered disk or a quadruple-layered short rod with consumption of A-proteins, accompanied by a small number of multi-layered short rods. The SAXS results indicate that the A-protein and the multilayered short rods are polydisperse with respect to size and shape, i.e. the mixture of A-protein, double-layered disks and multi-layered short rods coexists in the equilibrium state without pressure-induced partial dissociation of TMPV as observed during normal ultracentrifugation, and even under solution conditions in which the formation of double-layered disks or higher-order aggregates is favored.     

Supramolecular structure of lipopolysaccharide and free lipid A under physiological conditions as determined by synchrotron small-angle X-ray diffraction

Lipopolysaccharides, the major amphiphilic components of the outer leaflet of the outer membrane of Gram-negative bacteria, may assume various three-dimensional supramolecular structures depending on molecular properties (e.g. chemical structure) and on ambient conditions (e.g. temperature, concentration of divalent cations). We applied synchrotron small-angle X-ray diffraction to investigate the supramolecular structures of natural and synthetic Escherichia-coli-type lipid A, of lipid A from Salmonella minnesota, and of rough mutant lipopolysaccharides of E. coli and S. minnesota under physiological water content (greater than 90%) at different temperatures (20, 37, and 55 degrees C) and at different lipid/divalent cation molar ratios (20:1 to 1:1). We found that in the absence of divalent cations rough mutant lipopolysaccharide and free lipid A form unilamellar structures with the main reflections centered around 4.50 nm for free lipid A, 4.80 nm for Re lipopolysaccharide, and 5.90 nm for Rd1 lipopolysaccharide at 20 degrees C, i.e. below the beta----alpha acyl-chain-melting transition temperature. Above this temperature, the reflections are shifted to 4.30 nm for free lipid A (at 55 degrees C), 4.60 nm for Re lipopolysaccharide (at 37 degrees C), and to 5.50 nm for Rd1 lipopolysaccharide (at 37 degrees C). The addition of divalent cations leads (at lower concentrations, i.e. lipid/cation molar ratios 20:1 to 5:1) to sharper reflections expressing a higher state of order and to a shift of the center of the main reflections lying now at 5.10 nm for free lipid A, 6.40 nm for Re and 7.20 nm for Rd1 lipopolysaccharide at 20 degrees C. At higher concentrations of divalent cations (e.g. lipid/cation molar ratio 1:1), an increasing tendency to form nonlamellar, inverted cubic structures is observed which is indicated by the occurrence of another main periodicity and/or of reflections with spacing ratios 1: square root of 2, 1: square root of 3 of the main periodicity. The tendency to assume inverted cubic structures is only weakly pronounced for rough mutant lipopolysaccharides but dominant for free lipid A even at physiological temperature and divalent cation concentration.     

Quaternary structure of flavorubredoxin as revealed by synchrotron radiation small-angle X-ray scattering

Flavodiiron proteins (FDP) are modular enzymes which function as NO and/or O(2) reductases. Although the majority is composed of two structural domains, the homolog found in Escherichia coli, flavorubredoxin, possesses an extra C-terminal module consisting of a linker and a rubredoxin (Rd) domain necessary for interprotein redox processes. In order to investigate the location of the Rd domain with respect to the flavodiiron structural core, small-angle X-ray scattering was used to construct low-resolution structural models of flavorubredoxin. Scattering patterns from the Rd domain, the FDP core, and full-length flavorubredoxin were collected. The latter two species were found to be tetrameric in solution. Ab initio shape reconstruction and rigid-body modeling indicate a peripheral location for the Rd domains, which appear to have weak contacts with the FDP core. This finding suggests that Rd behaves as an independent domain and is freely available to participate in redox reactions with protein partners.     

Temperature change of the ripple structure in fully hydrated dimyristoylphosphatidylcholine/cholesterol multibilayers.

The ripple structure was studied as a function of temperature in fully hydrated dimyristoylphosphatidylcholine (DMPC)/cholesterol multibilayers using synchrotron x-ray small-angle diffraction and freeze-fracture electron microscopy. In the presence of cholesterol, the ripple structure appears below the pretransition temperature of pure DMPC multibilayers. In this temperature range the ripple periodicity is relatively large (25-30 nm) and rapidly decreases with increasing temperature. In this region, defined as region I, we observed coexistence of the P beta' phase and the L beta' phase. The large ripple periodicity is caused by the formation of the P beta' phase region in which cholesterol is concentrated and the L beta' phase region from which cholesterol is excluded. An increase in ripple periodicity also takes place in the narrow temperature range just below the main transition temperature. We define this temperature region as region III, where the ripple periodicity increases dramatically toward the main transition temperature. In region II, between regions I and III, the ripple periodicity decreases gradually with temperature. This behavior is quite similar to that of pure DMPC. Temperature-versus-ripple periodicity curves are parallel among pure DMPC and DMPCs with various cholesterol contents. We explain this behavior in terms of a model proposed by other workers.     

The structure of DNA-DOPC aggregates formed in presence of calcium and magnesium ions: A small-angle synchrotron X-ray diffraction study

The structure of aggregates formed due to DNA interaction with dioleoylphosphatidylcholine (DOPC) vesicles in presence of Ca²⁺ and Mg²⁺ cations was investigated using synchrotron small-angle X-ray diffraction. For DOPC/DNA = 1:1 mol/base and in the range of concentration of the cation²⁺ 0-76.5 mM, the diffractograms show the coexistence of two lamellar phases: L^x phase with repeat distance d_Lx ∼ 8.26-7.39 nm identified as a phase where the DNA strands are intercalated in water layers between adjacent lipid bilayers, and L_DOPC phase with repeat distance d_DOPC ∼ 6.45-5.65 nm identified as a phase of partially dehydrated DOPC bilayers without any divalent cations and DNA strands. The coexistence of these phases was investigated as a function of DOPC/DNA molar ratio, length of DNA fragments and temperature. If the amount of lipid increases, the fraction of partially dehydrated L_DOPC phase is limited, depends on the portion of DNA in the sample and also on the length of DNA fragments. Thermal behaviour of DOPC + DNA + Ca²⁺ aggregates was investigated in the range 20-80 °C. The transversal thermal expansivities of both phases were evaluated.     

The structure of DNA-DOPC aggregates formed in presence of calcium and magnesium ions: a small-angle synchrotron X-ray diffraction study

The structure of aggregates formed due to DNA interaction with dioleoylphosphatidylcholine (DOPC) vesicles in presence of Ca(2+) and Mg(2+) cations was investigated using synchrotron small-angle X-ray diffraction. For DOPC/DNA=1:1 mol/base and in the range of concentration of the cation(2+) 0-76.5 mM, the diffractograms show the coexistence of two lamellar phases: L(x) phase with repeat distance d(Lx) approximately 8.26-7.39 nm identified as a phase where the DNA strands are intercalated in water layers between adjacent lipid bilayers, and L(DOPC) phase with repeat distance d(DOPC) approximately 6.45-5.65 nm identified as a phase of partially dehydrated DOPC bilayers without any divalent cations and DNA strands. The coexistence of these phases was investigated as a function of DOPC/DNA molar ratio, length of DNA fragments and temperature. If the amount of lipid increases, the fraction of partially dehydrated L(DOPC) phase is limited, depends on the portion of DNA in the sample and also on the length of DNA fragments. Thermal behaviour of DOPC+DNA+Ca(2+) aggregates was investigated in the range 20-80 degrees C. The transversal thermal expansivities of both phases were evaluated.     

Structural characterization of lactate dehydrogenase dissociation under high pressure studied by synchrotron high-pressure small-angle X-ray scattering.

The effect of high pressure on lactate dehydrogenase (LDH) was studied using small-angle X-ray scattering (SAXS). The SAXS results are interpreted in terms of the dissociation and association of LDH within a compression and decompression cycle and its temperature dependence. LDH consists of four identical subunits. At 120 MPa and 25 degrees C, 50% of the LDH dissociates into subunits, while at 10 degrees C, this occurs at 78 MPa. The hysteresis in the dissociation and association under pressure was confirmed in terms of the radius of gyration and was seen to be more conspicuous at low temperature. Forward scattering, I(0)/C, which is proportional to molecular weight, showed that LDH dissociated into dimer (not monomer) subunits under pressure. The application of high pressure to dissociated dimers induced irreversible aggregation. This result is in sharp contrast with the result of fluorescence spectroscopy suggesting a dissociated monomer [King, L., and Weber, G. (1986) Biochemistry 25, 3637-3640]. As for structural change after reassociation, there was little structural difference between native and drifted LDH. The difference was smaller than the structure change by ligand binding. At 200 MPa, the presence of five scattering peaks in the medium-angle region indicates that the dissociated dimer does not have a molten globule-like structure but a core structure. We propose a model of the dissociated dimer, based on the SAXS profile, in which the volume is reduced without disrupting the core structure.     

Histological structure of human nail as studied by synchrotron X-ray microdiffraction.

Three layers (characterized by different orientations of the keratin molecules) from the outer to the inner side of human nail were observed by synchrotron X-ray microdiffraction. These layers are associated with the histological dorsal, intermediate and ventral plates. The hair-like type alpha-keratin filaments (81 A in diameter), are only present in the intermediate layer (accounting for approximately 2/3 of the nail width) and are perfectly oriented perpendicular to the growth axis, in the nail plane. Keratin filaments of stratum corneum (epidermis) type, found in the dorsal and ventral cells, are oriented in two privileged directions; parallel and perpendicular to the growth axis. This "sandwich" structure in the corneocytes and the strong intercellular junctions, gives the nail high mechanical rigidity and hardness, both in the curvature direction and in the growth direction. Lipid bilayers (49 A thick) parallel to the nail surface fill certain ampullar dilations of the dorsal plate and intercellular spaces in the ventral plate. Using X-ray micro-diffraction, we show that onychomycosis disrupts the keratin structure, probably during the synthesis phase.     

The solution structure and activation of visual arrestin studied by small-angle X-ray scattering.

Visual arrestin is converted from a 'basal' state to an 'activated' state by interaction with the phosphorylated C-terminus of photoactivated rhodopsin (R*), but the conformational changes in arrestin that lead to activation are unknown. Small-angle X-ray scattering (SAXS) was used to investigate the solution structure of arrestin and characterize changes attendant upon activation. Wild-type arrestin forms dimers with a dissociation constant of 60 micro m. Small conformational changes, consistent with local movements of loops or the mobile N- or C-termini of arrestin, were observed in the presence of a phosphopeptide corresponding to the C-terminus of rhodopsin, and with an R175Q mutant. Because both the phosphopeptide and the R175Q mutation promote binding to unphosphorylated R*, we conclude that arrestin is activated by subtle conformational changes. Most of the arrestin will be in a dimeric state in vivo. Using the arrestin structure as a guide [Hirsch, J.A., Schubert, C., Gurevich, V.V. & Sigler, P.B. (1999) Cell 97, 257-269], we have identified a model for the arrestin dimer that is consistent with our SAXS data. In this model, dimerization is mediated by the C-terminal domain of arrestin, leaving the N-terminal domains free for interaction with phosphorylated R*.     

Structural properties of a phosphatidylcholine-cholesterol system as studied by small-angle neutron scattering: ripple structure and phase diagram

Small-angle neutron scattering has been used to study structural features of lamellar bilayer membranes of dimyristoylphosphatidylcholine (DMPC) and DMPC mixed with various amount of cholesterol. The studies were recorded at a fixed hydration level of 17% 2H2O, i.e. just below saturation. Bragg reflections gives information on the ripple structure and on the bilayer periodicity. The crystalline Lc phase, which was stabilized after long time storage at low temperature, exhibits major small angle scattering when cholesterol is mixed into the membrane. The intermediate P beta' gel-phase, which is characteristic by the rippled structure, is dramatically stabilized by the introduction of cholesterol. The ripple structure depends significantly both on the cholesterol content and on the temperature. At high temperatures, T greater than 15 degrees C, the inverse ripple periodicity varies basically linearly with cholesterol content, and approach zero (i.e. periodicity goes to infinite) at 20 mol% cholesterol, approximately. At lower temperatures the correlation is more complex. The data indicate additional phase boundaries below 2 mol% and at approx. 8 mol%. Secondary rippled structures are observed in the low temperature L beta'-phase for cholesterol content below approx. 8 mol%. The data gives detailed insight into the phosphatidylcholine cholesterol phase diagram, which is discussed on the basis of a simple model in which the cholesterol complexes are fixed to the defect stripes of the rippled structure.     

Kinetics of pretransition in multilamellar dimyristoylphosphatidylcholine vesicle X-ray diffraction study

The time course of transformation between P′_β phase and L′_β phase in a multilamellar dimyristoylphosphatidylcholine vesicle containing 50% water was followed by small-angle X-ray diffraction. The transformation of P′_β → L′_β was 90% complete at 7 min after the temperature jump, although imperfections remained for a long time. Transformation in the opposite direction was fast as compared with P′_β → L′_β.     

Influence of local anesthetics on the phosphatidylcholine model membrane: small-angle synchrotron X-ray diffraction and neutron scattering study

The phase preferences of egg yolk phosphatidylcholine (EYPC) have been examined in the presence of tertiary amine anesthetics [2-(propyloxy)phenyl]-2-(1-piperidinyl)ethyl ester of carbamic acid (C3A) and [2-(heptyloxy)phenyl]-2-(1-piperidinyl)ethyl ester of carbamic acid (C7A, heptacaine). Using the synchrotron small-angle X-ray diffraction (SAXD), it is shown that the C3A anesthetic induces the cubic and hexagonal (H(I)) phases at 2 > or = C3A:EYPC > 0.5 and H2O:EYPC < or = 40 molar ratios. In contrast, longer alkyloxy chain homolog C7A has no effect on the bilayer arrangement of EYPC at C7A:EYPC < = 1 molar ratios as observed by SAXD in C7A + EYPC mixtures hydrated at H2O:EYPC < = 40 molar ratios, as well as in sonicated C7A + EYPC mixtures hydrated in excess water as proved by the small-angle neutron scattering (SANS). The bilayer thickness d(L) decreases and the bilayer C7A surface area SC7A increases with the increase of C7A:EYPC molar ratio. It is suggested that the ability of tertiary amine local anesthetics to influence the dL and SC7A values and EYPC polymorphism is caused by their effective molecular shape and by charge. The possibility that anesthetic molecules may exert some of their biological effects by virtue of these properties is discussed.     

Interactions of hydrophobin proteins in solution studied by small-angle X-ray scattering

Hydrophobins are a group of very surface-active, fungal proteins known to self-assemble on various hydrophobic/hydrophilic interfaces. The self-assembled films coat fungal structures and mediate their attachment to surfaces. Hydrophobins are also soluble in water. Here, the association of hydrophobins HFBI and HFBII from Trichoderma reesei in aqueous solution was studied using small-angle x-ray scattering. Both HFBI and HFBII exist mainly as tetramers in solution in the concentration range 0.5-10 mg/ml. The assemblies of HFBII dissociate more easily than those of HFBI, which can tolerate changes of pH from 3 to 9 and temperatures in the range 5°C-60°C. The self-association of HFBI and HFBII is mainly driven by the hydrophobic effect, and addition of salts along the Hofmeister series promotes the formation of larger assemblies, whereas ethanol breaks the tetramers into monomers. The possibility that the oligomers in solution form the building blocks of the self-assembled film at the air/water interface is discussed.     

pH-dependent unfolding of aspergillopepsin II studied by small-angle X-ray scattering

Aspergillopepsin II (EC 3.4.23.6) secreted from the fungus Aspergillus niger var. macrosporus is a non-pepsin-type acid proteinase. It consists of two polypeptide chains (i.e., a heavy chain and a light chain), which are bound noncovalently to each other. The pH titration analysis using small-angle X-ray scattering (SAXS) as well as circular dichroism (CD) and gel filtration indicated that the enzyme was unfolded around a neutral pH with concomitant dissociation of the two chains. Detailed analyses showed that the midpoint pH values for the unfolding are not coincident with one another (pH 6.1 in circular dichroism and gel filtration, pH 6.4 in zero-angle intensity of SAXS, pH 6.8 in radius of gyration). The difference between these values suggested the existence of an intermediate state during the unfolding. Further analyses of the SAXS data showed that the heavy chain just after the dissociation still kept molecular compactness and that it gradually increased its dimensions as the pH was further raised. Noncoincidence of the two phenomena (i.e., chain dissociation and swelling) led to elucidation of a novel intermediate state during unfolding, which was confirmed by the subsequent singular value decomposition (SVD) analysis.     

Dynamic mechanism of the self-assembly process of tobacco mosaic virus protein studied by rapid temperature-jump small-angle X-ray scattering using synchrotron radiation

The self-assembly process of tobacco mosaic virus protein (TMVP) was observed by rapid temperature-jump time-resolved solution X-ray small-angle scattering using synchrotron radiation. The temperature-jump device used for the X-ray measurements is rapid enough to cope with even the fastest-assembling process of TMVP, and accumulates data of reasonable signal-to-noise ratios with a minimum total counting time of 7.5 seconds. The measurements suggested that the 20 S disk of TMVP polymerized to stacked disks (short rods). The time to complete stacking varied from approximately 25 seconds to approximately 1200 seconds, depending on the solution condition and magnitude of the temperature gap. Higher protein concentration, ionic strength and temperature favoured faster association. The results were analysed in terms of a set of kinetic equations that describe the two-stage aggregation of TMVP with an equilibrium constant K1, and two rate constants k+2 and k-2 for association and dissociation of disks, respectively. The consistency of the analysis suggests that the TMVP assembly proceeds in two steps of: (1) the aggregation of A-proteins into double-layered disks; and (2) the stacking of double-layered disks. The kinetic analysis indicated that the stacking belongs to the lowest range of protein-protein interaction system.     

The complete VS ribozyme in solution studied by small-angle X-ray scattering

We have used small-angle X-ray solution scattering to obtain ab initio shape reconstructions of the complete VS ribozyme. The ribozyme occupies an electron density envelope with an irregular shape, into which helical sections have been fitted. The ribozyme is built around a core comprising a near-coaxial stack of three helices, organized by two three-way helical junctions. An additional three-way junction formed by an auxiliary helix directs the substrate stem-loop, juxtaposing the cleavage site with an internal loop to create the active complex. This is consistent with the current view of the probable mechanism of trans-esterification in which adenine and guanine nucleobases contributed by the interacting loops combine in general acid-base catalysis.     

Conformations of variably linked chimeric proteins evaluated by synchrotron X-ray small-angle scattering

We constructed chimeric proteins that consist of two green fluorescent protein variants, EBFP and EGFP, connected by flexible linkers, (GGGGS)n (n = 3 approximately 4), and helical linkers, (EAAAK)n (n = 2 approximately 5). The conformations of the chimeric proteins with the various linkers were evaluated using small-angle X-ray scattering (SAXS). The SAXS experiments showed that introducing the short helical linkers (n = 2 approximately 3) causes multimerization, while the longer linkers (n = 4 approximately 5) solvate monomeric chimeric proteins. With the moderate-length linkers (n = 4), the observed radius of gyration (R(g)) and maximum dimension (D(max)) were 38.8 A and 120 A with the flexible linker, and 40.2 A and 130 A with the helical linker, respectively. The chimeric protein with the helical linker assumed a more elongated conformation as compared to that with the flexible linker. When the length of the helical linker increased (n = 5), R(g) and D(max) increased to 43.2 A and 140 A, respectively. These results suggest that the longer helix effectively separates the two domains of the chimeric protein. Considering the connectivity of the backbone peptide of the protein, the helical linker seems to connect the two domains diagonally. Surprisingly, the chimeric proteins with the flexible linker exhibited an elongated conformation, rather than the most compact side-by-side conformation expected from the fluorescence resonance energy transfer (FRET) analysis. Furthermore, the SAXS analyses suggest that destabilization of the short helical linker causes multimerization of the chimeric proteins. Information about the global conformation of the chimeric protein is thus be necessary for optimization of the linker design.     

Solution structure of phosphorylase kinase studied using small-angle X-ray and neutron scattering.

Small-angle X-ray and neutron scattering have been used to characterize the solution structure of rabbit skeletal phosphorylase kinase. The radius of gyration of the unactivated holoenzyme determined from neutron scattering is 94 A, and its maximum dimension is approximately 275-295 A. A planar model has been constructed that is in general agreement with the dimensions of the transmission electron microscope images of negatively stained phosphorylase kinase and that gives values for the radius of gyration, maximum linear dimension, and a pair distribution function for the structure that are consistent with the scattering data.