Fractionation of Regenerated Silk Fibroin and Characterization of the Fractions.

The molecular weight (MW) of regenerated silk fibroin (RSF) decreases during degumming and dissolving processes. Although MW and the MW distribution generally affect polymer material processability and properties, few reports have described studies examining the influences of MW and the distribution on silk fibroin (SF) material. To prepare different MW SF fractions, the appropriate conditions for fractionation of RSF by ammonium sulfate (AS) precipitation process were investigated. The MW and the distribution of each fraction were found using gel permeation chromatography (GPC) and SDS-polyacrylamide electrophoresis (SDS-PAGE). After films of the fractionated SFs formed, the secondary structure, surface properties, and cell proliferation of films were evaluated. Nanofiber nonwoven mats and 3D porous sponges were fabricated using the fractionated SF aqueous solution. Then, their structures and mechanical properties were analyzed. The results showed AS precipitation using a dialysis membrane at low temperature to be a suitable fractionation method for RSF. Moreover, MW affects the nanofiber and sponge morphology and mechanical properties, although no influence of MW was observed on the secondary structure or crystallinity of the fabricated materials.

Etherification of Wood-Based Hemicelluloses for Interfacial Activity.

We present wetting, hygroscopicity, and interfacial activity of hemicellulose with respect to etherification and contrast it to their potential as interfacial modifiers, which is demonstrated by oil-in-water emulsification containing up to 60 vol% of the oil phase. Tunable amphiphilicity of hardwood and softwood hemicelluloses, xylans, and galactoglucomannans, respectively, was accomplished via controlled etherification. A series of degree of substitution (DS) of hydroxypropylated and 3-butoxy-2-hydroxypropylated ("butylated") grades was synthesized. The hemicellulose ethers were characterized by gel permeation chromatography, spectroscopic techniques, such as NMR, and contact angle measurements. An attenuated total reflectance infrared method was developed for fast identification of the DS. Near infrared analysis was utilized to explore the hygroscopicity of the material and to perform principle component analysis. The modification to butylated grades decreased the hygroscopicity, whereas the hydroxypropylated grades bound moisture. All of the hemicellulose ethers were water-soluble. The interfacial tension of the aqueous hemicellulose solutions was determined by pendant-drop tensiometer, and it was demonstrated to be dependent on the degree of modification.

Development of a flexible optical fiber based high resolution integrated PET∕MRI system.

PURPOSE: The simultaneous measurement of PET and magnetic resonance imaging (MRI) is an emerging field for molecular imaging research. Although optical fiber based PET∕MRI systems have advantages on less interference between PET and MRI, there is a drawback in reducing the scintillation light due to the fiber. To reduce the problem, the authors newly developed flexible optical fiber bundle based block detectors and employed them for a high resolution integrated PET∕MRI system. METHODS: The flexible optical fiber bundle used 0.5 mm diameter, 80 cm long double clad fibers which have dual 12 mm × 24 mm rectangular inputs and a single 24 mm × 24 mm rectangular output. In the input surface, LGSO scintillators of 0.025 mol.% (decay time: ∼31 ns: 0.9 mm × 1.3 mm × 5 mm) and 0.75 mol.% (decay time: ∼46 ns: 0.9 mm × 1.3 mm × 6 mm) were optically coupled in depth direction to form depth-of-interaction detector, arranged in 11 × 13 matrix and optically coupled to the fiber bundle. The two inputs of the bundle are bent for 90°, bound to one, and are optically coupled to a Hamamatsu 1-in. square position sensitive photomultiplier tube. RESULTS: Light loss due to the fiber bundle could be reduced and the performance of the block detectors was improved. Eight optical fiber based block detectors (16 LGSO blocks) were arranged in a 56 mm diameter ring to form a PET system. Spatial resolution and sensitivity were 1.2 mm full-width at half-maximum and 1.2% at the central field-of-view, respectively. Sensitivity change was less than 1% for 2 °C temperature changes. This PET system was integrated with a 0.3 T permanent magnet MRI system which has 17 cm diameter hole at the yoke area for insertion of the PET detector ring. There was no observable interference between PET and MRI. Simultaneous imaging of PET and MRI was successfully performed for small animal studies. CONCLUSIONS: The authors confirmed that the developed high resolution PET∕MRI system is promising for molecular imaging research.

Metal artifacts in MRI from non-magnetic dental alloy and its FEM analysis.

Artifacts in MR(Magnetic Resonance) images of oral cavity produced from non-magnetic metal restorations was verified by measuring the image of index finger and a cylinder of fat test piece with a type 4 gold alloy ring using a compact MRI equipment. In the images of finger, portion around the ring disappeared. However, it was nearly restored with a cut ring. In the cylinder of fat test piece, obvious artifacts appeared when circumferential surface of the ring was placed perpendicular to RF(Radio Frequency) field of MRI equipment's excitation/detection coil. However, in other directions or with a cut ring, artifact disappeared. The cause was simulated with FEM(Finite Element Method) electromagnetic field analysis, and alternating magnetic field was shown to induce surface current on the continuous gold ring. Magnetic field produced by that current interfered with the field from excitation coil. This demonstrated the characteristics and cause of artifacts by non-magnetic dental metals.

Automated system for high-throughput protein production using the dialysis cell-free method.

High-throughput protein production systems have become an important issue, because protein production is one of the bottleneck steps in large-scale structural and functional analyses of proteins. We have developed a dialysis reactor and a fully automated system for protein production using the dialysis cell-free synthesis method, which we previously established to produce protein samples on a milligram scale in a high-throughput manner. The dialysis reactor was designed to be suitable for an automated system and has six dialysis cups attached to a flat dialysis membrane. The automated system is based on a Tecan Freedom EVO 200 workstation in a three-arm configuration, and is equipped with shaking incubators, a vacuum module, a robotic centrifuge, a plate heat sealer, and a custom-made tilting carrier for collection of reaction solutions from the flat-bottom cups with dialysis membranes. The consecutive process, from the dialysis cell-free protein synthesis to the partial purification by immobilized metal affinity chromatography on a 96-well filtration plate, was performed within ca. 14h, including 8h of cell-free protein synthesis. The proteins were eluted stepwise in a high concentration using EDTA by centrifugation, while the resin in the filtration plate was washed on the vacuum manifold. The system was validated to be able to simultaneously and automatically produce up to 96 proteins in yields of several milligrams with high well-to-well reliability, sufficient for structural and functional analyses of proteins. The protein samples produced by the automated system have been utilized for NMR screening to judge the protein foldedness and for structure determinations using heteronuclear multi-dimensional NMR spectroscopy. The automated high-throughput protein production system represents an important breakthrough in the structural and functional studies of proteins and has already contributed a massive amount of results in the structural genomics project at the RIKEN Structural Genomics/Proteomics Initiative (RSGI).

Structural and functional differences of SWIRM domain subtypes.

SWIRM is a conserved domain found in several chromatin-associated proteins. Based on their sequences, the SWIRM family members can be classified into three subfamilies, which are represented by Swi3, LSD1, and Ada2. Here we report the SWIRM structure of human MYb-like, Swirm and Mpn domain-containing protein-1 (MYSM1). The MYSM1 SWIRM structure forms a compact HTH-related fold comprising five alpha-helices, which best resembles the Swi3 SWIRM structure, among the known SWIRM structures. The MYSM1 and Swi3 SWIRM structures are more similar to the LSD1 structure than the Ada2alpha structure. The SWIRM domains of MYSM1 and LSD1 lacked DNA binding activity, while those of Ada2alpha and the human Swi3 counterpart, SMARCC2, bound DNA. The dissimilarity in the DNA-binding ability of the MYSM1 and SMARCC2 SWIRM domains might be due to a couple of amino acid differences in the last helix. These results indicate that the SWIRM family has indeed diverged into three structural subfamilies (Swi3/MYSM1, LSD1, and Ada2 types), and that the Swi3/MYSM1-type subfamily has further diverged into two functionally distinct groups. We also solved the structure of the SANT domain of MYSM1, and demonstrated that it bound DNA with a similar mode to that of the c-Myb DNA-binding domain.

Solution structure of the SWIRM domain of human histone demethylase LSD1.

SWIRM is an evolutionarily conserved domain involved in several chromatin-modifying complexes. Recently, the LSD1 protein, which bears a SWIRM domain, was found to be a demethylase for Lys4-methylated histone H3. Here, we report a solution structure of the SWIRM domain of human LSD1. It forms a compact fold composed of 6 alpha helices, in which a 20 amino acid long helix (alpha4) is surrounded by 5 other short helices. The SWIRM domain structure could be divided into the N-terminal part (alpha1-alpha3) and the C-terminal part (alpha4-alpha6), which are connected to each other by a salt bridge. While the N-terminal part forms a SWIRM-specific structure, the C-terminal part adopts a helix-turn-helix (HTH)-related fold. We discuss a model in which the SWIRM domain acts as an anchor site for a histone tail.

Solution structure of the zinc finger HIT domain in protein FON.

The zinc finger HIT domain is a sequence motif found in many proteins, including thyroid hormone receptor interacting protein 3 (TRIP-3), which is possibly involved in maturity-onset diabetes of the young (MODY). Novel zinc finger motifs are suggested to play important roles in gene regulation and chromatin remodeling. Here, we determined the high-resolution solution structure of the zinc finger HIT domain in ZNHIT2 (protein FON) from Homo sapiens, by an NMR method based on 567 upper distance limits derived from NOE intensities measured in three-dimensional NOESY spectra. The structure yielded a backbone RMSD to the mean coordinates of 0.19 A for the structured residues 12-48. The fold consists of two consecutive antiparallel beta-sheets and two short C-terminal helices packed against the second beta-sheet, and binds two zinc ions. Both zinc ions are coordinated tetrahedrally via a CCCC-CCHC motif to the ligand residues of the zf-HIT domain in an interleaved manner. The tertiary structure of the zinc finger HIT domain closely resembles the folds of the B-box, RING finger, and PHD domains with a cross-brace zinc coordination mode, but is distinct from them. The unique three-dimensional structure of the zinc finger HIT domain revealed a novel zinc-binding fold, as a new member of the treble clef domain family. On the basis of the structural data, we discuss the possible functional roles of the zinc finger HIT domain.

Solution structure of the general transcription factor 2I domain in mouse TFII-I protein.

The general transcription factor TFII-I, with the corresponding gene name GTF2I, is an unusual transcriptional regulator that associates with both basal and signal-induced transcription factors. TFII-I consists of six GTF2I repeat domains, called I-repeats R1-R6. The structure and function of the GTF2I domain are not clearly understood, even though it contains a helix-loop-helix motif, which is considered to be the protein-protein interaction area, based on biochemical analyses. Here, we report the solution structure of the fifth repeat of the six GTF2I repeat domains from murine TFII-I, which was determined by heteronuclear multidimensional NMR spectroscopy (PDB code 1Q60). The three-dimensional structure of the GTF2I domain is classified as a new fold, consisting of four helices (residues 8-24, 34-39, 63-71, and 83-91), two antiparallel beta strands (residues 44-47 and 77-80), and a well-defined loop containing two beta-turns between sheet 1 and helix 3. All of the repeats probably have similar folds to that of repeat 5, because the conserved residues in the GTF2I repeat domains are assembled on the hydrophobic core, turns, and secondary structure elements, as revealed by a comparison of the sequences of the first through the sixth GTF2I repeats in TFII-I.

Solution structure of the kinase-associated domain 1 of mouse microtubule-associated protein/microtubule affinity-regulating kinase 3.

Microtubule-associated protein/microtubule affinity-regulating kinases (MARKs)/PAR-1 are common regulators of cell polarity that are conserved from nematode to human. All of these kinases have a highly conserved C-terminal domain, which is termed the kinase-associated domain 1 (KA1), although its function is unknown. In this study, we determined the solution structure of the KA1 domain of mouse MARK3 by NMR spectroscopy. We found that approximately 50 additional residues preceding the previously defined KA1 domain are required for its proper folding. The newly defined KA1 domain adopts a compact alpha+beta structure with a betaalphabetabetabetabetaalpha topology. We also found a characteristic hydrophobic, concave surface surrounded by positively charged residues. This concave surface includes the highly conserved Glu-Leu-Lys-Leu motif at the C terminus, indicating that it is important for the function of the KA1 domain.

An Arabidopsis SBP-domain fragment with a disrupted C-terminal zinc-binding site retains its tertiary structure.

SQUAMOSA promoter-binding proteins (SBPs) form a major family of plant-specific transcription factors, mainly related to flower development. SBPs share a highly conserved DNA-binding domain of approximately 80 amino acids (SBP domain), which contains two non-interleaved zinc-binding sites formed by eight conserved Cys or His residues. In the present study, an Arabidopsis SPL12 SBP-domain fragment that lacks a Cys residue involved in the C-terminal zinc-binding pocket was found to retain a folded structure, even though only a single Zn2+ ion binds to the fragment. Solution structure of this fragment determined by NMR is very similar to the previously determined structures of the full SBP domains of Arabidopsis SPL4 and SPL7. Considering the previous observations that chelating all the Zn2+ ions of SBPs resulted in the complete unfolding of the structure and that a mutation of the Cys residue equivalent to that described above impaired the DNA-binding activity, we propose that the Zn2+ ion at the N-terminal site is necessary to maintain the overall tertiary structure, while the Zn2+ ion at the C-terminal site is necessary for the DNA binding, mainly by guiding the basic C-terminal loop to correctly fit into the DNA groove.

Solution structure of the major DNA-binding domain of Arabidopsis thaliana ethylene-insensitive3-like3.

Ethylene-insensitive3 (EIN3) and EIN3-like (EIL) proteins are essential transcription factors in the ethylene signaling of higher plants. The EIN3/EIL proteins bind to the promoter regions of the downstream genes and regulate their expression. The location of the DNA-binding domain (DBD) in the primary structure was unclear, since the proteins show no sequence similarity to other known DBDs. Here, we identify the major DBD of an EIN3/EIL protein, Arabidopsis thaliana EIL3, containing a key mutational site for DNA binding and signaling (ein3-3 site), and determine its solution structure by NMR spectroscopy. The structure consists of five alpha-helices, possessing a novel fold dissimilar to known DBD structures. By a chemical-shift perturbation analysis, a region including the ein3-3 site is suggested to be involved in DNA binding.

The CAP-Gly domain of CYLD associates with the proline-rich sequence in NEMO/IKKgamma.

CYLD was originally identified as the human familial cylindromatosis tumor suppressor. Recently, it was reported that CYLD directly interacts with NEMO/IKKgamma and TRAF2 in the NF-kappaB signaling pathway. The two proteins bind to a region of CYLD that contains a Cys-box motif and the third cytoskeleton-associated protein-glycine conserved (CAP-Gly) domain. Here we report that the third CAP-Gly domain of CYLD specifically interacts with one of the two proline-rich sequences of NEMO/IKKgamma. The tertiary structure of the CAP-Gly domain shares the five-stranded beta sheet topology with the SH3 domain, which is well known as a proline-rich sequence-recognition domain. However, chemical shift mapping revealed that the peptide binding site of the CAP-Gly domain is formed without the long peptide binding loop characteristic of the SH3 domain. Therefore, CAP-Gly is likely to be a novel proline-rich sequence binding domain with a mechanism different from that of the SH3 domain.

Development of an ESR/MR dual-imaging system as a tool to detect bioradicals.

A system combining electron spin resonance imaging (ESRI) with another imaging modality capable of enabling visualization of the distribution of bioradicals on an anatomical map of the specimens would be a superior biomedical imaging system. We describe the development of an ESR/MR dual-imaging system with one permanent magnet and the biomedical applications of this system. The magnetic circuit developed for the ESR/MR dual-imaging system consisted of the permanent magnet made of Fe-Nd-B, pole pieces, and poke. The permanent magnet was installed on the MR side only, and the ESR side was made of pole pieces only. The magnetic field was adjusted to 0.5T at MR and to 0.042T at ESR. The overall dimensions of the magnet developed for the ESR/MR imaging system were 460 (W)x440 (D)x460 (H) mm, and it weighed 220 kg. The distance of each center for the magnet for ESR and MR imaging could be set as close as 200 mm. The entire ESR/MR imaging system can be installed in a common laboratory without magnetic shielding. MR images of plants (myoga) and small animals (mice and rats) were successfully acquired with or without ESR operation. ESR spectra of nitroxyl spin probes were also measured, even with MRI operation. ESR signals of triarylmethyl derivatives with narrow line-width (0.026 mT) were observed in living mice while MRI was operating. The ESR/MR imaging dual functions work properly with no electric or magnetic interference. The ESR/MR dual images demonstrate that this system enables visualization of the distribution of bioradicals on the anatomical map of the object.

Solution structure of the PWWP domain of the hepatoma-derived growth factor family.

Among the many PWWP-containing proteins, the largest group of homologous proteins is related to hepatoma-derived growth factor (HDGF). Within a well-conserved region at the extreme N-terminus, HDGF and five HDGF-related proteins (HRPs) always have a PWWP domain, which is a module found in many chromatin-associated proteins. In this study, we determined the solution structure of the PWWP domain of HDGF-related protein-3 (HRP-3) by NMR spectroscopy. The structure consists of a five-stranded beta-barrel with a PWWP-specific long loop connecting beta2 and beta3 (PR-loop), followed by a helical region including two alpha-helices. Its structure was found to have a characteristic solvent-exposed hydrophobic cavity, which is composed of an abundance of aromatic residues in the beta1/beta2 loop (beta-beta arch) and the beta3/beta4 loop. A similar ligand binding cavity occurs at the corresponding position in the Tudor, chromo, and MBT domains, which have structural and probable evolutionary relationships with PWWP domains. These findings suggest that the PWWP domains of the HDGF family bind to some component of chromatin via the cavity.

A novel zinc-binding motif revealed by solution structures of DNA-binding domains of Arabidopsis SBP-family transcription factors.

SQUAMOSA promoter binding proteins (SBPs) form a major family of plant-specific transcription factors related to flower development. Although SBPs are heterogeneous in primary structure, they share a highly conserved DNA-binding domain (DBD) that has been suggested to be zinc binding. Here we report the NMR solution structures of DBDs of two SBPs of Arabidopsis thaliana, SPL4 and SPL7. The two share essentially the same structural features. Each structure contains two zinc-binding sites consisting of eight Cys or His residues in a Cys3HisCys2HisCys or Cys6HisCys sequence motif in which the first four residues coordinate to one zinc and the last four coordinate to the other. These structures are dissimilar to other known zinc-binding structures, and thus represent a novel type of zinc-binding motif. The electrostatic profile on the surface suggested that a continuous region, including all the conserved basic residues, is involved in the DNA binding, the mode of which is likely to be novel as well.

Solution structure of a BolA-like protein from Mus musculus.

The BolA-like proteins are widely conserved from prokaryotes to eukaryotes. The BolA-like proteins seem to be involved in cell proliferation or cell-cycle regulation, but the molecular function is still unknown. Here we determined the structure of a mouse BolA-like protein. The overall topology is alphabetabetaalphaalphabetaalpha, in which beta(1) and beta(2) are antiparallel, and beta(3) is parallel to beta(2). This fold is similar to the class II KH fold, except for the absence of the GXXG loop, which is well conserved in the KH fold. The conserved residues in the BolA-like proteins are assembled on the one side of the protein.

Solution structure of the RWD domain of the mouse GCN2 protein.

GCN2 is the alpha-subunit of the only translation initiation factor (eIF2alpha) kinase that appears in all eukaryotes. Its function requires an interaction with GCN1 via the domain at its N-terminus, which is termed the RWD domain after three major RWD-containing proteins: RING finger-containing proteins, WD-repeat-containing proteins, and yeast DEAD (DEXD)-like helicases. In this study, we determined the solution structure of the mouse GCN2 RWD domain using NMR spectroscopy. The structure forms an alpha + beta sandwich fold consisting of two layers: a four-stranded antiparallel beta-sheet, and three side-by-side alpha-helices, with an alphabetabetabetabetaalphaalpha topology. A characteristic YPXXXP motif, which always occurs in RWD domains, forms a stable loop including three consecutive beta-turns that overlap with each other by two residues (triple beta-turn). As putative binding sites with GCN1, a structure-based alignment allowed the identification of several surface residues in alpha-helix 3 that are characteristic of the GCN2 RWD domains. Despite the apparent absence of sequence similarity, the RWD structure significantly resembles that of ubiquitin-conjugating enzymes (E2s), with most of the structural differences in the region connecting beta-strand 4 and alpha-helix 3. The structural architecture, including the triple beta-turn, is fundamentally common among various RWD domains and E2s, but most of the surface residues on the structure vary. Thus, it appears that the RWD domain is a novel structural domain for protein-binding that plays specific roles in individual RWD-containing proteins.

A fluorescent-based high-throughput screening assay for small molecules that inhibit the interaction of MdmX with p53.

A fluorescent-based high-throughput screening (HTS) assay for small molecules that inhibit the interaction of MdmX with p53 was developed and applied to identify new inhibitors. The assay evaluated the MdmX-p53 interaction by detecting the quenching of the fluorescence of green fluorescent protein (GFP) fused to the MdmX protein, after its interaction with a p53 peptide labeled with a fluorescence quencher. In this report, the developed HTS assay was applied to about 40 000 compounds, and 255 hit compounds that abrogated the GFP quenching were selected. Next, the obtained hits were reevaluated by other assays. First, their effects on the diffusion time of a fluorescently-labeled p53 peptide after incubation with the MdmX protein were tested by measuring the diffusion time using fluorescence correlation spectroscopy, and six stable hit compounds with IC(50) values less than 5 µM were selected. Next, we further confirmed their inhibition of the MdmX-p53 interaction by surface plasmon resonance. To indicate the efficacy of the hit compound as a candidate anticancer drug, we showed that the hit compound triggered apoptosis after p53 and p21 accumulation in cultured MV4;11 leukemia cells. Thus, the new HTS assay is effective for obtaining novel MdmX-p53 interaction inhibitors that are valuable as candidate compounds for cancer treatment.

Simultaneous imaging using Si-PM-based PET and MRI for development of an integrated PET/MRI system.

The silicon photomultiplier (Si-PM) is a promising photo-detector for PET for use in magnetic resonance imaging (MRI) systems because it has high gain and is insensitive to static magnetic fields. Recently we developed a Si-PM-based depth-of-interaction PET system for small animals and performed simultaneous measurements by combining the Si-PM-based PET and the 0.15 T permanent MRI to test the interferences between the Si-PM-based PET and an MRI. When the Si-PM was inside the MRI and installed around the radio frequency (RF) coil of the MRI, significant noise from the RF sequence of the MRI was observed in the analog signals of the PET detectors. However, we did not observe any artifacts in the PET images; fluctuation increased in the count rate of the Si-PM-based PET system. On the MRI side, there was significant degradation of the signal-to-noise ratio (S/N) in the MRI images compared with those without PET. By applying noise reduction procedures, the degradation of the S/N was reduced. With this condition, simultaneous measurements of a rat brain using a Si-PM-based PET and an MRI were made with some degradation in the MRI images. We conclude that simultaneous measurements are possible using Si-PM-based PET and MRI.