Tetsuro Fujisawa

Conformational landscape of cytochrome c folding studied by microsecond-resolved small-angle x-ray scattering

To investigate protein folding dynamics in terms of compactness, we developed a continuous-flow mixing device to make small-angle x-ray scattering measurements with the time resolution of 160 μs and characterized the radius of gyration (Rg) of two folding intermediates of cytochrome c (cyt c). The early intermediate possesses ≈20 Å of Rg, which is smaller by ≈4 Å than that of the acid-unfolded state. The Rg of the later intermediate is ≈18 Å, which is close to that of the molten globule state. Considering the α-helix content (fH) of the intermediates, we clarified the folding pathway of cyt c on the conformational landscape defined by Rg and fH. Cyt c folding proceeds with a collapse around a specific region of the protein followed by a cooperative acquisition of secondary structures and compactness.

Small-angle X-ray scattering station at the SPring-8 RIKEN beamline

RIKEN beamline I (BL45XU) is an undulator beamline with two branches. One is for protein crystallography (PX) and the other is for small-angle x-ray scattering (SAXS). The beam is split into the two branches by a diamond monochromator so that two experiments can be done simultaneously [Yamamoto et al. (1995) Rev. Sci. Instrum. 66, 1833-1835]. The SAXS branch was designed for studying the weak interaction of proteins or subunits of fibrous or protein solutions especially using hydrostatic pressure. The optics makes use of the good parallelism of the undulator beam in order to reduce parasitic scattering. The beamline consists of a double crystal monochromator and a K-B type focusing mirror system. In order to cope with the high flux of the beam, an x-ray image intensifier (Hamamatsu Photonics, V5445P) with a cooled CCD camera (C4880-82) was used. As a result, decreases in both collection time and sample amount were realized in standard static experiments. These improvements will greatly facilitate SAXS experiments under high pressure.

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∼4), and helical linkers, (EAAAK)n (n = 2∼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∼3) causes multimerization, while the longer linkers (n = 4∼5) solvate monomeric chimeric proteins. With the moderate‐length linkers (n = 4), the observed radius of gyration (Rg) and maximum dimension (Dmax) were 38.8 Å and 120 Å with the flexible linker, and 40.2 Å and 130 Å 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), Rg and Dmax increased to 43.2 Å and 140 Å, 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. Proteins 2004.

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.

Expansion of Polyglutamine Induces the Formation of Quasi-aggregate in the Early Stage of Protein Fibrillization

We examined the effects of the expansion of glutamine repeats on the early stage of protein fibrillization. Small-angle x-ray scattering (SAXS) and electron microscopic studies revealed that the elongation of polyglutamine from 35 to 50 repeats in protein induced a large assembly of the protein upon incubation at 37 °C and that its formation was completed in ∼3 h. A bead modeling procedure based on SAXS spectra indicated that the largely assembled species of the protein, quasi-aggregate, is composed of 80 to ∼90 monomers and a bowl-like structure with long and short axes of 400 and 190 Å, respectively. Contrary to fibril, the quasi-aggregate did not show a peak at S = 0.21 Å–1 corresponding to the 4.8-Å spacing of β-pleated sheets in SAXS spectra, and reacted with a monoclonal antibody specific to expanded polyglutamine. These results imply that β-sheets of expanded polyglutamines in the quasi-aggregate are not orderly aligned and are partially exposed, in contrast to regularly oriented and buried β-pleated sheets in fibril. The formation of non-fibrillary quasi-aggregate in the early phase of fibril formation would be one of the major characteristics of the protein containing an expanded polyglutamine.

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 μm 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.

Static and Dynamic X-Ray Diffraction Recordings from Living Mammalian and Amphibian Skeletal Muscles

Static and time-resolved two-dimensional x-ray diffraction patterns, recorded from the living mouse diaphragm muscle, were compared with those from living frog sartorius muscle. The resting pattern of mouse muscle was similar to that of frog muscle, and consisted of actin- and myosin-based reflections with spacings basically identical to those of frog. As a notable exception, the sampling pattern of the myosin layer lines (MLLs) indicated that the mouse myofilaments were not organized into a superlattice as in frog. The intensity changes of reflections upon activation were also similar. The MLLs of both muscles were markedly weakened. Stereospecific (rigorlike) actomyosin species were not significantly populated in either muscle, as was evidenced by the 6th actin layer line (ALL), which was substantially enhanced but without a shift in its peak position or a concomitant rise of lower order ALLs. On close examination of the mouse pattern, however, a few lower order ALLs were found to rise, slightly but definitely, at the position expected for stereospecific binding. Their quick rise after the onset of stimulation indicates that this stereospecific complex is generated in the process of normal contraction. However, their rise is still too small to account for the marked enhancement of the 6th ALL, which is better explained by a myosin-induced structural change of actin. Since the forces of the two muscles are comparable regardless of the amount of stereospecific complex, it would be natural to consider that most of the force of skeletal muscle is supported by nonstereospecific actomyosin species.

The use of a Hamamatsu X-ray image intensifier with a cooled CCD as a solution X-ray scattering detector

CCD detectors are now widely used in many synchrotron small-angle X-ray scattering beamlines. The use of an X-ray image intensifier with cooled CCD (XR-II + CCD) was studied, especially for use in synchrotron solution X-ray scattering. Two samples, polystyrene latex and apoferritin, were used. These two samples have fine structure in the solution scattering profile due to symmetry and narrow size distribution. The recorded scattering profile, in comparison with that obtained by a position-sensitive proportional counter (PSPC), showed that XR-II + CCD has a much smaller practical dynamic range (100:1) than that of a pixel well (7500:1). This limited dynamic range was overcome by placing various-size masks on the detection plane, thereby eliminating the high-intensity region. The images recorded with various masks were combined, and the reconstituted solution scattering profile was submitted to various analyses, including Guinier analysis, power-law analysis, size distribution analysis and calculation of radial density distributions. The results were the same as those obtained with the PSPC. This indicates that spatial distortion as well as shading, a decrease in sensitivity from the centre to the edge of the detecting region [Amemiya, Ito, Yagi, Asano, Wakabayashi, Ueki & Endo (1995). Rev. Sci. Instrum. 66, 2290–2294], have very little effect on the SAXS results. This paper presents a practical protocol for obtaining a reliable solution scattering profile given the limitations of XR-II + CCD for synchrotron solution X-ray scattering.

The shapes and sizes of two domains of tropomodulin, the P‐end‐capping protein of actin‐tropomyosin

Tropomodulin, the P‐end (slow‐growing end)‐capping protein of the actin‐tropomyosin filament, and its fragment (C20) of the C‐terminal half were studied by synchrotron small‐angle X‐ray scattering, restoring low‐resolution shapes using an ab initio shape‐determining procedure. Tropomodulin is elongated (115 Å long) and consists of two domains, one of 65 Å in length and the other being similar to C20 in shape and size if the long axes of the two are tilted by about 40° relative to each other. We propose a model for tropomodulin in association with tropomyosin and actin: the N‐terminal half of tropomodulin, a rod, binds to the N‐terminus of tropomyosin and the C‐terminal triangle domain protrudes from the P‐end being slightly bent towards the actin subunit at the end, thereby blocking the P‐end.

Redesign of artificial globins: effects of residue replacements at hydrophobic sites on the structural properties.

Artificial sequences of the 153 amino acids have been designed to fit the main-chain framework of the sperm whale myoglobin (Mb) structure based on a knowledge-based 3D-1D compatibility method. The previously designed artificial globin (DG1) folded into a monomeric, compact, highly helical and globular form with overall dimensions similar to those of the target structure, but it lacked structural uniqueness at the side-chain level [Isogai, Y., Ota, M., Fujisawa, T. , Izuno, H., Mukai, M., Nakamura, H., Iizuka, T., and Nishikawa, K. (1999) Biochemistry 38, 7431-7443]. In this study, we redesigned hydrophobic sites of DG1 to improve the structural specificity. Several Leu and Met residues in DG1 were replaced with beta-branched amino acids, Ile and Val, referring to the 3D profile of DG1 to produce three redesigned globins, DG2-4. These residue replacements resulted in no significant changes of their compactness and alpha-helical contents in the absence of denaturant, whereas they significantly affected the dependence of the secondary structure on the concentration of guanidine hydrochloride. The analyses of the denaturation curves revealed higher global stabilities of the designed globins than that of natural apoMb. Among DG1-4, DG3, in which 11 Leu residues of DG1 are replaced with seven Ile and four Val residues, and one Met residue is replaced with Val, displayed the lowest stability but the most cooperative folding-unfolding transition and the most dispersed NMR spectrum with the smallest line width. The present results indicate that the replacements of Leu (Met) with the beta-branched amino acids at appropriate sites reduce the freedom of side-chain conformation and improve the structural specificity at the expense of stability.