TheBiophysicalSociety of Japan General IncorporatedAssociation experience deepens the user's understanding of the complicated 3D protein strueture and e]ucidates ligand binding and protein-protein interactions. This model would be an effeetive discussion tool for the classroom or laboratory that stimulates inspired learning in this study field, IPT137 eHfilefiMet[NIiVzacoeeue Effects ef sugars on the thermal stability of proteins Hiraku Oshimai, Ken-ichi Amano2, Masahiro Kineshitai (]Inst. Adv.

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... EneFgy, Kl,oto Univ., 2Dep. C7tem., Kbbe Uhiv.) It is experimentally known that the heat-denaturation temperature of a pTotein 71. is raised by the sugar addition. The magnitude ofthis effect stabilizing the protein depends on the sugar speeies [ 1 ]. In earlicr work, we proposed a measure ofthe thermal stability ofa protein, which is defined as the solvent-entropy gain at 298 K upon protein folding S normatized by the number ofresidues [2,3]. S was calculated using a hybrid of the angle-dependent integral equation theory combinedwiththemulipolarwaterrnodelandthemorphornetricapproach.Here we show that S can be calculated using the haTd-sphere so]vent whose number densiry and molecular diarneter are set at those ofwater. We then investigate the effects of sugar addition on the thermal stability by considering water-sugar solutien rnodeled as a binaiy mixture of hard spheres. The three-dimensiona] integral equation theory is employed. The therma] stabi]ity is determined by a eomplex interplay ofthe molecular size of the sugar D and the total packing fraction ofthe solution n, D is estimated from the voJurne per molecule in the sugar erystal, and n is catculated using the expeT{menta] data of the solution density. We find that the therma] stability is considerably enhanced by addition ofsucrose.Inthepresentation,wewilldiscusstheeffectsofadditionofnotonly sugars other than sucrose but also osmo]ytes. (iGrad. Sch. imt. Sci., MdLS7L 2Graduate School qfLifle Science, Ilbkkaido U}iivensity, ]Osaka Uhiversity qfPharmaceutical Seiences) Human calcitonin (hCT) is a 32-residue peptide honmonc. hCT is used as a medicine for osteoporosis, but it easi]y forms amyloid fibrits under physiological condjtion, which is a problem on medjcation. Salrnon calcitonin (sCT) is known to inhibit amyloid formation ofhCT. Combinationa] use ofhCT and sCT is expected to improve the pharmaco]ogical actMty. While the Nterrninalregion(9-19)ofsCTtakesana-helix,theC-terminathalfofthehelixis diserdered in hCT. Although an interaction between the disordered region and the C-terminal tail of hCT is proposed to play a role on the amyloid formation, the detailed mechanisin of the inhibition of the amyloid formation by sCT has not been revealed. In this study, we investigated amyloid fibrll formation ofhCT in the presence of each of two chimera peptides, which are cemprised of the 1-15residues ofhCT and the l6-32residues of sCT and, the reverse cernbination. The former region (1-1S) contains the stable a-he]ix and the latteT the flexib]e region in hCT. Amyloid fibri] formation was monitored by fluorescence intensity from Thioflavin T. While the fiuorescence intensity raised about 30--40 hours after incubation of hCT alone, the amyloid fibril formation was inhibited in the presence of sCT, or each ofthe two chimera peptides. indicating that each of the N-termina] and the C-terminal regions of sCT possesses the inhibitory abiHty. From these results, we conclude that both the C-termina] region and the N-terminal stable cr-helix are engaged {n the inhibition of the amyloid formation. 1PTI39 immense]y ]arge minimum at 207 nm on CD spectmm at pH 2.9 (named pH2.9 fibrils), but aggregate-tike fibrils with a minimum at 220 nm were formed at pH 7.5 (named pH7.S fibrils) near its isoelectric pojnt. In this study, flrstly single pHjump from 2.9 to 7.5 was performed. As a resu]t, the CD spectrurn showed that the pH2.9 fibrils instantly changed to pH7.5-like fibrils with a minimum at 218 nm, but the e]]ipticities were certainly distinct between pH7.5-like and pH7,5 fibrils, implying that the conformation ofpH2,9 fibri]s partially remains even at pH 7,5, Moreover, the pH7.5-like fibrils almost retunned to the pH2,9 fibrits by restoring the solution pH from 7.5 to 2.9. FT-IR spectra indicated that theseconfbrmationalchangeswerecausedbythedisruptionofhighlyorderedP -sheet and B-turn confomiations, and the subsequent their reconstructions. In addition, kinetic conformational changes of the fibriLs after the single and doub]e pHjumps were examined using ANS fluorescence stopped-flow. As a resutt, these conversions of the fibrils composed of a few phases were accomp]ished within several seconds. Thus, amyloid fibrils can be changed readi]y between the distinct conformations separated by a low energy barrier almost reversib]y. IPT140 Kazuya Iwama] Sch.Sbi,U}liv.Mbiji. efivaal[S6ltFVUt:,9;,JWffopA;7fFMhOMvagtt Conformation change in peptide fragments of human priofi proteill caused by metai binding , Takahito Irnakii, Masahiro Yagi2, Wakako Hiraoka2 (LGrad ?Sch.Sci,Univ.Meiji) y rJvpH yvyJlca67:-o・t' Fnvol:liajmacozammg{t oasth Analysis of Almost Reyersible Conformationa] Change of Amyloid Fibri]s by Double pH-jump Keiichi Yamagllchii, Yaji O, Kamatari2, Mayuko Fukuokai, Reiji Miyaji3, Kazuo Kuwatai (i Unit. Grad. Sch. of'Drug Dis. and Mbd. InjT Sci., Gifu U}iiv., 2Lijle thi. Res. Center, Gijit Uhiv., 3Sup. andDev. Center.fbr Jlech, Ed., Flrc. ofEng., Gifu U}iiv.) To obtain insights into the mechanism of arnyloid fibril conversion, pHjump experiments were perfbrmed using a H2 peptide of mouse prion protein. Previous;y, we reported that H2 peptide formed ordered amyloid fibrils wjth an Prion diseases are transmissib]e spongiform encephalopathies (TSE) and fiethal infectious diseases caused by misfolding of prion protein (PrP). Hurnan prion protein (huPrP) binds metal jon at the residues 60-91 {His61,His69,His77, His85) and residues 90-126 (His96,Hist 1 1) in N-terminal. Histidine residue are known as high-aennity sites to metal binding. Metal binding to PrP is considered as an important proeess for misfolding of PrP. This report aims to investigate conformation change in huPrP caused by meta] binding. huPrP Peptide fragments conteining His96 andtor Hisl11 were exarnined for binding metal such as Cu!', Ni!' and Coi'. pKa values were determined with eleetro titrimetric analysis (Metrohm, 848 titrino plus). Meta] binding to peptide fragmcnt was observed using visib]e absorption spectra (Hitachi. U-2800 Spectrophotometcr) and Circular Dichroism (CD)(JASCO,J-g20). Secondary structure ofpeptide fragment was observed using Far-UV CD. Addition ofCu2' and Ni?' to huPrP (93-102/GGTHSQWN) showed the shift ofpKa value. Vis absorption and Vis CD indicated the characteristic spectTa resu]ting from meta1 binding, It was dcrived from imidazole N bound to rneta] ion and from d-d transitionofmetalion.Far-UVCDspectrumshowedtheconformationalchange of secendary structure induced by metal binding. We wi]1 a]so investigate metal binding to peptide fragments of huPrP containing Hisl11 by using UVfVis spectroscopy, CD and electfon spin resonance (ESR) to further understand about metal binding te huPrP, tPT141 Ziflf;iopin.S-ffh-eM=*JV\-wwpa Temperature-pressure-free energy landscape of ubiquitin Tsubasa YamamotoT, Minoru Kato2 {tGraduate school of Liji] Science, Ritsumeikan bhiversity, ?Department of'Pharmae.v, Ritsumeikan U}iiversity) To understand the change of packing andlor solvation of protein denaturation ftom quantitative point, volume change AV and isothermal compressibility change A6T, and therrnal expansion coefficient change Aa are needed. These themiodynamic parameters at givcn temperature and pressure are determined ftorn ternperature(T)-pressureCp)-free energy change(AG) landscape,which is obtained fraom cornbined series of pressurelternperature variable denaturation experiments. In this study, we explore the (TL p, AG) landscape of ubiquitin. FTIR spectra of ubiquitinwasTeversiblychangedbytemperaturelpressureperturbationatpD2.0 and pD S.5. To deteFmined AG at given temperature and pressure, temperaturelpressure transition curves were obtained from change in FTIR peak intensity at 1673 em'i which is assigned to native B-strand structure. Threedimensional (Z p, AG) tandscapes and thermodynamic parameters (A V, A5T, A a, entropy change AS and heat capacity change ACp,) at ambient temperature and pressurc (298K, O.1MPa) were obtained.