Thermodynamic and kinetic analysis on oligomeric protein dissociation using high-pressure native PAGE velocity method.

High pressure is known to dissociate several oligomeric proteins, and regarded as an important tool to shift the oligomerization equilibrium. Native polyacrylamide gel electrophoresis (native PAGE) at high pressure can characterize the dissociates and clearly discriminate the aggregates. However, a band smearing of migration profiles often hinders more detailed analyses (Miwa et al., High Pressure Res. (2019) 39, 218-224). In this paper, we focused on the band smearing dependent on the migration velocity so as to extract both thermodynamic and kinetic parameters. We systematically perturbed the migration velocity by changing the gel concentration and carried out numerical analysis for a series of the migration profiles based on a simple dissociation reaction scheme with limited thermodynamic and kinetic parameters. Then, complete volumetric properties on oligomerization process can be available. We term the present analysis method as a high-pressure native PAGE velocity method. We also report the application of this method to revisit the pressure dissociation of tetrameric lactate dehydrogenase (LDH) from pig heart.

Simple Thermodynamic Description of the Micellar-Bilayer State Transition of Assemblies Composed of n-Octyl-ß-D-glucopyranoside and 1,2-Dioleolyl-sn-glycero-3-phosphocholine Dispersed in Aqueous Media or Supported on Solid Substrates.

In the preceding paper, we investigated a mixed assembly composed of a nonionic surfactant, n-octyl-ß-D-glucopyranoside (OG), and an amphoteric lipid, 1,2-dioleolyl-sn-glycero-3-phosphocholine (DOPC), formed on hydrophilized solid substrates immersed in aqueous solutions containing OG and DOPC. The experimental data could be interpreted in terms of the phase equilibrium; thus, the partition equilibrium profile of OG between the bulk solution phase and the supported assembly phase was obtained, as well as that between the bulk solution and the dispersed assembly. The partition equilibrium profiles suggested that micellar-bilayer state transitions occur both in the supported assembly and in the dispersed one in a roughly synchronized manner, even though there are significant discrepancies between them. In this paper, we propose a simple thermodynamic model for the micellar-bilayer transition of the dispersed and supported assembly of OG and DOPC, assuming that the micellar and bilayer states are also pseudo-phases distinct from each other. Using this model, we analyzed these partition equilibrium profiles and concluded that the transition in the supported assembly should mainly be attributed to the transition in the dispersed assembly, which is partly modified by the interaction energy between the supported assembly and the substrate.

Highly Collapsed Conformation of the Initial Folding Intermediates of ß-Lactoglobulin with Non-Native α-Helix.

In the folding of ß-lactoglobulin (ßLG), a predominantly ß-sheet protein, a transient intermediate possessing an excess amount of non-native α-helix is formed within a few milliseconds. To characterize the early folding dynamics of ßLG in terms of secondary structure content and compactness, we performed submillisecond-resolved circular dichroism (CD) and small-angle X-ray scattering (SAXS) measurements. Time-resolved CD after rapid dilution of urea showed non-native α-helix formation within 200µs. Time-resolved SAXS showed that the radius of gyration (R(g)) of the intermediate at 300 µs was 23.3±0.7 Å, indicating a considerable collapse from the unfolded state having R(g) of 35.1±7.1 Å. Further compaction to R(g) of 21.2±0.3 Å occurred with a time constant of 28±11 ms. Pair distribution functions showed that the intermediate at 300 µs comprises a single collapsed domain with a small fluctuating domain, which becomes more compact after the second collapse. Kinetic measurements in the presence of 2,2,2-trifluoroethanol showed that the intermediate at several milliseconds possessed an increased amount of α-helix but similar R(g) of 23.0±0.8 Å, suggesting similarity of the shape of the intermediate in different solvents. Consequently, the initial collapse occurs globally to a compact state with a small fluctuating domain irrespective of the non-native α-helical contents. The second collapse of the fluctuating domain occurs in accordance with the reported stabilization of the non-native helix around strand A. The non-native helix around strand A might facilitate the formation of long-range contacts required for the folding of ßLG.

High Pressure Small-Angle X-Ray Scattering.

Small-angle scattering, solution scattering from proteins in solution, reflects the shape of the scatter as a spread of electron density, which is common to protein crystallography. Although the obtained resolution of small-angle scattering is inferior to that of crystallography, it shows the global image of protein structure in solution without constraints of neighboring molecules in crystal lattice. At ambient pressure, data collection technology and analyses of small-angle scattering method developed so greatly in recent 10 years that it is recognized as one of the powerful method of structural biology. In parallel, many efforts have been made to apply this technique under high pressure. The instrumentation and interpretation of small-angle scattering under pressure, however, requires special considerations. The present chapter reviews the technological aspect of scattering from protein solution especially optimized for synchrotron X-ray sources.

Negative thermal expansibility change for dissociation of lysozyme variant amyloid protofibril.

A disulfide-deficient variant of hen lysozyme, 0SS, is known to form an amyloid protofibril spontaneously, and to dissociate into monomers at high hydrostatic pressure. We carried out native PAGE at various temperatures (20-35°C) and pressures (0.1-200 MPa), to characterize the dissociation equilibrium of disulfide-deficient variant of hen lysozyme amyloid protofibril. Based on the density profiles, the partial molar volume and thermal expansibility changes for dissociation, ΔvD and ΔeD , were obtained to be -74 cm(3) /mol at 25°C and -2.3 cm(3) mol(-1) K(-1) , respectively. The dissociation of amyloid fibril destroys the cross ß-structure, and such conformational destruction in native protein fold rarely accompanies negative thermal expansibility change. We discussed the negative thermal expansibility change in terms of hydration and structural packing of the amyloid protofibril.

Salt-induced changes in the subunit structure of the Bacillus stearothermophilus lipoate acetyltransferase.

The Bacillus stearothermophilus lipoate acetyltransferase (E2), composed of sixty identical, subunits is the core component of the pyruvate dehydrogenase complex (PDC). E2 polypeptide is composed of LD, PSBD, and CD domains. Most studies had focused on a truncated E2 that is deficient in LD and PSBD, because CD mainly contributes to maintaining the multimeric structure. We examined salt-induced changes in E2 without truncation and constructed reaction models. We speculate that in the presence of KCl, E2 is dissociated into a monomer and then assembled into an aggregative complex (C(A)) and a quasi-stable complex (C(Q)). C(A) was larger than C(Q), but smaller than intact E2. C(A) and C(Q), were dominant complexes at about neutral pH and at basic pH respectively. PDC, in which PSBD is occupied by other components, and a truncated E2 undergo dissociation only. LD-PSBD region besides CD might then contribute to the partial association of dissociated E2.

A clinical case of severe anemia in a sheep coinfected with Mycoplasma ovis and 'Candidatus Mycoplasma haemovis' in Hokkaido, Japan.

A 2-year-old East Friesian sheep imported from Australia exhibited severe anemia after contagious pustular dermatitis in Hokkaido, Japan. Hemoplasma infection was confirmed in blood smears. Both Mycoplasma ovis and 'Candidatus Mycoplasma haemovis' were detected by PCR and sequence analyses. In the epidemiological analysis, dual pathogens were detected in 6 of 12 (50.0%) sheep imported from Australia with the infected ewe at the same time, 1 of 5 (20.0%) sheep introduced from a domestic farm in Hokkaido, and in 1 of 16 (6.3%) sheep from an epidemiologically unrelated ranch. It is the first clinical case of sheep to confirm coinfection of these pathogens in Japan.

Time-resolved small-angle X-ray scattering study of the folding dynamics of barnase.

Structural changes of barnase during folding were investigated using time-resolved small-angle X-ray scattering (SAXS). The folding of barnase involves a burst-phase intermediate, sometimes designated as the denatured state under physiological conditions, D(phys), and a second hidden intermediate. Equilibrium SAXS measurements showed that the radius of gyration (R(g)) of the guanidine unfolded state (U) is 26.9±0.7 Å, which remains largely constant over a wide denaturant concentration range. Time-resolved SAXS measurements showed that the R(g) value extrapolated from kinetic R(g) data to time zero, R(g,0), is 24.3±0.1 Å, which is smaller than that of U but which is expanded from that of folding intermediates of other proteins with similar chain lengths (19 Å). After the burst-phase change, a single-exponential reduction in R(g)(2) was observed, which corresponds to the formation of the native state for the major component containing the native trans proline isomer. We estimated R(g) of the minor component of D(phys) containing the non-native cis proline isomer (D(phys,cis)) to be 25.7±0.6 Å. Moreover, R(g) of the major component of D(phys) containing the native proline isomer (D(phys,tra)) was estimated as 23.9±0.2 Å based on R(g,0). Consequently, both components of the burst-phase intermediate of barnase (D(phys,tra) and D(phys,cis)) are still largely expanded. It was inferred that D(phys) possesses the N-terminal helix and the center of the ß-sheet formed independently and that the formation of the remainder of the protein occurs in the slower phase.

Structural properties of silkworm small heat-shock proteins: sHSP19.9 and sHSP20.8.

sHSP20.8 and sHSP19.9 are silkworm small-heat shock proteins (sHSPs) comprising a number of polypeptides of molecular sizes of several tens of kilodaltons as subunits. The structural properties of sHSPs were investigated. sHSP19.9 was found to be aggregated by itself during incubation at 60 degrees C. Aggregation was suppressed in the presence of dithiothreitol and at high ionic strength. In contrast, sHSP20.8 was not aggregated. Aggregation of sHSP19.9 was partially suppressed by sHSP20.8 and in the presence of catalase as a target protein. Based on changes in small-angle X-ray scattering, it is possible that the molecular size of sHSP19.9 is larger than that of sHSP20.8, and that their molecular sizes increase with increasing temperature in a reversible, biphasic manner. sHSPs did not protect catalase from thermal inactivation, but protected it from precipitation by forming a soluble complex. sHSP20.8 and sHSP19.9 with dithiothreitol were stable against lyophilization, autoclaving at 120 degrees C, and boiling.

Filament structure, organization, and dynamics in MreB sheets.

In vivo fluorescence microscopy studies of bacterial cells have shown that the bacterial shape-determining protein and actin homolog, MreB, forms cable-like structures that spiral around the periphery of the cell. The molecular structure of these cables has yet to be established. Here we show by electron microscopy that Thermatoga maritime MreB forms complex, several mum long multilayered sheets consisting of diagonally interwoven filaments in the presence of either ATP or GTP. This architecture, in agreement with recent rheological measurements on MreB cables, may have superior mechanical properties and could be an important feature for maintaining bacterial cell shape. MreB polymers within the sheets appear to be single-stranded helical filaments rather than the linear protofilaments found in the MreB crystal structure. Sheet assembly occurs over a wide range of pH, ionic strength, and temperature. Polymerization kinetics are consistent with a cooperative assembly mechanism requiring only two steps: monomer activation followed by elongation. Steady-state TIRF microscopy studies of MreB suggest filament treadmilling while high pressure small angle x-ray scattering measurements indicate that the stability of MreB polymers is similar to that of F-actin filaments. In the presence of ADP or GDP, long, thin cables formed in which MreB was arranged in parallel as linear protofilaments. This suggests that the bacterial cell may exploit various nucleotides to generate different filament structures within cables for specific MreB-based functions.

Polymeric structures and dynamic properties of the bacterial actin AlfA.

AlfA is a recently discovered DNA segregation protein from Bacillus subtilis that is distantly related to actin and the bacterial actin homologues ParM and MreB. Here we show that AlfA mostly forms helical 7/3 filaments, with a repeat of about 180 A, that are arranged in three-dimensional bundles. Other polymorphic structures in the form of two-dimensional rafts or paracrystalline nets were also observed. Here AlfA adopted a 16/7 helical symmetry, with a repeat of about 387 A. Thin polymers consisting of several intertwining filaments also formed. Observed helical symmetries of AlfA filaments differed from those of other members of the actin family: F-actin, ParM, or MreB. Both ATP and guanosine 5'-triphosphate are able to promote rapid AlfA filament formation with almost equal efficiencies. The helical structure is only preserved under physiological salt concentrations and at a pH between 6.4 and 7.4, the physiological range of the cytoplasm of B. subtilis. Polymerization kinetics are extremely rapid and compatible with a cooperative assembly mechanism requiring only two steps: monomer activation followed by elongation, making AlfA one of the most efficient polymerizing motors within the actin family. Phosphate release lags behind polymerization, and time-lapse total internal reflection fluorescence images of AlfA bundles are consistent with treadmilling rather than dynamic microtubule-like instability. High-pressure small angle X-ray scattering experiments reveal that the stability of AlfA filaments is intermediate between the stability of ParM and the stability of F-actin. These results emphasize that actin-like polymerizing machineries have diverged to produce a variety of filament geometries with diverse properties that are tailored for specific biological processes.

RecR forms a ring-like tetramer that encircles dsDNA by forming a complex with RecF.

In the RecFOR pathway, the RecF and RecR proteins form a complex that binds to DNA and exerts multiple functions, including directing the loading of RecA onto single-stranded (ss) DNA regions near double-stranded (ds) DNA-ssDNA junctions and preventing it from forming a filament beyond the ssDNA region. However, neither the structure of the RecFR complex nor its DNA-binding mechanism was previously identified. Here, size-exclusion chromatography and small-angle X-ray scattering data indicate that Thermus thermophilus (tt) RecR binds to ttRecF to form a globular structure consisting of four ttRecR and two ttRecF monomers. In addition, a low resolution model shows a cavity in the central part of the complex, suggesting that ttRecR forms a ring-like tetramer inside the ttRecFR complex. Mutant ttRecR proteins lacking the N- or C-terminal interfaces that are required for tetramer formation are unable to form a complex with ttRecF. Furthermore, a ttRecFR complex containing the DNA-binding deficient ttRecR K23E/R27E double mutant, which contains mutations lying inside the ring, exhibits significantly reduced dsDNA binding. Thus, we propose that the ring-like ttRecR tetramer has a key role in tethering the ttRecFR complex onto dsDNA and that the ring structure may function as a clamp protein.

Molecular structure of the ParM polymer and the mechanism leading to its nucleotide-driven dynamic instability.

ParM is a prokaryotic actin homologue, which ensures even plasmid segregation before bacterial cell division. In vivo, ParM forms a labile filament bundle that is reminiscent of the more complex spindle formed by microtubules partitioning chromosomes in eukaryotic cells. However, little is known about the underlying structural mechanism of DNA segregation by ParM filaments and the accompanying dynamic instability. Our biochemical, TIRF microscopy and high-pressure SAX observations indicate that polymerization and disintegration of ParM filaments is driven by GTP rather than ATP and that ParM acts as a GTP-driven molecular switch similar to a G protein. Image analysis of electron micrographs reveals that the ParM filament is a left-handed helix, opposed to the right-handed actin polymer. Nevertheless, the intersubunit contacts are similar to those of actin. Our atomic model of the ParM-GMPPNP filament, which also fits well to X-ray fibre diffraction patterns from oriented gels, can explain why after nucleotide release, large conformational changes of the protomer lead to a breakage of intra- and interstrand interactions, and thus to the observed disintegration of the ParM filament after DNA segregation.

Curvature-dependent recognition of ethanolamine phospholipids by duramycin and cinnamycin.

Duramycin is a 19-amino-acid tetracyclic lantibiotic closely related to cinnamycin (Ro09-0198), which is known to bind phosphatidylethanolamine (PE). The lipid specificity of duramycin was not established. The present study indicates that both duramycin and cinnamycin exclusively bind to ethanolamine phospholipids (PE and ethanolamine plasmalogen). Model membrane study indicates that the binding of duramycin and cinnamycin to PE-containing liposomes is dependent on membrane curvature, i.e., the lantibiotics bind small vesicles more efficiently than large liposomes. The binding of the lantibiotics to multilamellar liposomes induces tubulation of membranes, as revealed by electron microscopy and small-angle x-ray scattering. These results suggest that both duramycin and cinnamycin promote their binding to the PE-containing membrane by deforming membrane curvature.

Diversity of structural behavior in vertebrate conventional myosins complexed with actin.

Low-resolution three-dimensional structures of acto-myosin subfragment-1 (S1) complexes were retrieved from X-ray fiber diffraction patterns, recorded either in the presence or absence of ADP. The S1 was obtained from various myosin-II isoforms from vertebrates, including rabbit fast-skeletal and cardiac, chicken smooth and human non-muscle IIA and IIB species, and was diffused into an array of overstretched, skinned skeletal muscle fibers. The S1 attached to the exposed actin filaments according to their helical symmetry. Upon addition of ADP, the diffraction patterns from acto-S1 showed an increasing magnitude of response in the order as listed above, with features of a lateral compression of the whole diffraction pattern (indicative of increased radius of the acto-S1 complex) and an enhancement of the fifth layer-line reflection. The structure retrieval indicates that these changes are mainly due to the swing of the light chain (LC) domain in the direction consistent with the cryo-electron microscopic results. In the non-muscle isoforms, the swing is large enough to affect the manner of quasi-crystal packing of the S1-decorated actin filaments and their lattice dimension, with a small change in the twist of actin filaments. Variations also exist in the behavior of the 50K-cleft, which apparently opens upon addition of ADP to the non-muscle isoforms but not to other isoforms. The fast-skeletal S1 remains as the only isoform that does not clearly exhibit either of the structural changes. The results indicate that the "conventional" myosin-II isoforms exhibit a wide variety of structural behavior, possibly depending on their functions and/or the history of molecular evolution.

Molecular shape of palytoxin in aqueous solution.

Palytoxin, one of the most toxic non-peptide substances, formed an associated dimer of 5 nm length in aqueous solution.

pH-dependent formation of membranous cytoplasmic body-like structure of ganglioside G(M1)/bis(monoacylglycero)phosphate mixed membranes.

Membrane structures of the mixtures of ganglioside G(M1) and endosome specific lipid, bis (monoacylglycero) phosphate (BMP, also known as lysobisphosphatidic acid) were examined at various pH conditions by freeze-fracture electron microscopy and small-angle x-ray scattering. At pH 8.5-6.5, a G(M1)/BMP (1:1 mol/mol) mixture formed small vesicular aggregates, whereas the mixture formed closely packed lamellar structures under acidic conditions (pH 5.5, 4.6) with the lamellar repeat distance of 8.06 nm. Since BMP alone exhibits a diffuse lamellar structure at a broad range of pH values and G(M1) forms a micelle, the results indicate that both G(M1) and BMP are required to produce closely stacked multilamellar vesicles. These vesicles resemble membranous cytoplasmic bodies in cells derived from patients suffering from G(M1) gangliosidosis. Similar to G(M1) gangliosidosis, cholesterol was trapped in BMP vesicles in G(M1)- and in a low pH-dependent manner. Studies employing different gangliosides and a G(M1) analog suggest the importance of sugar chains and a sialic acid of G(M1) in the pH-dependent structural change of G(M1)/BMP membranes.

Autoxidized phospholipids in hexane: nano-self-assemblies studied by synchrotron small-angle X-ray scattering.

Synchrotron small-angle X-ray scattering (SAXS) was used to analyze the structure of self-assembled autoxidized phospholipids in a very dilute solution of hexane. In addition, it was used to build a self-consistent model of the aggregates, taking into account their inner heterogeneities and polydispersity. The scattering intensity from a dilute mixture of different types of noninteracting components of the phospholipid system was represented as a linear combination of partial intensities from the components weighted by their volume fractions. Applying this approach the final model of the system was described as a mixture of polydisperse reverse micelles and aggregates with spherical and cylindrical shapes. Spherical aggregates were represented as hollow spheres with inner radius 0.7 nm (occupied by water or hexane) and outer radius 1.5 nm. Geometrical parameters of the aggregates did not change much during the oxidation process, while the ratio of reverse micelles and aggregates in solution varied. The amount of the reverse micelles increased from very low to about 80%, whereas the content of other aggregates constantly reduced. The analysis performed in this study helps one to better understand the processes of phospholipid oxidation, which may occur in biological membranes.

The signaling pathway in histidine kinase and the response regulator complex revealed by X-ray crystallography and solution scattering.

The structure of a histidine kinase (ThkA) complexed with a response regulator (TrrA) in the two-component regulatory system from hyperthermophile Thermotoga maritima was determined by a combination of X-ray crystallography at a resolution of 4.2 A and small-angle X-ray scattering (SAXS). The boundary of the three component domains (PAS-sensor, dimerization and catalytic domains) of ThkA and the bound TrrA molecule were unambiguously assigned in the electron density map at 4.2 A resolution. ThkA forms a dimer with crystallographic 2-fold symmetry and two monomeric TrrAs bind to the ThkA dimer. SAXS experiments also confirmed this association state in solution and specific binding between ThkA and TrrA (Kd=8.2x10(-11) M(-2)). The association interface between ThkA and TrrA contains the phosphotransfer His residue in the ThkA, indicative of an efficient receipt of the phosphoryl group. One Per-Arnt-Sim (PAS) domain does not interact with the other PAS domain, but with the catalytic domain of the same polypeptide chain and with one TrrA molecule. Observed inter-domain and inter-molecular interactions reveal a definite pathway of signal transduction in the kinase/regulator complex. In addition, we propose a responsible role of TrrA for the feedback regulation of sensing and/or kinase activities of ThkA.