TheBiophysical Society of Japan General IncorporatedAssociation approximately divided into 1 1 independent re]axational modes, about ha]f or five ofwhich wjth larger amplitudes contribute to 76% ofthe whole fluctuation. (3) Each of these major modes originates in correlated fluctuations of the hydration states of severa] specific PGs. Their relaxation times range widely from 1O ps to several tens of ns. lpTolo di!rem"veavmlcdi6tru-uyMmco*mmnf Hydration study of oligophosphates by dielectric

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... xation spectroscopy Kazuki Ishimori, George Mogami, Nobuyuki Merimoto. Makoto Suzuki (Grad. Sbh. Eng., Uhiv. Tbhoku} Adenosine triphosphate (ATP) is an energy mediator in biological systems. Recently, the importance ofthe hydration effects on ATP hydrolysis free energy is being reaiized widety. In this study, we performed high-reso]ution djelectric relaxation spectroscopy (DRS) for o]igophosphates aqueous so[utjon (orthophosphate, pyrophosphate and triphosphate; S20mM) as a rnodel of adenosine oligophosphates. The present DRS analysis can dividc dielcctric properties of the so]ution into muttip]e retaxation components, Experiments were performed by microwave network analyzer en O.2-26 GHz frequency range with the sample solution controlled at 20.0 ± O.O1 eC. Firstly. we found two types ofwater that have different relaxation frequency (5 GHz, 20 GHz) from bulk water (17 GHz) around solute ions. The 5-GHz component was attributed to the constrained water, and the 20-GHz component to the hyper-mobile water; HMW. Secondly, with increasing valence of sotute ions, the dispersion intensity ef HMW increased significantly. It suggested that the hydration state of oligophosphates was strongly influenced by the valence rather than the polymerized structure ofphosphate unit, ATP, ADP, Pi oj*rsnbi5E8orM7:-:,ItOS7-JbrsueAs ogfi : t ¢ ee"\rmeptn=xA ThermodynamicsandmechanismoftransferofATP,ADPand Pi from aqueous solutioll to fatty acid aminetoctanol solvent (Dept. Bioscience & Bioin,formatics, 1<yushu inst. Tlech.) shows rather different responses to ch]oride ions and ternperature. In particular, the heat ofoxygenation is found to be much lower in the rnammoth than in the elephant protein, which appears to be an adaptation to the harsh Siberlan winter. Many Arctic anima}s such as reindeer show a sirnilarly low temperature dependence ofoxygen affinity in order to ensurc oxygen transport to the limbs, which may experience much ]ower temperatuFes than the body core, even in ]arge, active and fur-covered mammals. In order to discover the structural basis for the a]tered heterotropie effect we have determined the crystal structures of the Hb in the deoxy, carbenmonoxy and aquo-met forms. These modets, the first protein structures from any extinct species, show many features reminiscent of human Hb, but underline how delicate control ofoxygen aennity relies on inuch more than overall quaternary structure changes. &'h. Eng., NLrgqva Univ., SAll)S Res, Center, NilD, 4Grad. Sch. Med., Nbgo.va Univ.) 1PTOI1 Hideyuki Komatsu ATP hydrolysis is an important exergonic reaction driving the thermodynamically unfavorable reactions in living systems, Altheugh hydration is consideTed to contribute significantly to energy profi]es of chernica] reactions in aqueous condition, the extent of its contribution to ATP hydrolysis remains to be experirnentatEy assessed. In order to evaiuate the effect of hydratien on the thermodynamics ofATP hydre]ysis, ATP hydrolysis in non aqueous condition (i,e" organic solvent) has been studied, For this purpose, fatty acid amjnecontaining octano] was used as a mode] so]vent, in which ATP, ADP and Pi are highly dissolved. ApproKimate hydration energies were estimated from the partition ceerncients as Gibbs energy changes (AG) for transfer of ATP, ADP and Pi from aqueous solution to the fatly acid amine-containing oetano], and compared to AG for hydrolysis in the sotvent. AG for the transfer was srnaller than that for hydrolysjs in the solvent, Thus, the hydration energy may modest]y modulate the Gibbs energy of ATP hydrolysis in aqueous solution. This energy profile is going to be refined by consideratien of the entha]py changes for the transfers, which can be estimated from van't Hoff plots ef temperature-dependencies of the panition cocfficicnts. rn addition, in order to elueidate how ATP, ADP and Pi are stabiHzed in the solvent. ionizations of ATP, ADP and Pi and reversed mice]]e foTmation wi]] be investigated by pH titration and dynamic light scattering. respectively. IPTIOI Darunavir (DRV) is the rnost potent protease (PR) inhibitoT (Pl) current]y avai]ab]e for anti-HIV therapy, Jt has a higher genetic banier to resistance deve]oprnent than the other PIs. HoweveT, it has been reported that DRV is vu]nerab]e to the resistance acquisition, especiatty for the patients who have previously failed the PI-containing treatments. Here, in ordeT to understand the basis of DRV-resistunce, we ana]yzed structures ofthe DRV-resistant PR. We first induced DRV-resistant virus in the culture system with the DRV concentration increased over IS4 days. On day IS4, we ise]ated the DRVresistant virus with 55.5 times higher IC50 against DRV than the wild-type (WT) c]ene. The analysis ofthe viral sequences revealed that the escape mutant with five mutations in PR was dominantly induced during the virat replications, Next, we determined a high-reso]ution (1.8 A) crystal stnicture of the DRVresistant PR. It turns out that the DRV-resistant PR has an open configuration at its fiap region, which has not been observed in WT PR. Furthermore, the motional properties and DRV-binding modes of the PR were analyzed by mo]ecular dynamics (MD) sjmu[ations. MD sjmu]ations showed that the flap region of DRV-Tesistant PR is more fiexib]e than WT, and that the DRV-inteTact{on medes aTe dist{nct. The resutts suggest that struetural change at the flap region is involved in the resistance development ofHIV-1 PR. Our findings will provide usefu1 information to develop effective PIs even to the DRVresistant HIV-1. ATP is known as the vilal energy source involved in energy metabolism, biosynthetic reactions and active transport. It is also an essential extTace]lular signaling molecule that activates two distinct families of ATP reeeptors/ ionotropic P2X and G-protein-coupled P2Y receptors. P2X receptors are trimeric ATP-activated jon channels perrneable to Na' . K' and Ca]', expressed throughout the human body ineluding the nervous, cardiovascular and lmmune systems and are implicated in various physio]ogica] processes such as synaptic transmission, smooth musc]e contraction, taste and pain sensing and inflammation. A rccent crystal structure of zebrafish P2X4 receptor in an apo, clesed state, revealed the chalice-shaped trimeric architecture of P2X receptors. However, due to the lack ofa crystal structure in complex with ATP, the ATP binding site and the mechanism of channe] activation rcmained une]ear. Here wc report the crysta] structures ofzebrafish P2X4 receptor in the presence and absence of ATP at 2.8 and 2.9 A reso]ution, respectively. The agonistbound structure reveals a novet ATP binding motif and un open channcl porc conformation. A structural comparison oftwo states suggests that ATP binding induces clefi c]osure in an intersubunit agonist binding site. which expands the region of the receptor proximal to the transmembrane domain, causing an irislike opening of the ehannel pore. This work provides the first structural insights into how ATP binding activates P2X receptors based on atomic resolution structures, Hiroki Noguchii parki, Jeremy U}iiversity, J4pan canado, 3Department Usu)We have recreated the hemoglobin (Hb) of the extinct wool[y mamrnoth using artifieiaL genes expressed in Eycherichia coti. The globin sequences were previously determined using DNA rccovered from frozen cadavers. Although highly simi]ar to the Hb of existing elephants, the wool]y rnammoth protein vtivFtsattmaNu;Ftssttmaoptr ± ti?;,Ex.Niv"E'yo snmmThe crystal structures of hemogLobin from the wool]y mammoth in ]iganded and unliganded states