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Volume 7
Showing 1-15 articles out of 15 articles from the selected issue
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Regular Article
  • Rumi Shiba, Mika Umeyama, Sayaka Tsukasa, Hironari Kamikubo, Yoichi Ya ...
    Volume 7 (2011) Pages 1-10
    Released: January 19, 2011
    JOURNALS FREE ACCESS
    Decoding sequence information is equivalent to elucidating the design principles of proteins. For this purpose, we conducted systematic alanine insertion analysis to reveal the regions in the primary structure where the sequence continuity cannot be disrupted. We applied this method to dihydrofolate reductase (DHFR), and examined the effects of alanine insertion on structure and the enzymatic activity by solubility assay and trimethoprim resistance, respectively. We revealed that DHFR is composed of “Structure Elements”, “Function Elements” and linkers connecting these elements. The “Elements” are defined as regions where the alanine insertion caused DHFR to become unstructured or inactive. Some “Structure Elements” overlap with “Function Elements”, indicating that loss of structure leads to loss of function. However, other “Structure Elements” are not “Function Elements”, in that alanine insertion mutants of these regions exhibit substrate- or inhibitor-induced folding. There are also some “Function Elements” which are not “Structure Elements”; alanine insertion into these elements deforms the catalytic site topology without the loss of tertiary structure. We hypothesize that these elements are involved essential interactions for structure formation and functional expression. The “Elements” are closely related to the module structure of DHFR. An “Element” belongs to a single module, and a single module is composed of some number of “Elements.” We propose that properties of a module are determined by the “Elements” it contains. Systematic alanine insertion analysis is an effective and unique method for deriving the regions of a sequence that are essential for structure formation and functional expression.
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  • Hajime Honda, Shin'ichi Ishiwata
    Volume 7 (2011) Pages 11-19
    Released: February 04, 2011
    JOURNALS FREE ACCESS
    We found that a solution of actin filaments can form a periodic texture in the process of drying on a flat glass surface in the air; the periodic texture was composed of smooth meandering bundles of actin filaments. We also found that a branched salt crystal grows in the space between the meandering bundles of actin filaments. The distance between the adjacent striae (striation period) in the resulting dried two-dimensional pattern of striation decreased from about 50 to 2 μm, as the ambient temperature was increased from 4 to 40°C at 1 mg/ml actin, and showed an increasing tendency from a few to several tens μm with the increase in the initial concentration of actin filaments from 0.6 to 2.0 mg/ml at room temperature. As the speed of drying is increased at a certain temperature, the striation period was also found to decrease. We propose that the formation of the two-dimensional striation pattern of bundles of actin filaments is the result of condensation of proteins due to dehydration, and suggest that the solvent flow from the center to the periphery of the sample causes the meandering of actin filaments.
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Review Article
  • Hiroyuki Iwamoto
    Volume 7 (2011) Pages 21-28
    Released: February 17, 2011
    JOURNALS FREE ACCESS
    Insects, the largest group of animals on the earth, owe their prosperity to their ability of flight and small body sizes. The ability of flight provided means for rapid translocation. The small body size allowed access to unutilized niches. By acquiring both features, however, insects faced a new problem: They were forced to beat their wings at enormous frequencies. Insects have overcome this problem by inventing asynchronous flight muscle, a highly specialized form of striated muscle capable of oscillating at >1,000 Hz. This article reviews the structure, mechanism, and molecular evolution of this unique invention of nature.
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Regular Article
  • You Jia, Masaaki Kuroda
    Volume 7 (2011) Pages 29-34
    Released: April 29, 2011
    JOURNALS FREE ACCESS
    N-terminal actin-binding domain of α-actinin is connected to central rod domain through flexible neck region that is susceptible to proteolysis. It is suggested that the neck region assumes variable orientations by actin binding. In order to examine the effect of actin binding to α-actinin, we carried out limited digestion of α-actinin by chymotrypsin in the presence and absence of F-actin. Although the cleavage process was retarded when bound to F-actin, digestion to 32 kDa-head and 55 kDa-rod domains occurred through the same intermediate products as the digestion in the absence of F-actin. N-terminal sequencing of 55 kDa-fragment showed the neck region was cleaved at 276-Leu. The cleavage site was not affected by binding to F-actin nor ionic strength of the solvent. It was also indicated that α-actinin was cleaved at 15-Tyr by chymotrypsin. Quantitation of the cleavage products by densitometry of the SDS-gels suggested the conformational change of α-actinin at domain-connecting regions by F-actin binding.
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  • Chihiro Kitagawa, Akiko Nakatomi, Dasol Hwang, Issey Osaka, Hiroki Fuj ...
    Volume 7 (2011) Pages 35-49
    Released: May 21, 2011
    JOURNALS FREE ACCESS
    Electrospray ionization mass spectrometry (ESI-MS), circular dichroism (CD), nuclear magnetic resonance (NMR) spectroscopy, flow dialysis, and bioactivity measurements were employed to investigate the roles of the C-terminal residues of calmodulin (CaM). In the present study, we prepared a series of truncated mutants of chicken CaM that lack four (CCMΔ4) to eight (CCMΔ8) residues at the C-terminal end. It was found that CCMΔ4, lacking the last four residues (M145 to K148), binds four Ca2+ ions. Further deletion gradually decreased the ability to bind the fourth Ca2+ ion, and CCMΔ8 completely lost the ability. Interestingly, both lobes of Ca2+-sturated CCMΔ5 showed instability in the conformation, although limited part in the C-lobe of Ca2+-saturated CCMΔ4 was instable. Moreover, unlike CCMΔ4, structure of the C-lobe in CCMΔ5 bound to the target displayed dissimilarity to that of CaM, suggesting that deletion of M144 changes the binding manner. Deletion of the last five residues (M144 to K148) and further truncation of the C-terminal region decreased apparent capacity for target activation. Little contribution of the last four residues including M145 was observed for structural stability, Ca2+-binding, and target activation. Although both M144 and M145 have been recognized as key residues for the function, the present data suggest that M144 is a more important residue to attain Ca2+ induced conformational change and to form a proper Ca2+-saturated conformation.
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  • Yuki Sudo, Rikou Tanaka, Toshitatsu Kobayashi, Naoki Kamo, Toshiyuki K ...
    Volume 7 (2011) Pages 51-58
    Released: June 18, 2011
    JOURNALS FREE ACCESS
    An approach of cell-free synthesis is presented for the functional expression of transmembrane proteins without the need of refolding. The transmembrane region of the pharaonis halobacterial transducer protein, pHtrII, was translated with various large soluble tags added (thioredoxin, glutathione S-transferase, green fluorescent protein and maltose binding protein). In this system, all fusion pHtrII were translated in a soluble fraction, presumably, forming giant micelle-like structures. The detergent n-dodecyl-β-D-maltoside was added for enhancing the solubilization of the hydrophobic region of pHtrII. The activity of the expressed pHtrII, having various tags, was checked using a pull-down assay, using the fact that pHtrII forms a signaling complex with pharaonis phoborhodopsin (ppR) in the membrane, as also in the presence of a detergent. All tagged pHtrII showed a binding activity with ppR. Interestingly, the binding activity with ppR was positively correlated with the molecular weight of the soluble tags. Thus, larger soluble tags lead to higher binding activities. We could show, that our approach is beneficial for the preparation of active membrane proteins, and is also potentially applicable for larger membrane proteins, such as 7-transmembrane proteins.
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  • Masafumi Koike, Noriko Nishioka, Seiji Kojima, Michio Homma
    Volume 7 (2011) Pages 59-67
    Released: September 07, 2011
    JOURNALS FREE ACCESS
    The polar flagellum of Vibrio alginolyticus is driven by sodium ion flux via a stator complex, composed of PomA and PomB, across the cell membrane. The interaction between PomA and the rotor component FliG is believed to generate torque required for flagellar rotation. Previous research reported that a GFP-fused FliG retained function in the Vibrio flagellar motor. In this study, we found that N-terminal or C-terminal fusion of GFP has different effects on both torque generation and the switching frequency of the direction of flagellar motor rotation. We could detect the GFP-fused FliG in the basal-body (rotor) fraction although its association with the basal body was less stable than that of intact FliG. Furthermore, the fusion of GFP to the C-terminus of FliG, which is believed to be directly involved in torque generation, resulted in very slow motility and prohibited the directional change of motor rotation. On the other hand, the fusion of GFP to the N-terminus of FliG conferred almost the same swimming speed as intact FliG. These results are consistent with the premise that the C-terminal domain of FliG is directly involved in torque generation and the GFP fusions are useful to analyze the functions of various domains of FliG.
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  • Takafumi Mizuno, Yuji Sekiguchi
    Volume 7 (2011) Pages 69-75
    Released: September 27, 2011
    JOURNALS FREE ACCESS
    Fish epidermal keratocytes locomote along surfaces without overall cell size or shape changes, as kinematically described by the graded radial extension (GRE) model. We found that the cell size increased during locomotion after the addition of a low dose of staurosporine or K-252a, broad-spectrum protein-serine/threonine kinase inhibitors. Quantitative shape analysis showed that the cell size increase resulted from an increase in lamellipodial width, the maximal length perpendicular to the direction of the cell locomotion, whereas the lamellipodial length, along the locomotion direction, remained constant. Importantly, the gradient of radial extension in the leading edge disappeared during lamellipodial width increase. These results suggest that a special mechanism for producing graded radial extension of lamellipodia exists to keep cell size constant, and that a protein-serine/threonine kinase plays an important role in regulating this mechanism.
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  • Takeshi Sugawara, Kunihiko Kaneko
    Volume 7 (2011) Pages 77-88
    Released: November 09, 2011
    JOURNALS FREE ACCESS
    Biological units such as macromolecules, organelles, and cells are directed to a proper location by gradients of chemicals. We consider a macroscopic element with surface binding sites where chemical adsorption reactions can occur and show that a thermodynamic force generated by chemical gradients acts on the element. By assuming local equilibrium and adopting the grand potential used in thermodynamics, we derive a formula for the “chemophoresis” force, which depends on chemical potential gradients and the Langmuir isotherm. The conditions under which the formula is applicable are shown to occur in intracellular reactions. Further, the role of the chemophoresis in the partitioning of bacterial chromosomal loci/plasmids during cell division is discussed. By performing numerical simulations, we demonstrate that the chemophoresis force can contribute to the regular positioning of plasmids observed in experiments.
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Review Article
  • Tatsuya Iwata, Satoru Tokutomi, Hideki Kandori
    Volume 7 (2011) Pages 89-98
    Released: November 09, 2011
    JOURNALS FREE ACCESS
    Phototropin (Phot), a blue-light photoreceptor in plants, consists of two FMN-binding domains (named LOV1 and LOV2) and a serine/threonine (Ser/Thr) kinase domain. We have investigated light-induced structural changes of LOV domains, which lead to the activation of the kinase domain, by means of light-induced difference FTIR spectroscopy. FTIR spectroscopy revealed that the reactive cysteine is protonated in both unphotolyzed and triplet-excited states, which is difficult to detect by other methods such as X-ray crystallography. In this review, we describe the light-induced structural changes of hydrogen-bonding environment of FMN chromophore and protein backbone in Adiantum neo1-LOV2 in the C=O stretching region by use of 13C-labeled samples. We also describe the comprehensive FTIR analysis of LOV2 domains among Arabidopsis phot1, phot2, and Adiantum neo1 with and without Jα helix domain.
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Hypotheses and Perspectives
  • Christoph Gerle
    Volume 7 (2011) Pages 99-104
    Released: November 09, 2011
    JOURNALS FREE ACCESS
    The membrane domain of rotary ATPases (Fo/Vo/Ao) contains a membrane-embedded rotor ring which rotates against an adjacent cation channel-forming subunit during catalysis. The mechanism that allows stabilization of the highly mobile and yet tightly connected domains during operation while not impeding rotation is unknown. Remarkably, all known ATPase rotor rings are filled by lipids. In the crystal structure of the rotor ring of a V-ATPase from Enterococcus hirae the ring filling lipids form a proper membrane that is lower with respect to the embedding membrane surrounding both subunits. I propose first, that a vertical shift between lumenal lipids and embedding outside membrane is a general feature of rotor rings and second that it leads to a radial potential fall-off between rotor ring and cation channel, creating attractive forces that impact rotor-stator interaction in Fo/Vo/Ao during rotation.
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Note
  • Noriyuki Yoshii, Tomomi Emoto, Emiko Okamura
    Volume 7 (2011) Pages 105-111
    Released: November 18, 2011
    JOURNALS FREE ACCESS
    The kinetics of binding, the diffusivity, and the binding amount of a neuropeptide, leucine-enkephalin (L-Enk) to lipid bilayer membranes are quantified by pulsed-field-gradient (PFG) 1H NMR in situ. The peptide signal is analyzed by the solution of the Bloch equation with exchange terms in the presence of large unilamellar vesicles (LUVs) as confined, but fluid model cell membranes. Even in the case that the membrane-bound and the free states of L-Enk cannot be distinguished in the one-dimensional NMR spectrum, the PFG technique unveils the bound component of L-Enk after the preferential decay of the free component at the high field gradient. In 100-nm diameter LUVs consisting of egg phosphatidylcholine, the rate constants of the peptide binding and dissociation are 0.040 and 0.40 s–1 at 303 K. This means that the lifetime of the peptide binding is of the order from second to ten-second. The diffusivity of the bound L-Enk is 5×10–12 m2/s, almost 60 times as restricted as the movement of free L-Enk at 303 K. One-tenth of 5 mM L-Enk is bound to 40 mM LUV. The binding free energy is calculated to be −2.9 kJ/mol, the magnitude close to the thermal fluctuation, 2.5 kJ/mol. The result demonstrates the potential of PFG 1H NMR to quantify molecular dynamics of the peptide binding to membranes.
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Review Article
  • Takashi Yoshidome
    Volume 7 (2011) Pages 113-122
    Released: November 18, 2011
    JOURNALS FREE ACCESS
    We briefly review our theoretical study on the rotation scheme of F1-ATPase. In the scheme, the key factor is the water entropy which has been shown to drive a variety of self-assembly processes in biological systems. We decompose the crystal structure of F1-ATPase into three sub-complexes each of which is composed of the γ subunit, one of the β subunits, and two α subunits adjacent to them. The βE, βTP, and βDP subunits are involved in the sub-complexes I, II, and III, respectively. We calculate the hydration entropy of each sub-complex using a hybrid of the integral equation theory for molecular liquids and the morphometric approach. It is found that the absolute value of the hydration entropy follows the order, sub-complex I > sub-complex II > sub-complex III. Moreover, the differences are quite large, which manifests highly asymmetrical packing of F1-ATPase. In our picture, this asymmetrical packing plays crucially important roles in the rotation of the γ subunit. We discuss how the rotation is induced by the water-entropy effect coupled with such chemical processes as ATP binding, ATP hydrolysis, and release of the products.
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  • Takuya Miyakawa, Masaru Tanokura
    Volume 7 (2011) Pages 123-128
    Released: November 23, 2011
    JOURNALS FREE ACCESS
    Abscisic acid (ABA) is a major phytohormone that mediates the adaptation of plants to environmental stresses such as drought and regulates developmental signals such as seed maturation. Studies on ABA signaling have progressed rapidly since the recent discovery of PYR/PYL receptor proteins as soluble ABA receptors. In plant cells, the receptor receives ABA to inhibit the phosphatase activity of type 2C protein phosphatase (PP2C), which is the major negative regulator in ABA signaling. SNF1-related protein kinase 2 (SnRK2) is then released from negative regulation by PP2C, turning on ABA signals by the phosphorylation of downstream factors. Insights into the regulation of PYR/PYL receptor proteins is therefore required in order to control drought-stress tolerance in plants. This article reviews the regulatory mechanism of the ABA receptor by ABA and its selective agonist. Structural analyses of PYR/PYL receptors have clearly elucidated the mechanism of ABA perception of the receptor or the mechanism of interaction with PP2C that leads to inhibition of its phosphatase activity. Moreover, the structures of PYR/PYL receptors complexed with pyrabactin, a selective ABA agonist, have provided the structural basis of ABA agonism and antagonism.
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  • Tomoya Tsukazaki, Osamu Nureki
    Volume 7 (2011) Pages 129-133
    Released: November 30, 2011
    JOURNALS FREE ACCESS
    Protein transport across membranes is a fundamental and essential cellular activity in all organisms. In bacteria, protein export across the cytoplasmic membrane, driven by dynamic interplays between the protein-conducting SecYEG channel (Sec translocon) and the SecA ATPase, is enhanced by the proton motive force (PMF) and a membrane-integrated Sec component, SecDF. However, the structure and function of SecDF have remained unclear. We solved the first crystal structure of SecDF, consisting of a pseudo-symmetrical 12-helix transmembrane domain and two protruding periplasmic domains. Based on the structural features, we proposed that SecDF functions as a membrane-integrated chaperone, which drives protein movement without using the major energetic currency, ATP, but with remarkable cycles of conformational changes, powered by the proton gradient across the membrane. By a series of biochemical and biophysical approaches, several functionally important residues in the transmembrane region have been identified and our model of the SecDF function has been verified.
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