Fumiaki Yumoto

The Scripps Research Institute, لا هویا, California, United States

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Publications (34)118.72 Total impact

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    ABSTRACT: The Dlx5 homeodomain is a transcription factor related to the Drosophila Distal-less gene that is associated with breast and lung cancer, lymphoma, Rett syndrome and osteoporosis in humans. Mutations in the DLX5 gene have been linked to deficiencies in craniofacial and limb development in higher eukaryotes, including Split Hand and Foot Malformation-1 (SHFM-1) in humans. Our characterization of a Dlx5 homeodomain–(CGACTAATTAGTCG)2 complex by NMR spectroscopy paved the way for determination of its crystal structure at 1.85 Å resolution that enabled rationalization of the effects of disease-related mutations on the protein function. A remarkably subtle mutation, Q186H, is linked to SHFM-1; this change likely affects affinity of DNA binding by disrupting water-mediated interactions with the DNA major groove. A more subtle effect is implicated for the Q178P mutation, which is not in direct contact with the DNA. Our data indicate that these mutations diminish the ability of the Dlx5 homeodomain to recognize and bind target DNAs, and likely destabilize the formation of functional complexes.
    No preview · Article · Jan 2016 · Journal of Molecular Biology
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    ABSTRACT: FKBP52 and β-catenin have emerged in recent years as attractive targets for prostate cancer treatment. β-catenin interacts directly with the androgen receptor (AR) and has been characterized as a co-activator of AR-mediated transcription. FKBP52 is a positive regulator of AR in cellular and whole animal models and is required for the development of androgen-dependent tissues. We previously characterized an AR inhibitor termed MJC13 that puta-tively targets the AR BF3 surface to specifically inhibit FKBP52-regulated AR signaling. Predictive modeling suggests that β-catenin interacts with the AR hormone binding domain on a surface that overlaps with BF3. Here we demonstrate that FKBP52 and β-catenin interact directly in vitro and act in concert to promote a synergistic up-regulation of both hormone independent and-dependent AR signaling. Our data demonstrate that FKBP52 promotes β-catenin interaction with AR and is required for β-catenin co-activation of AR activity in prostate cancer cells. MJC13 effectively blocks β-catenin interaction with the AR LBD and the synergistic up-regulation of AR by FKBP52 and β-catenin. Our data suggest that co-regulation of AR by FKBP52 and β-catenin does not require FKBP52 PPIase catalytic activity, nor FKBP52 binding to Hsp90. However, the FKBP52 proline-rich loop that overhangs the PPIase pocket is critical for synergy.
    Full-text · Article · Jul 2015 · PLoS ONE
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    ABSTRACT: NANOG (from Irish mythology Tír na nÓg) transcription factor plays a central role in maintaining pluripotency, cooperating with OCT4 (also known as POU5F1 or OCT3/4), SOX2, and other pluripotency factors. Although the physiological roles of the NANOG protein have been extensively explored, biochemical and biophysical properties in relation to its structural analysis are poorly understood. Here we determined the crystal structure of the human NANOG homeodomain (hNANOG HD) bound to an OCT4 promoter DNA, which revealed amino acid residues involved in DNA recognition that are likely to be functionally important. We generated a series of hNANOG HD alanine substitution mutants based on the protein-DNA interaction and evolutionary conservation and determined their biological activities. Some mutant proteins were less stable, resulting in loss or decreased affinity for DNA binding. Overexpression of the orthologous mouse NANOG (mNANOG) mutants failed to maintain self-renewal of mouse embryonic stem cells without leukemia inhibitory factor. These results suggest that these residues are critical for NANOG transcriptional activity. Interestingly, one mutant, hNANOG L122A, conversely enhanced protein stability and DNA-binding affinity. The mNANOG L122A, when overexpressed in mouse embryonic stem cells, maintained their expression of self-renewal markers even when retinoic acid was added to forcibly drive differentiation. When overexpressed in epiblast stem cells or human induced pluripotent stem cells, the L122A mutants enhanced reprogramming into ground-state pluripotency. These findings demonstrate that structural and biophysical information on key transcriptional factors provides insights into the manipulation of stem cell behaviors and a framework for rational protein engineering.
    Full-text · Article · Apr 2015 · Proceedings of the National Academy of Sciences
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    ABSTRACT: SRY(Sex determining Region Y)-box or SOX transcription factors are important in early development and maintenance of different cell pools after birth. Of the ~20 SOX proteins (SRY, SOX1-SOX15, SOX17, SOX18, SOX21 and SOX30), SOX2, SOX9 and SOX10 mutations are primarily disease-associated: SOX2 with Combined Pituitary Hormone Deficiency, Microphthalmia, Septo-optic dysplasia and anophthalmic syndrome; SOX9 with Campomelic Dysplasia (affects development of the reproductive and skeletal system); and SOX10 (~94% sequence identity to SOX9) with Waardenburg Syndrome (affects audition and pigmentation in hair, eyes and skin; and specifically with WS types 2 and 4). As part of our Protein Structure Initiative (PSI)-Biology partnership, we performed structural and mutational analyses including x-ray crystallography and surface plasmon resonance assays, on the DNA-binding HMG domain of SOX9 with duplex DNA. Crystals were obtained in C222 space group and the structure was determined by molecular replacement to 2.77 Å resolution with final Rcryst/Rfree of 24.8/27.8%. The overall structure of the SOX9-DNA complex is similar to other SOX/SRY protein complexes. The SOX9-DNA protein-DNA interactions suggested a panel of mutations to assay for biochemical activity, which allowed us to understand the molecular basis of five mutations identified in Campomelic Dysplasia. These mutated residues have direct contact with DNA as well as indirect contacts, i.e., these mutations lead to allosteric secondary structure changes in the protein, which affect residues in direct contact with DNA. Due to the very high sequence identity between SOX9 and SOX10, our crystal structure also helps to rationalize the effect of SOX10 mutations in Waardenburg Syndrome. This work is supported by NIH grants U54 GM094586 and U01 GM094614. SSRL operations are funded by DOE BES, and the SSRL SMB program by DOE BER, NIH NCRR BTP and NIH NIGMS.
    No preview · Article · Aug 2014 · Acta Crystallographica Section A: Foundations and Advances
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    ABSTRACT: Plastins are Ca(2+)-regulated actin-bundling proteins, and essential for developing and stabilizing actin cytoskeletons. T-plastin is expressed in epithelial and mesenchymal cells of solid tissues, whereas L-plastin is expressed in mobile cells such as hemopoietic cell lineages and cancer cells. Using various spectroscopic methods, gel-filtration chromatography, and isothermal titration calorimetry, we here demonstrate that the EF-hand motifs of both T- and L-plastin change their structures in response to Ca(2+), but the sensitivity to Ca(2+) is lower in T-plastin than in L-plastin. These results suggest that T-plastin is suitable for maintaining static cytoskeletons, whereas L-plastin is suitable for dynamic rearrangement of cytoskeletons.
    No preview · Article · Nov 2012 · Biochemical and Biophysical Research Communications
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    ABSTRACT: The human nuclear factor related to kappa-B-binding protein (NFRKB) is a 1299-residue protein that is a component of the metazoan INO80 complex involved in chromatin remodeling, transcription regulation, DNA replication and DNA repair. Although full length NFRKB is predicted to be around 65% disordered, comparative sequence analysis identified several potentially structured sections in the N-terminal region of the protein. These regions were targeted for crystallographic studies, and the structure of one of these regions spanning residues 370-495 was determined using the JCSG high-throughput structure determination pipeline. The structure reveals a novel, mostly helical domain reminiscent of the winged-helix fold typically involved in DNA binding. However, further analysis shows that this domain does not bind DNA, suggesting it may belong to a small group of winged-helix domains involved in protein-protein interactions.
    Full-text · Article · Sep 2012 · PLoS ONE
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    ABSTRACT: Abstract Troponin C (TnC) is the Ca2+-sensing subunit of troponin that triggers the contraction of striated muscles. In scallops, the striated muscles consume little ATP energy in sustaining strong contractile forces. The N-terminal domain of TnC works as the Ca2+ sensor in vertebrates, whereas scallop TnC uses the C-terminal domain as the Ca2+ sensor, suggesting that there are differences in the mechanism of the Ca2+-dependent regulation of muscles between invertebrates and vertebrates. Here, we report the crystal structure of the Akazara scallop adductor muscle TnC C-terminal domain (asTnCC) complexed with a short troponin I fragment (asTnIS) and Ca2+. The electron density of a Ca2+ ion is observed in only one of the two EF-hands. The EF-hands of asTnCC can only be in the fully open conformation with the assistance of asTnIS. The number of hydrogen bonds between asTnCC and asTnIS is markedly lower than the number in the vertebrate counterparts. The Ca2+ modulation on the binding between asTnCC and asTnIS is weaker, but structural change of the complex depending on Ca2+ concentration was observed. Together, these findings provide a detailed description of the distinct molecular mechanism of contractile regulation in the scallop adductor muscle from that of vertebrates.
    No preview · Article · Aug 2012 · Biological Chemistry
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    ABSTRACT: We report the three-dimensional structure of a β-catenin armadillo repeat in complex with the liver receptor homolog-1 (LRH-1) ligand binding domain at 2.8 Å resolution as the first structure of β-catenin in complex with any nuclear receptor. The surface of β-catenin that binds LRH-1 partly overlaps defined contact sites for peptide segments of β-catenin partners, including T-cell factor-4. The surface of LRH-1 that engages β-catenin is comprised of helices 1, 9, and 10 and is distinct from known interaction surfaces of LRH-1, including corepressor and coactivator binding sites. Targeted mutagenesis of amino acids forming both sides of the LRH-1/β-catenin interface reveals that they are essential for stable interactions between these proteins in solution. The LRH-1 binding site in β-catenin is also required for association with androgen receptor, providing evidence that the observed LRH-1/β-catenin interaction may be prototypic.
    Preview · Article · Dec 2011 · Proceedings of the National Academy of Sciences
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    ABSTRACT: Catechins, biologically active polyphenols in green tea, are known to have a protective effect against cardiovascular diseases. In this study, we investigated direct actions of green tea catechins on cardiac muscle function to explore their uses as potential drugs for cardiac muscle disease. The effects of catechins were systematically investigated on the force-pCa relationship in skinned cardiac muscle fibres to determine their direct effects on cardiac myofilament contractility. The mechanisms of action of effective catechins were investigated using troponin exchange techniques, quartz crystal microbalance, nuclear magnetic resonance and a transgenic mouse model. (-)-Epicatechin-3-gallate (ECg) and (-)-epigallocatechin-3-gallate (EGCg), but not their stereoismers (-)-catechin-3-gallate and (-)-gallocatechin-3-gallate, decreased cardiac myofilament Ca(2+) sensitivity probably through its interaction with cardiac troponin C. EGCg restored cardiac output in isolated working hearts by improving diastolic dysfunction caused by increased myofilament Ca(2+) sensitivity in a mouse model of hypertrophic cardiomyopathy. The green tea catechins, ECg and EGCg, are Ca(2+) desensitizers acting through binding to cardiac troponin C. These compounds might be useful compounds for the development of therapeutic agents to treat the hypertrophic cardiomyopathy caused by increased Ca(2+) sensitivity of cardiac myofilaments.
    Full-text · Article · Nov 2010 · British Journal of Pharmacology
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    ABSTRACT: Despite >50 years of research work since the discovery of sliding filament mechanism in muscle contraction, structural details of the coupling of cyclic cross-bridge movement to ATP hydrolysis are not yet fully understood. An example would be whether lever arm tilting on the myosin filament backbone will occur in the absence of actin. The most direct way to elucidate such movement is to record ATP-induced cross-bridge movement in hydrated thick filaments. Using the hydration chamber, with which biological specimens can be kept in an aqueous environment in an electron microscope, we have succeeded in recording ATP-induced cross-bridge movement in hydrated thick filaments consisting of rabbit skeletal muscle myosin, with gold position markers attached to the cross-bridges. The position of individual cross-bridges did not change appreciably with time in the absence of ATP, indicating stability of time-averaged cross-bridge mean position. On application of ATP, individual cross-bridges moved nearly parallel to the filament long axis. The amplitude of the ATP-induced cross-bridge movement showed a peak at 5-7.5 nm. At both sides of the filament bare region, across which the cross-bridge polarity was reversed, the cross-bridges were found to move away from, but not toward, the bare region. Application of ADP produced no appreciable cross-bridge movement. Because ATP reacts rapidly with the cross-bridges (M) to form complex (M x ADP x Pi) with an average lifetime >10 s, the observed cross-bridge movement is associated with reaction, M + ATP --> M x ADP x Pi. The cross-bridges were observed to return to their initial position after exhaustion of ATP. These results constitute direct demonstration of the cross-bridge recovery stroke.
    Full-text · Article · Dec 2008 · Proceedings of the National Academy of Sciences
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    Masayuki Nara · Fumiaki Yumoto · Hiroyuki Kagi · Masaru Tanokura
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    ABSTRACT: Akazara scallop striated adductor muscle troponin C (TnC) binds only one Ca2+ because the three EF-hand motifs are short of critical residues for the coordination of Ca2+. Fourier-transform infrared spectroscopy was applied to study coordination structures of M2+ (= Mg2+, Ca2+, Sr2+, and Ba2+) bound in an Akazara scallop TnC mutant (E142D) and the wild-type TnC C-lobe in D2O solution. The region of the COO− antisymmetric stretch provides information regarding the coordination modes of a COO− group to a metal ion. The side chain COO− group of Asp142 did not bind to Ca2+ in the bidentate coordination mode, suggesting that the absence of a methylene group is critical for the Ca2+ coordination structure of Akazara scallop TnC (Nara et al., Vib Spect 2006, 42, 188–191). The present study has shown that the absence of a methylene group is not compensated for by a larger metal ion such as Sr2+ or Ba2+. CD spectra showed that the secondary structures are conserved between M2+-free (apo), Mg2+-loaded, Ca2+-loaded, Sr2+-loaded, and Ba2+-loaded states, which was consistent with the results estimated from their amide I band patterns. The metal-ligand interaction at position 12 of site IV is discussed in comparison with the coordination mode of the side chain COO− group of the wild-type TnC C-lobe. © 2008 Wiley Periodicals, Inc. Biopolymers 89: 595–599, 2008. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com
    Full-text · Article · Jul 2008 · Biopolymers
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    ABSTRACT: Plant respiratory burst oxidase homolog (rboh) proteins, which are homologous to the mammalian 91-kDa glycoprotein subunit of the phagocyte oxidase (gp91(phox)) or NADPH oxidase 2 (NOX2), have been implicated in the production of reactive oxygen species (ROS) both in stress responses and during development. Unlike mammalian gp91(phox)/NOX2 protein, plant rboh proteins have hydrophilic N-terminal regions containing two EF-hand motifs, suggesting that their activation is dependent on Ca(2+). However, the significance of Ca(2+) binding to the EF-hand motifs on ROS production has been unclear. By employing a heterologous expression system, we showed that ROS production by Arabidopsis thaliana rbohD (AtrbohD) was induced by ionomycin, which is a Ca(2+) ionophore that induces Ca(2+) influx into the cell. This activation required a conformational change in the EF-hand region, as a result of Ca(2+) binding to the EF-hand motifs. We also showed that AtrbohD was directly phosphorylated in vivo, and that this was enhanced by the protein phosphatase inhibitor calyculin A (CA). Moreover, CA itself induced ROS production and dramatically enhanced the ionomycin-induced ROS production of AtrbohD. Our results suggest that Ca(2+) binding and phosphorylation synergistically activate the ROS-producing enzyme activity of AtrbohD.
    No preview · Article · May 2008 · Journal of Biological Chemistry
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    ABSTRACT: Akazara scallop (Chlamys nipponensis akazara) troponin C (TnC) of striated adductor muscle binds only one Ca(2+) ion at the C-terminal EF-hand motif (Site IV), but it works as the Ca(2+)-dependent regulator in adductor muscle contraction. In addition, the scallop troponin (Tn) has been thought to regulate muscle contraction via activating mechanisms that involve the region spanning from the TnC C-lobe (C-lobe) binding site to the inhibitory region of the TnI, and no alternative binding of the TnI C-terminal region to TnC because of no similarity between second TnC-binding regions of vertebrate and the scallop TnIs. To clarify the Ca(2+)-regulatory mechanism of muscle contraction by scallop Tn, we have analyzed the Ca(2+)-binding properties of the complex of TnC C-lobe and TnI peptide, and their interaction using isothermal titration microcalorimetry, nuclear magnetic resonance, circular dichroism, and gel filtration chromatography. The results showed that single Ca(2+)-binding to the Site IV leads to a structural transition not only in Site IV but also Site III through the structural network in the C-lobe of scallop TnC. We therefore assumed that the effect of Ca(2+)-binding must lead to a change in the interaction mode between the C-lobe of TnC and the TnI peptide. The change should be the first event of the transmission of Ca(2+) signal to TnI in Tn ternary complex.
    No preview · Article · May 2008 · Biochemical and Biophysical Research Communications
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    ABSTRACT: Troponin C (TnC) is the Ca(2+)-binding component of troponin and triggers muscle contraction. TnC of the invertebrate Akazara scallop can bind only one Ca(2+) at the C-terminal EF-hand motif. Recombinant TnC was expressed in Escherichia coli, purified, complexed with a 24-residue synthetic peptide derived from scallop troponin I (TnI) and crystallized. The crystals diffracted X-rays to 1.80 A resolution and belonged to space group P2(1)2(1)2(1), with unit-cell parameters a = 32.1, b = 42.2, c = 60.0 A. The asymmetric unit was assumed to contain one molecular complex of the Akazara scallop TnC C-lobe and TnI fragment, with a Matthews coefficient of 1.83 A(3) Da(-1) and a solvent content of 33.0%.
    Full-text · Article · Jul 2007 · Acta Crystallographica Section F Structural Biology and Crystallization Communications
  • Fumiaki Yumoto · Masaru Tanokura
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    ABSTRACT: The Ca2+-regulation of scallop striated muscle contraction, a Ca2+-regulation mechanism that is linked to myosin, was first discovered by A. G. Szent-Györgyi and his colleagues. 1,2 In myosin-linked Ca2+-regulation, the Ca2+ -receptive site is the essential light chain of myosin, and the ATPase of the scallop myofibrils has been found to be desensitized to Ca2+ by removal of the regulatory light chain (RLC) of myosin in response to treatment with a divalent cation chelator (EDTA). At the same time, three components of troponin and tropomyosin have also been isolated from scallop striated muscle, and several of their biochemical properties have been investigated.3–5 In this troponin-linked Ca2+-regulation, the concurrent presence of all three components of troponin (troponins C, I, and T; TnC, TnI, and TnT) and tropomyosin are necessary for the regulation of actomyosin ATPase activity.6–10 The action of Ca2+ on TnC ultimately induces actomyosin ATPase activity. Troponin-linked Ca2+ -regulation is also desensitized by the removal of TnC in response to treatment with divalent cation chelators such as EDTA or CDTA. The mutual relation of these two types of Ca2+-regulations in scallop myofibrils was then investigated as follows.11 Desensitized scallop myofibrils were prepared by removing both RLC and TnC by treatment with a divalent cation chelator, CDTA, and the effects of reconstitution with RLC and/or TnC on the ATPase activity of the desensitized myofibrils were examined.
    No preview · Article · Feb 2007 · Advances in Experimental Medicine and Biology
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    ABSTRACT: In summary, we have shown that the TnI-TnC-TnT2 ternary complex (-52 kDa) has a mobile actin-binding domain (-6.1 kDa) that tumbles independently of the core domain. By docking the mobile domain and the core domain into the cryo-EM map obtained for thin filaments at low Ca2+, a model for actin-troponin interaction has been obtained. This model shows the atomic details of interactions of actin with the mobile domain and suggests the mechanism by which troponin generates a shift in the azimuthal position of tropomyosin in response to changes in Ca2+ levels. In this model the mobile domain of troponin interacts with three actins and one troponin interacts with four actin molecules. The relationship between myosin and the mobile domain suggests that the latter may work as a fail-safe latch to secure a relaxed state. The model also provides insights into many mutations associated with human cardiomyopathy and has implications for the function of other actin-binding proteins. Coordinates of the mobile domain have been deposited in the Protein Data Bank under accession codes 1VDI (low Ca2+) and 1VDJ (high Ca2+). Chemical shifts of the mobile domain have been deposited in the BMRB under accession ID 18140.
    No preview · Article · Feb 2007 · Advances in Experimental Medicine and Biology
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    ABSTRACT: Fourier-transform infrared spectroscopy (FT-IR) was applied to study the coordination structure of Ca2+ bound in Akazara scallop troponin C (TnC) and its site-directed mutant possessing inactivated Site IV (E142D mutant) in D2O solution. The COO− antisymmetric stretching region provides information about the coordination modes of a COO− group to a metal ion. The wild type exhibits a band at 1543cm−1 in the Ca2+-bound state, indicating that the side-chain COO− group of Glu142 (the position 12 of Site IV) serves as the ligand for Ca2+ in the bidentate coordination mode [F. Yumoto, M. Nara, H. Kagi, W. Iwasaki, T. Ojima, K. Nishita, K. Nagata, M. Tanokura, Eur. J. Biochem. 268 (2001) 6284–6290]. However, the E142D mutant showed no band around 1543cm−1 in the Ca2+-loaded state, indicating that the side-chain COO− group of Asp142 does not bind to Ca2+ in the bidentate coordination mode. This result suggests that the absence of a methylene group is critical for the Ca2+ coordination structure of Akazara scallop TnC. The Ca2+-ligand interaction at Site IV is discussed in comparison with the results of synthetic peptide analogues of Site IV of Akazara scallop TnC.
    No preview · Article · Nov 2006 · Vibrational Spectroscopy
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    ABSTRACT: The coordination structures of Ca2+ ion bound to synthetic peptide analogues of the calcium-binding site III of rabbit skeletal muscle troponin C (TnC) were investigated by Fourier transform infrared (FTIR) spectroscopy. The region of the COO− antisymmetric stretching vibration provides information about the coordination modes of a COO− group to a metal ion. The 34-residue peptide corresponding to the EF hand motif (helix–loop–helix) showed a band at 1552 cm−1 in the Ca2+-loaded state, indicating that the side-chain COO− group of Glu at the 12th position serves as a ligand for Ca2+ in the bidentate coordination mode. On the other hand, the 13-residue peptide (Ac-DRDADGYIDAEEL-NH2) containing the Ca2+-binding site III (DRDADGYIDAEE) did not show such spectral patterns in the Ca2+-loaded state, meaning that shorter synthetic peptide corresponding to the site III has less or no affinity for Ca2+. It was found that the 17-residue peptide (Ac-DRDADGYIDAEELAEIF-NH2) is the minimum peptide necessary for the interaction of side-chain COO−of Glu at the 12th position with Ca2+ in the bidentate coordination mode. We discuss the relationship between the amino acid length of synthetic peptide analogues and the formation of Ca2+-bound coordination structure. © 2006 Wiley Periodicals, Inc. Biopolymers 82: 339–343, 2006 This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com
    No preview · Article · Jul 2006 · Biopolymers
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    ABSTRACT: The zona pellucida is the acellular transparent envelope surrounding the mammalian oocyte. An analysis of the changes in the structures of zona pellucida proteins is essential for understanding the molecular mechanisms underlying the important physiological roles of the zona during fertilization and preimplantation. The hardening of the zona caused by the structural changes during fertilization is generally accepted to be responsible for blocking polyspermy. In this study, we analyzed changes in the secondary structure of the zona during fertilization by Fourier transform infrared (FTIR) spectroscopy and transmission electron microscopy. The predominance of beta-sheet structure in porcine ovarian egg zona proteins in water was ascertained using FTIR spectra. Alpha-helix structure was also present. The attenuated total reflection (ATR)-FTIR spectrum of intact, unsolubilized porcine zonae pellucidae from ovarian eggs indicated that the zona proteins in the native zona pellucida also have beta-structure as the main constituent. Attenuated total reflection-FTIR spectroscopy of intact bovine zona pellucida obtained from ovarian and fertilized eggs at the blastocyst stage revealed that the beta-structure content increased during fertilization. Furthermore, a reduction of the thickness of the zona during fertilization was observed using transmission electron microscopy. Therefore, the change in the zona architecture that causes hardening of the zona during fertilization is accompanied by changes in the secondary structure of the zona proteins.
    Full-text · Article · Mar 2006 · Experimental Biology and Medicine
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    ABSTRACT: Six missense mutations in human cardiac troponin I (cTnI) were recently found to cause restrictive cardiomyopathy (RCM). We have bacterially expressed and purified these human cTnI mutants and examined their functional and structural consequences. Inserting the human cTnI into skinned cardiac muscle fibers showed that these mutations had much greater Ca2+-sensitizing effects on force generation than the cTnI mutations in hypertrophic cardiomyopathy (HCM). The mutation K178E in the second actin-tropomyosin (Tm) binding region showed a particularly potent Ca2+-sensitizing effect among the six RCM-causing mutations. Circular dichroism and nuclear magnetic resonance spectroscopy revealed that this mutation does not extensively affect the structure of the whole cTnI molecule, but induces an unexpectedly subtle change in the structure of a region around the mutated residue. The results indicate that the K178E mutation has a localized effect on a structure that is critical to the regulatory function of the second actin-Tm binding region of cTnI. The present study also suggests that both HCM and RCM involving cTnI mutations share a common feature of increased Ca2+ sensitivity of cardiac myofilament, but more severe change in Ca2+ sensitivity is associated with the clinical phenotype of RCM.
    Full-text · Article · Jan 2006 · Biochemical and Biophysical Research Communications

Publication Stats

696 Citations
118.72 Total Impact Points

Institutions

  • 2015
    • The Scripps Research Institute
      • Department of Integrative Structural and Computational Biology
      لا هویا, California, United States
  • 2011-2015
    • University of California, San Francisco
      • Department of Biochemistry and Biophysics
      San Francisco, California, United States
  • 2003-2012
    • The Jikei University School of Medicine
      • • Department of Cell Physiology
      • • Laboratory of Neurophysiology
      Edo, Tōkyō, Japan
  • 2001-2008
    • The University of Tokyo
      • Department of Applied Biological Chemistry
      Tōkyō, Japan
  • 2007
    • Teikyo University
      • Department of Biosciences
      Tokyo, Tokyo-to, Japan