TheBiophysicalSociety of Japan General IncorporatedAssociation HypF are involved in the biosynthesis of the nitrile group as a precursor ofthe cyario groups. HypF catalyzes S-carbamoylation ofthe C-terminal cysteine of HypE via three steps using caTbamoy]phosphate and ATP, producing two unstable intermediates/ carbamate and carbamoyladenylate, It remains uncleaT how the censecutive reactions occur without the ]oss ofunstable intermediates during the preposed reaction scheme. We have recent]y

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... ermined the crystat structures of fu11-length HypF both a]one and in comptex with HypE at reso]utiens of 2.0 ancl 2.6 A, respectively. Three cata]ytic sites of the structures ofthe HypF "ucleotide-and phosphate-bound forms have been identified, with each site connected via channels inside the protein, This finding suggests that the first twe consecutlve reactions occur without the release of caTbamate or carbamoyladenylate from the enzyme. The structure of HypF in complex with HypE revealed that HypF can associate with HypE without disturbing its homodimeric interaction and that the binding manner al]ows the C-termina] Cys351 ofHypE to aecess the S-carbamoylation active site in HypF, suggesting that the third step can also proceed without the release of carbamoyladenylate. 3B0924 Dynamic structurat and antigen binding analyses of antibody single-chain Fvs Yllsllke Tanakai, Hiroshi Sekiguchi2, YLlji C. Sasaki3, Takachika Azuma4, Masayuki Oda] (]Grad. Sch. ofLijle and Environ. Sci., 1<voto Pref Uhiv., 2.ipn. byn. Rad. Res. Jnst, 3Grad. Sch, ofFron. and Sci., Univ. of 7bdy'o., 4Res, Ins. for Biol. Sci., 7blyo Uhiv. oj'Sci.) We generated single-chain Fvs (scFvs) of anti-(4-hydroxy-3-nitrophenyl)acetyl (NP) antibodies, N1G9 and C6, which are germline and aMnity maturated IgG 1 antibodies, to analyze the change of structural fiuctuation upon the antigen binding and through the affinity maturation. We overexpTessed the scFvs in E. eoli, and refolded by stepwise-diatysis method, fo11owed by purification using NP-coniugated co]urnn. The scFv-antigen interactions were analyzed using surface plasmon resonanee biosensor, Biacore, in which NP corijugated proteins were immobilized on sensor chip. The equilibrium association constant of C6 scFv for NP was about 1O times higher than that ofNIG9 scFv, mainly due to the decreased dissociation rate constant, The kinetie paTameters were similar to those of corresponding Fab and intact antibody, indicating that the scFvs are refolded correctly. The structural fluctuations of scFvs were measured using difftacted X-ray tracking (DXT) system at single-rnolecule level. Upon the binding ofNP-e-amino caproicacid, the fiuctuations were decreased in either NIG9 or C6 scFv, indicating that the antigen binding stabilizes the scFv structure. Ill comparison of N1G9 and C6 scFvs in the absence of antigen, the fluctuation ofC6 was lower than that ofN1G9, which could be correlated with the different antigen binding mechanism such as "induced-fit" and "lock-andkey" types. On the advantage of scFv, whose amino acid couid be mutated. we try to obtain more quantitative and site-specific information. 3B0948 Shuya Ishiii ofPh.vs., FZieulty ofSei. Eng., }PZisedke a-catenin is known as a linker protein between cell adhesion and the actin cytoskeleton, It has been be]ieved tbat menemer ef a-catenin forms a complex with S-catenin and E-cadherin in cell-eell aclherens junctions and plays ari essential role as an anchor, directly or indirectly, of E-cadherin-mediated adherens junctions and cytoskeleta[ F-actin netwoTks. Dimer ofa-catenin, which is formed by the dissociation from the complex, binds to F-actin with -M aMnity and bund]es it. Iocally inhibitlng the Arp2/3 complex. This reorgariization ofactin dynamics decreases membrane activity at sites ofeell-cell contact and stabilizes cadherin-mediated cell-cell adliesion. In addition, inhibition studies of myosin ATPase activity suggested that the intracellular actomyosin contractile force a]so contributes to the assembly and extension ofadherens junctions, In present study, we report that a-catenin inhibits actomyosin contraction in the dose-dependent rnanner, which was characterized by an in vitro F-actin gliding assay with myosin II HMM. Furthermore, ne direct significant association between a-catenin and myosin II was observed in immunoprecipitatien assay, indicating that a-catenin may compete with myosin II for binding to actin, resulting in a decrease in the number ofmyosin heads able to propel actin fi1aments, or may indirectly, through an actin fi]ament, inhlbit myosin activity. Herein, we will discuss how cr-catenin inhibits actomyesin contraction and the implications of that in maintaining adhercnsjunctions. ah7=ylc s67o Fz-tv7mmom-Inhibition of actomyosin contractility by a-catenin, a component of adherens junctions , Takashi Ohkii, Hiroaki Kubotat, Shin'ichi Ishiwatai'2'3 (iDept. and Eng., Pi2iseda Uhiv., 2Adv. Res. inst. ,for Scie, and Uhiv., 3PPL4BIOS PPZisedo Uhiv.) 3B0936 PYPop)tlllMfittoplretuen"nvpm Mechanical controt of light-induced protein conformational change of photoactiye yellow protein Yasushi Imamoto, Take Matsuyama, Yoshinori Shichida {Grad. Sch. Sci., Kyoto U}tiv.) Proteins are formed frorn polypeptides by folding into the specific cenformations, by which the physiological roles ofthe proteins are determined. The stmcture of the protein closely corre]ates with the physio]ogical activity, and the activity of the protein is regulatecl by conformational change. For example, the receptor proteins alter their conformations in response to the stimuli to activate the ce]]ular transduction system, Thus, if the protein conformation is artificially regulated, one would control the protein actlvity. Here we tried to establish the technique to regu]ate the protein activity by changing the conformatjon. Photoactive yellow protein (PYP), a bacterial blue-light sensor protein, was used as the mode] protein, Two amino acid residues present in the surface ef PYP rnolecule were replaced by cysteines and crosslinked by an azobenzene linker, whose length is reversib)y changed by translcis photoisomerization. We found that the velocity ofphotocycle was varied by 1O-1Oe times by the isomerization of azobenzene when Met]OO loop and pCA loop were crosslinked, Since the global protein conformational change involving MetlOO loop takes place in the photocycle, it is likely that the protein conformational changes were perturbed by the isomerization of azobenzene linker. This technique is pessibly applied to switch the enzymatic activity of the protein by light. 3Blolo Kurisut, Haruki Nakamura] (]lnstitute for Protein Research, Osako V}tiv., 'BMC ALS7) Dyneins are large microtubule motor proteins that play important roles in various biological movements. Cytoplasmie dynein is responsible for cell division, cell migration and othcr basic cellular functions. The motor domain of dynein consists ofa ring-shaped six ATPases called AAA' modu]es. Recently, an ADP-bound high-resolution stmctures of cytoplasmic dynein have revealed the organizatien of the motor domain that cornprises the AAA' ring, the linker, stalklstrut and C sequence {PDB IDs = 3vkh, 3vkg), However, the highreso]ution structure ofan ATP-bound dynein remains unclear, Here, we canied out melecular dynamics (MD) simulations ofboth ADP and ATP-bound forms to examine their stmctures and dynamics. We bui]t initial structures for MD as fo11owing. A higher resolution structure (3vkg), which is a tmaneation mutant, was chosen. Then, we modeled missing residues and added a truncated domain from the wild type structure (3vkh). Four ADP rnolecules were placed to their original pesitions in the ADP bound form. One of the ADP molecules. bound to the main ATPase site (AAAI), was replaced to ATP in the ATP bound form, A rectangular water box was placed around dynein, FinatEy, the systems consisted of approximateiy one million atoms. Electrostatic interactions were treated with zero-dipole surnmation method, and their computation was accelerated by using GPGPU. We will discuss the effeet of ATP on the structure and dyiiamics of dynein by comparing the trajecteries between the ADPand ATP-bound forms, 3BI022 Pai-ChiResearchinstitute. A computational Inyestigation into the MHC-I Recognition Mechanism of MIR2 from Kaposi's Sarcoma-Associated Herpesvirus Lii'2, Naoyuki Miyashita3, Satoshi Ishidoi, yaji Sugita2 (iRfi(EN Center for Allergy and Immunolqgy, !Rll(EN Advanced Sbience 3RIKEN Quantitative Biology Center) Kapesi's sarcoma-assocjated herpesvirus, a human tumor virus, encodes two membrane-bound E3 ubiquitin ligases, modulator ef immune recognition l (MIRI) and MIR2, to evade host imrnune system through the ubiquitinationmediated endocytosis and lysosomal degradation of the proteins involved in immune recognition. MIR2 dowuregulates the surface expression of MHC I (major histocompatibilty complex class I), co-stimulatory proteins {B7-2, ICAM-1), and platelet endothelial cell adhesion mo]ecu]e 1 (PECAM-1). Recent -S5S-NII-Electronic