TheBiophysicalSociety of Japan General IncorporatedAssociation agonist and non-agonistic mAbs have distinct bjnding sites on the CD3Ey heteredimer.AgonisticmAbsbinddiagonallytothemembranedista1CD3alobe adjacent to the lever-like CP FG loop that facilitates antigcn (pMHC)-triggered activation, ln contrast a non-agonist mAb targets the clefi between CI)3s and CD3v, in a perpendicu]ar mode. Subsequent biological and biophysiea] experiments confirmed that the difference {n eeT1 triggering is not

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... ked to mAb affinity or CD3E binding stoichiometry per TCR but to the differenee in thc binding cpitepe on CD3sv. Morc importantly, non-agonistic mAbs is stimulatory only subsequent to the application ofan external tangential force C20 pN) generated by an optical t",izzer technique. These tlndings indicate that a dynarnic bLLt coordinated receptor quaternary structurc changc in T cell receptor is important for T cel1 activation. Corninon quatermary stnicture change can better faciLitate structurat signal initiation, given the vast array ef TCRs and their specific pMHC ligands. nE"NMRhi6fi6eefiopfi=*JVi-neecoNes High pressure NMR discloses a rich world of high-energy structures of proteins Ryo Kjtahara (Collage cifPharmacettticat Sciences, Ritsumeikan Univensity) A protein in soiution is a thermodynamic entity exist{ng in dynamic equilibriurn of multiple conformers. The collformational fluctuatioll could be coupled with protein functions and misfolding events lcading to amyolid diseases. NMR is a technique that can, in princip]e, detect signa[s from all fluctuating conformers in solution. However, such high-Gibbs energy conformers are not directTy obse",able in conventional NMR techniques, either because of the conformatiena] fluctuation is rapid (less than ms) or the population ef highenergy conformers is toe ]ow under physio]ogica] condition. In order to vi sualize the actual existence of high-energy conformers of proteins, we use the highprcssurc NMR technique. Sinee the volume is strongly coupled to conformation, pressure shifts the confonnation within the range ofits fluctuation, Surprisingly, in many proteins, various high-energy conformers with fairty distinct structures have beell detected in adclition to a fu11y unfolded one; these include i,arious altemately folded, locally unfolded and molten globule conformers, disclosing a rich world eC protcin structurc beyond the basic folded one. Interestingly, the pressurc-stabMzcd eonfonners have sknilar eharaeteristics with those accessi]ig by the theirnal fiuctuation ut physiolegical cendition. No-" we challenge a rational design for high-energy conformers ofproteins for furthcr undcrstandjng of their struct-'e, dynamics and functional importance. lsD-o4 luaJv-vAvog#esunuKefimEtitt:-oxopgm Protein structural dynamics modulaiton induced by a mutation to flexible loop Yoji Wada. Eiji Ohmae. Kunihiko Gekko, Shin-ichi Tate (th'roshima Unive,:sdy, MLS) Protein structure is maintained in a subtle energy balance, therefore its spatial stmcture is essentially fiuctuating at the physiotogical temperature. Protein stmcture dynamics contain a various motiolls in wide time regimes. from psee to rnsec. In part{cular, the motions in the range of -secmsec contain rather 1argeatnplitude motion that correlates the residues in tong distances to each other. The mutation to a residue in a cerrelatcd motion shou]d be expected to cause motional changes to the other residues in correlated to the mutated Tesidue, This intuitive view prompted us to explore the protein dyiunllics change caused by mutation to a residue in fiexibte loop. Wc ana]yzed dihydrofolate Teductase (DHFR) on its structural dynamics modulation cased by the site-directed rnutation te G67 in a flcxible loop. The T2 relaxution dispersion experiments at three different tempeTatures using two magnetic ficlds were applied. The results showed that the Ioop mutation actually caused the dynamical modulation to the active loop motion that has the prirnal rote in the enzyme reaction, although the loop is far distant from the mutation site. In the presentation, the caused changes in the motion with its associating thermodynamic pararneters by the ]oop mutation and their functional significances will bc discussed. In eukaryotic ce]Is, each component of the protein complex assembles via weak interactions. These interactions are integrated, and it consequentty exerts highty regulated biological functions. I would like to present a NMR study ofa regulated protein-protein complex and its dynamical aspect ill a transcriptional passway. The transcriptional co-repressor SHARP contains a transcriptionaT repressor binding domain, SPOC which is known to bind to the C-tenninal region of corcpressor SMRT, SHA]IPISMRT complex recruits histone deacethylase to chromatin and regu]ates transcription negatively. We have detellnined the threedimensional structure of' SHARPISMRT eomplex to establish structura1 basis for the molecular recognition by using solutioll NMR. Wc found that phospherylated SMRT tightly bound to SPOC domain of Sf・[ARP, but the affinity was decreased about lOOO fold when it was not phosphorylated. Interestingly, a comparisoll of HSQC spectra showed that unphosphorylated SMRT also bound in almost identical manner to that of phosphorylated SMRT. We wM discuss detailed molccular recognition based on the determined stmcture, its dynannical aspect derived from LiN relaxation experiments. and their biological significance, ISD-06 U7JbS(AE=SUtrti[[S7TMtrnkAPOBEC3Gt[di6 DNA ± oppttregutex7・rf(:,crthvfJbuteyFy:/m 7:-7gmm Coupling between cytidine-deamination by APOBEC3G and its s]iding along DNA with polarity as revealed with real-time monitoring by NMR MasatoKatahirai,AyakoFurukawai,TakashiNagata2,HiroshiTakaku3,Ryo Morishita4, Akifumi TakaoriS, Akihidc Ryo6 Cifnst. Advtmeed Enetgy. K),oto Univ., ?Grad. Sch. Nanobio., lokohama Ciij, C,7iiv.. 3eniba inst. 7lech., 4CeilF}'ee Sbi., SGrad. S('h. Mbd., kyoto [J}tiv., 6arad. Sbh. Mbd., }lokohama City U)iiv.) Human APOBEC3G (A3G) exhibits anti-HIV-l activity by deaminating cytidines ofthe minus strand DNA of HIV-1 . We already demonstrated that the deaminatien reaction by A3G is suceessfu11y monitored usjng NMR signa]s in real-time. This method is supetior to conventional biochemica] methods to examine the enzymatie reaction in terms of its higher spatial and temporal resoZution. This time. deamination by A3G of DNA that contains multiple deamination sites has been cxamined by this method. We have found that the site Tocated eloser to the S' end of DNA is deaminated faster than that located ]ess close to the S' end. This obsen'ation can be rationalized by assuming that A3G slides along DNA with 3' to 5' polarity. Alternative explanation is deamination occurs only if A3G passes the deamination site with 3' to 5' polarity. '['hese tu'o possibilities will be discussed. Further improvement of spatial and temporal resolutien of our monitoring method with thc aid of iSC labeling wi]] also be presented. Development ofthe intelligent enzyme that switches its activity in response to the envirenmental K' coneentration, that was motivated by NMR study of the unique quadrup]ex structure of RNA, will also be presented. ISD-07 YoshihiroUnivei'sit.y) u),catt[[sDffrctrn6s;ilscofionevafftte ± ma\tsfits Phosphorylation induced conformation change and its biologicalimplication Kebashigawa, Hireyuki Kumeta, Fuyuhiko Inagaki (Rbkkaidb Phosphorylation is a major post-translational modification that regulates intracellular signal transduction. Phosphorylation induces conforrnation change that ptays a key roie in signaling. Atthough many X-ray works have been performed, the regulation meehanism by phosphorylation has remained elusive on the structura] basis, One reason is that signaling proteins aTe multi domain proteins and crysta]lization of signaling proteins is often d{tficu]t {n the phosphorylated state due to fiexibility. NMR is promising for structural study of signaling proteins in the phosphorylated state and gives an illsight into the biological impTjcations of phosphorylation. Structural studics require an ameunt of homogeneous phosphorylated pToteins, For this purpose, we devised a novel phosphorylation method that utilizes domain-domain interaction to enhance substrate local concentratioll. This enhances phosphorylation efficiencies by 1O-100 folds and thus, -,e preparecl an amount of homogeneous phosphoryIated samples. Using this preparation, we reveaTed tlie structure of sigrlaling molecules in the unphosphorylated and phosphory]ated states by NMR, We ",i11 discuss thc bio1ogical iTnplication of the phosphorylation on the structural basis. ISF-Ol Hitoshi Tatsumi "mumaEGIifrcE"nUmavapt,mavatfiifI[ )LIT : "kticoza"+ op:aasdivgeee Mechanical force-induced intrace]]ular signa]ing: assembly and disassembly of adhesion-related proteins in in vitro conditions (Dept Ph.vsioi, Nlrigaya Uhiv Seh Mbd) -S4-NII-Electronic