Novel inter-domain Ca2+-binding site in the gelsolin superfamily protein fragmin.

Gelsolin superfamily proteins, consisting of multiple domains (usually six), sever actin filaments and cap the barbed ends in a Ca2+-dependent manner. Two types of evolutionally conserved Ca2+-binding sites have been identified in this family; type-1 (between gelsolin and actin) and type-2 (within the gelsolin domain). Fragmin, a member in the slime mold Physarum polycephalum, consists of three domains (F1-F3) that are highly similar to the N-terminal half of mammalian gelsolin (G1-G3). Despite their similarities, the two proteins exhibit a significant difference in the Ca2+ dependency; F1-F3 absolutely requires Ca2+ for the filament severing whereas G1-G3 does not. In this study, we examined the strong dependency of fragmin on Ca2+ using biochemical and structural approaches. Our co-sedimentation assay demonstrated that Ca2+ significantly enhanced the binding of F2-F3 to actin. We determined the crystal structure of F2-F3 in the presence of Ca2+. F2-F3 binds a total of three calcium ions; while two are located in type-2 sites within F2 or F3, the remaining one resides between the F2 long helix and the F3 short helix. The inter-domain Ca2+-coordination appears to stabilize F2-F3 in a closely packed configuration. Notably, the F3 long helix exhibits a bent conformation which is different from the straight G3 long helix in the presence of Ca2+. Our results provide the first structural evidence for the existence of an unconventional Ca2+-binding site in the gelsolin superfamily proteins.

The sandwich structure of keratinous layers controls the form and growth orientation of chicken rhinotheca.

The upper beak bone of birds is known to be overlain by the rhinotheca, which is composed of the horny sheath of keratinous layers. However, the details of the structure and growth pattern of the rhinotheca are yet to be understood. In this study, the microstructure of the rhinotheca from chicken specimens of different growth stages (ranging from 1 to ~ 80 days old) was analyzed using a combination of thin section and scanning electron microscopy observations, and small-angle X-ray scattering analysis. We found that the rhinotheca comprises three different layers - outer, intermediate, and inner layers - throughout its growth. The outer layer arises from the proximal portion of the beak bone and covers the dorsal surface of the rhinotheca, whereas the intermediate and inner layers originate in the distal portion of the beak bone and underlie the outer layer. This tri-layered structure of the rhinotheca was also observed in wild bird specimens (grey wagtail, king quail, and brown dipper). On the median plane, micro-layers making up the outer and inner layers are bedded nearly parallel to the rostral bone at the base. However, more distally positioned micro-layers of the outer layer are more anteverted distally. The micro-layers of the intermediate layer are bedded nearly perpendicular to those of the outer and inner layers on the median plane. The growth of micro-layers in the intermediate layer adds thickness to the rhinotheca, which causes the difference in profile between the beak bone and the rhinotheca in the distal portion of the beak. Moreover, the entire intermediate layer grows distally as new proximal micro-layers form. The outer layer is dragged distally by the intermediate layer as a result of its distal growth, for the three layers are closely packed to each other at their boundaries. Furthermore, the occurrence of the intermediate and inner layers in the distal portion of the rostral bone may be because the distal end of the beak is frequently used and worn, and the rhinotheca therefore needs to be replaced more frequently at the distal end. The rhinotheca structure described here will be an important and useful factor in the reconstruction of the beaks of birds in extinct taxa.

Multimerization of small G-protein H-Ras induced by chemical modification at hyper variable region with caged compound.

The lipid-anchored small G protein Ras is a central regulator of cellular signal transduction processes, thereby functioning as a molecular switch. Ras forms a nanocluster on the plasma membrane by modifying lipids in the hypervariable region (HVR) at the C-terminus to exhibit physiological functions. In this study, we demonstrated that chemical modification of cysteine residues in HVR with caged compounds (instead of lipidation) induces multimerization of H-Ras. The sulfhydryl-reactive caged compound, 2-nitrobenzyl bromide, was stoichiometrically incorporated into the cysteine residue of HVR and induced the formation of the Ras multimer. Light irradiation induced the elimination of the 2-nitrobenzyl group, resulting in the conversion of the multimer to a monomer. Size-exclusion chromatography coupled with high-performance liquid chromatography and small-angle x-ray scattering analysis revealed that H-Ras forms a pentamer. Electron microscopic observation of the multimer showed a circular ring shape, which is consistent with the structure estimated from x-ray scattering. The shape of the multimer may reflect the physiological state of Ras. It was suggested that the multimerization and monomerization of H-Ras were controlled by modification with a caged compound in HVR under light irradiation.

Regulation of cysteine-rich protein 2 localization by the development of actin fibers during smooth muscle cell differentiation.

Cysteine-rich protein 2 (CRP2) is a cofactor for smooth muscle cell (SMC) differentiation. Here, we examined the mechanism of CRP2 distribution dynamics during SMC differentiation. CRP2 protein directly associated with F-actin through its N-terminal LIM domain and Gly-rich region, as determined by ELISA. In undifferentiated cells that contain few actin stress fibers, CRP2 was broadly distributed throughout the whole cell, including the nucleus. After induction of SMC differentiation, CRP2 localized to actin stress fibers as they formed. The stress fiber-localized CRP2 entered the nucleus because of induced actin depolymerization. These CRP2 dynamics were reproduced by in silico simulation. CRP2 localization dynamics, which affect CRP2 function, are regulated by the formation of actin stress fibers in conjunction with SMC differentiation.

Anisotropic structure of calcium-induced alginate gels by optical and small-angle X-ray scattering measurements.

It was more than 50 years ago that an appearance of birefringence in alginate gels prepared under cation flow was reported for the first time, however, the anisotropic structure of the alginate gel has not been studied in detail. In the present study, anisotropic Ca-alginate gels were prepared within dialysis tubing in a high Ca(2+)-concentration external bath, and optical and small-angle X-ray scattering (SAXS) measurements were performed to characterize the structure of the gel. The observations of the gel with crossed polarizers and with circular polarizers revealed the molecular orientation perpendicular to the direction of Ca(2+) flow. Analyses of the SAXS intensity profiles indicated the formation of rod-like fibrils consisting of a few tens of alginate molecules and that the anisotropy of the gel was caused by the circumferential orientation of the large fibrils. From the observed asymmetric SAXS pattern, it was found that the axis of rotational symmetry of the anisotropic structure was parallel to the direction of Ca(2+) flow. The alignment factor (A(f)) calculated from the SAXS intensity data confirmed that the orientation of the fibrils was perpendicular to the direction of Ca(2+) flow.

Infrared Laser-Induced Amyloid Fibril Dissociation: A Joint Experimental/Theoretical Study on the GNNQQNY Peptide.

Neurodegenerative diseases are usually characterized by plaques made of well-ordered aggregates of distinct amyloid proteins. Dissociating these very stable amyloid plaques is a critical clinical issue. In this study, we present a joint mid-infrared free electron laser experiment/nonequilibrium molecular dynamics simulation to understand the dissociation process of a representative example GNNQQNY fibril. By tuning the laser frequency to the amide I band of the fibril, the resonance takes place and dissociation is occurred. With the calculated and observed wide-angle X-ray scattering profiles and secondary structures before and after laser irradiation being identical, we can propose a dissociation mechanism with high confidence from our simulations. We find that dissociation starts in the core of the fibrils by fragmenting the intermolecular hydrogen bonds and separating the peptides and then propagates to the fibril extremities leading to the formation of unstructured expanded oligomers. We suggest that this should be a generic mechanism of the laser-induced dissociation of amyloid fibrils.

Quick shear-flow alignment of biological filaments for X-ray fiber diffraction facilitated by methylcellulose.

X-ray fiber diffraction is one of the most useful methods for examining the structural details of live biological filaments under physiological conditions. To investigate biologically active or labile materials, it is crucial to finish fiber alignment within seconds before diffraction analysis. However, the conventional methods, e.g., magnetic field alignment and low-speed centrifugations, are time-consuming and not very useful for such purposes. Here, we introduce a new alignment method using a rheometer with two parallel disks, which was applied to observe fiber diffractions of axonemes, tobacco mosaic tobamovirus, and microtubules. We found that fibers were aligned within 5 s by giving high shear flow (1000-5000 s(-1)) to the medium and that methylcellulose contained in the medium (approximately 1%) was essential to the accomplishment of uniform orientation with a small angular deviation (<5 degrees). The new alignment method enabled us to execute structure analyses of axonemes by small-angle x-ray diffraction. Since this method was also useful for the quick alignment of purified microtubules, as well as tobacco mosaic tobamovirus, we expect that we can apply it to the structural analysis of many other biological filaments.

Neutron diffraction measurements of skeletal muscle using the contrast variation technique: analysis of the equatorial diffraction patterns.

Among various methods for structural studies of biological macromolecules, neutron scattering and diffraction have a unique feature in that the contrast between the scattering length density of the molecules and that of the solvent can be varied easily by changing the D2O content in the solvent. This "contrast variation" technique enables one to obtain information on variations of scattering length density of the molecules of interest. Here, in order to explore the possibilities of the contrast variation technique in neutron fiber diffraction, neutron diffraction measurements of skeletal muscles were performed. The neutron fiber diffraction patterns from frog sartorius muscles were measured in various D2O concentrations in the relaxed state where no tension of muscle is produced, and in the rigor state where all myosin heads of the thick filaments bind tightly to actin in the thin filaments. It was shown that in both states, there were reflections having distinct contrast matching points, indicating a variation in the scattering length density of the protein regions in the unit cell of the muscle structure. Analysis of the equatorial reflections by the two-dimensional projected scattering length density map calculations by Fourier synthesis and model calculations provided the phase information of the equatorial reflections, with which the projected scattering length density maps of the unit cell of the hexagonal filament array in both states were calculated. The analysis showed that the scattering length density of the thick filament region was higher than that of the thin filament region, and that the scattering length density of the thick filament backbone changed as muscle went from the relaxed state into the rigor state.

Segmental Motions of Proteins under Non-native States Evaluated Using Quasielastic Neutron Scattering.

Characterization of the dynamics of disordered polypeptide chains is required to elucidate the behavior of intrinsically disordered proteins and proteins under non-native states related to the folding process. Here we develop a method using quasielastic neutron scattering, combined with small-angle X-ray scattering and dynamic light scattering, to evaluate segmental motions of proteins as well as diffusion of the entire molecules and local side-chain motions. We apply this method to RNase A under the unfolded and molten-globule (MG) states. The diffusion coefficients arising from the segmental motions are evaluated and found to be different between the unfolded and MG states. The values obtained here are consistent with those obtained using the fluorescence-based techniques. These results demonstrate not only feasibility of this method but also usefulness to characterize the behavior of proteins under various disordered states.

Dynamic Properties of Human α-Synuclein Related to Propensity to Amyloid Fibril Formation.

α-Synuclein (αSyn) is an intrinsically disordered protein that can form amyloid fibrils. Fibrils of αSyn are implicated with the pathogenesis of Parkinson's disease and other synucleinopathies. Elucidating the mechanism of fibril formation of αSyn is therefore important for understanding the mechanism of the pathogenesis of these diseases. Fibril formation of αSyn is sensitive to solution conditions, suggesting that fibril formation of αSyn arises from the changes in its inherent physico-chemical properties, particularly its dynamic properties because intrinsically disordered proteins such as αSyn utilize their inherent flexibility to function. Characterizing these properties under various conditions should provide insights into the mechanism of fibril formation. Here, using the quasielastic neutron scattering and small-angle x-ray scattering techniques, we investigated the dynamic and structural properties of αSyn under the conditions, where mature fibrils are formed (pH 7.4 with a high salt concentration), where clumping of short fibrils occurs (pH 4.0), and where fibril formation is not completed (pH 7.4). The small-angle x-ray scattering measurements showed that the extended structures at pH 7.4 with a high salt concentration become compact at pH 4.0 and 7.4. The quasielastic neutron scattering measurements showed that both intra-molecular segmental motions and local motions such as side-chain motions are enhanced at pH 7.4 with a high salt concentration, compared to those at pH 7.4 without salt, whereas only the local motions are enhanced at pH 4.0. These results imply that fibril formation of αSyn requires not only the enhanced local motions but also the segmental motions such that proper inter-molecular interactions are possible.

Structural changes of the regulatory proteins bound to the thin filaments in skeletal muscle contraction by X-ray fiber diffraction.

In order to clarify the structural changes related to the regulation mechanism in skeletal muscle contraction, the intensity changes of thin filament-based reflections were investigated by X-ray fiber diffraction. The time course and extent of intensity changes of the first to third order troponin (TN)-associated meridional reflections with a basic repeat of 38.4nm were different for each of these reflections. The intensity of the first and second thin filament layer lines changed in a reciprocal manner both during initial activation and during the force generation process. The axial spacings of the TN-meridional reflections decreased by approximately 0.1% upon activation relative to the relaxing state and increased by approximately 0.24% in the force generation state, in line with that of the 2.7-nm reflection. Ca(2+)-binding to TN triggered the shortening and a change in the helical symmetry of the thin filaments. Modeling of the structural changes using the intensities of the thin filament-based reflections suggested that the conformation of the globular core domain of TN altered upon activation, undergoing additional conformational changes at the tension plateau. The tail domain of TN moved together with tropomyosin during contraction. The results indicate that the structural changes of regulatory proteins bound to the actin filaments occur in two steps, the first in response to the Ca(2+)-binding and the second induced by actomyosin interaction.

Axial dispositions and conformations of myosin crossbridges along thick filaments in relaxed and contracting states of vertebrate striated muscles by X-ray fiber diffraction.

X-ray diffraction patterns from live vertebrate striated muscles were analyzed to elucidate the detailed structural models of the myosin crown arrangement and the axial disposition of two-headed myosin crossbridges along the thick filaments in the relaxed and contracting states. The modeling studies were based upon the previous notion that individual myosin filaments had a mixed structure with two regions, a "regular" and a "perturbed". In the relaxed state the distributions and sizes of the regular and perturbed regions on myosin filaments, each having its own axial periodicity for the arrangement of crossbridge crowns within the basic period, were similar to those reported previously. A new finding was that in the contracting state, this mixed structure was maintained but the length of each region, the periodicities of the crowns and the axial disposition of two heads of a crossbridge were altered. The perturbed regions of the crossbridge repeat shifted towards the Z-bands in the sarcomere without changing the lengths found in the relaxed state, but in which the intervals between three successive crowns within the basic period became closer to the regular 14.5-nm repeat in the contracting state. In high resolution modeling for a myosin head, the two heads of a crossbridge were axially tilted in opposite directions along the three-fold helical tracks of myosin filaments and their axial orientations were different from each other in perturbed and regular regions in both states. Under relaxing conditions, one head of a double-headed crossbridge pair appeared to be in close proximity to another head in a pair at the adjacent crown level in the axial direction in the regular region. In the perturbed region this contact between heads occurred only on the narrower inter-crown levels. During contraction, one head of a crossbridge oriented more perpendicular to the fiber axis and the partner head flared axially. Several factors that significantly influence the intensities of the myosin based-meridional reflections and their relative contributions are discussed.

Structural alterations of thin actin filaments in muscle contraction by synchrotron X-ray fiber diffraction.

Strong evidence has been accumulated that the conformational changes of the thin actin filaments are occurring and playing an important role in the entire process of muscle contraction. The conformational changes and the mechanical properties of the thin actin filaments we have found by X-ray fiber diffraction on skeletal muscle contraction are explored. Recent studies on the conformational changes of regulatory proteins bound to actin filaments upon activation and in the force generation process are also described. Finally, the roles of structural alterations and dynamics of the actin filaments are discussed in conjunction with the regulation mechanism and the force generation mechanism.

Small-angle X-ray and light scattering analysis of multi-layered Curdlan gels prepared by a diffusion method.

Curdlan, a microbial polysaccharide, forms a multi-layered gel consisting of four layers with different turbidity when its alkaline solution is dialyzed against aqueous solutions containing Ca2+ (diffusion-set gel). The present study clarified the microstructure of each layer of the diffusion-set Curdlan gel by small-angle X-ray scattering (SAXS) and small-angle light scattering (SALS). The SAXS data showed that Curdlan chains assume a helical ordered conformation in the gel and that the gel consists of the fibrils formed by the association of Curdlan chains and the aggregates of fibrils. The SAXS results also indicated that the gelation is induced by the formation of a network of Ca2+-cross-linked fibrils in the outer region of the gel, whereas by the network formation of the aggregation of fibrils in the neutralization process in the inner region of the gel. A structural anisotropy of the gel was investigated by analysis of two-dimensional SAXS images, showing that the fibril is oriented circumferentially in the outer region of the cylindrical gel, whereas it is oriented randomly in the inner region of the gel. The SALS data showed that a characteristic length of an inhomogeneous structure in the turbid layers is of the order of micrometers. The observed spatial variation of the microscopic structure is caused by the difference in the paths of pH and [Ca2+] traced in the gelation process.

Cysteine-rich protein 2 accelerates actin filament cluster formation.

Filamentous actin (F-actin) forms many types of structures and dynamically regulates cell morphology and movement, and plays a mechanosensory role for extracellular stimuli. In this study, we determined that the smooth muscle-related transcription factor, cysteine-rich protein 2 (CRP2), regulates the supramolecular networks of F-actin. The structures of CRP2 and F-actin in solution were analyzed by small-angle X-ray solution scattering (SAXS). The general shape of CRP2 was partially unfolded and relatively ellipsoidal in structure, and the apparent cross sectional radius of gyration (Rc) was about 15.8 Å. The predicted shape, derived by ab initio modeling, consisted of roughly four tandem clusters: LIM domains were likely at both ends with the middle clusters being an unfolded linker region. From the SAXS analysis, the Rc of F-actin was about 26.7 Å, and it was independent of CRP2 addition. On the other hand, in the low angle region of the CRP2-bound F-actin scattering, the intensities showed upward curvature with the addition of CRP2, which indicates increasing branching of F-actin following CRP2 binding. From biochemical analysis, the actin filaments were augmented and clustered by the addition of CRP2. This F-actin clustering activity of CRP2 was cooperative with α-actinin. Thus, binding of CRP2 to F-actin accelerates actin polymerization and F-actin cluster formation.

Incorporation of an azobenzene derivative into the energy transducing site of skeletal muscle myosin results in photo-induced conformational changes.

The region containing reactive cysteines, Cys 707 (SH1)-Cys 697 (SH2), of skeletal muscle myosin is thought to play a key role in the conformational changes of the myosin head during force generation coupled to ATP hydrolysis. In the present study, we synthesized a photochromic crosslinker, 4,4'-azobenzene-dimaleimide (ABDM), that undergoes reversible cis-trans isomerization upon ultra violet (UV) and visible (VIS) light irradiation resulting in a change in the crosslinking length from 5 to 17 A. The reactive cysteines, SH1 and SH2, of myosin subfragment 1 (S1) were crosslinked with ABDM, yielding an ABDM-S1 complex. The changes in absorbance induced by UV/VIS light irradiation of the complex were similar to those of free ABDM indicating that the incorporation of ABDM at the SH1 and SH2 sites did not disrupt the isomerization of crosslinked ABDM. Small-angle synchrotron X-ray scattering analysis of the ABDM-S1 complex in solution suggested that the localized conformational changes resulting from the cis to trans isomerization on ABDM crosslinking of SH1 and SH2 induced a small but significant swing in the lever arm portion of S1 in the opposite direction from that induced by ATP.

Modeling analysis of myosin-based meridional X-ray reflections from frog skeletal muscles in relaxed and contracting states.

Analysis of the myosin-based meridional intensity data in the X-ray diffraction patterns of live frog skeletal muscles was performed to propose a more precise model for a myosin crown periodicity and an axial disposition of two-headed crossbridges along the thick filament in a sarcomere. Modeling studies revealed that the thick filament has a mixed structure of two different periodicities of the myosin crossbridge crown arrangement and that the crown periodicity and the axial disposition of crossbridges are altered when muscle goes from the relaxed state to the contracting state. Factors that primarily affect the meridional intensities were examined.

Universality and specificity in molecular orientation in anisotropic gels prepared by diffusion method.

Molecular orientation in anisotropic gels of chitosan, Curdlan and DNA obtained by dialysis of those aqueous solutions in gelation-inducing solutions was investigated. In this diffusion method (or dialysis method), the gel formation was induced by letting small molecules diffuse in or out of the polymer solutions through the surface. For the gels of DNA and chitosan, the polymer chains aligned perpendicular to the diffusion direction. The same direction of molecular orientation was observed for the Curdlan gel prepared in the dialysis cell. On the other hand, a peculiar nature was observed for the Curdlan gel prepared in the dialysis tube: the molecular orientation was perpendicular to the diffusion direction in the outermost layer of the gel, while the orientation was parallel to the diffusion direction in the inner translucent layer. The orientation parallel to the diffusion direction is attributed to a small deformation of the inner translucent layer caused by a slight shrinkage of the central region after the gel formation. At least near the surface of the gel, the molecular orientation perpendicular to the diffusion direction is a universal characteristic for the gels prepared by the diffusion method.

Head-head interactions of resting myosin crossbridges in intact frog skeletal muscles, revealed by synchrotron x-ray fiber diffraction.

The intensities of the myosin-based layer lines in the x-ray diffraction patterns from live resting frog skeletal muscles with full thick-thin filament overlap from which partial lattice sampling effects had been removed were analyzed to elucidate the configurations of myosin crossbridges around the thick filament backbone to nanometer resolution. The repeat of myosin binding protein C (C-protein) molecules on the thick filaments was determined to be 45.33 nm, slightly longer than that of myosin crossbridges. With the inclusion of structural information for C-proteins and a pre-powerstroke head shape, modeling in terms of a mixed population of regular and perturbed regions of myosin crown repeats along the filament revealed that the myosin filament had azimuthal perturbations of crossbridges in addition to axial perturbations in the perturbed region, producing pseudo-six-fold rotational symmetry in the structure projected down the filament axis. Myosin crossbridges had a different organization about the filament axis in each of the regular and perturbed regions. In the regular region that lacks C-proteins, there were inter-molecular interactions between the myosin heads in axially adjacent crown levels. In the perturbed region that contains C-proteins, in addition to inter-molecular interactions between the myosin heads in the closest adjacent crown levels, there were also intra-molecular interactions between the paired heads on the same crown level. Common features of the interactions in both regions were interactions between a portion of the 50-kDa-domain and part of the converter domain of the myosin heads, similar to those found in the phosphorylation-regulated invertebrate myosin. These interactions are primarily electrostatic and the converter domain is responsible for the head-head interactions. Thus multiple head-head interactions of myosin crossbridges also characterize the switched-off state and have an important role in the regulation or other functions of myosin in thin filament-regulated muscles as well as in the thick filament-regulated muscles.

X-ray fiber diffraction modeling of structural changes of the thin filament upon activation of live vertebrate skeletal muscles.

In order to clarify the structural changes of the thin filaments related to the regulation mechanism in skeletal muscle contraction, the intensities of thin filament-based reflections in the X-ray fiber diffraction patterns from live frog skeletal muscles at non-filament overlap length were investigated in the relaxed state and upon activation. Modeling the structural changes of the whole thin filament due to Ca2+-activation was systematically performed using the crystallographic data of constituent molecules (actin, tropomyosin and troponin core domain) as starting points in order to determine the structural changes of the regulatory proteins and actin. The results showed that the globular core domain of troponin moved toward the filament axis by ∼6 Å and rotated by ∼16° anticlockwise (viewed from the pointed end) around the filament axis by Ca2+-binding to troponin C, and that tropomyosin together with the tail of troponin T moved azimuthally toward the inner domains of actin by ∼12° and radially by ∼7 Å from the relaxed position possibly to partially open the myosin binding region of actin. The domain structure of the actin molecule in F-actin we obtained for frog muscle thin filament was slightly different from that of the Holmes F-actin model in the relaxed state, and upon activation, all subdomains of actin moved in the direction to closing the nucleotide-binding pocket, making the actin molecule more compact. We suggest that the troponin movements and the structural changes within actin molecule upon activation are also crucial components of the regulation mechanism in addition to the steric blocking movement of tropomyosin.