TheBiophysical Society of Japan General IncorporatedAssociation 1)Qin et tl (2001)r[BSlett )07 299 302 2) Matsumura ct aT <2008)BToph}s ChLm T34 g4 92 3) Li ct al (2007) Biochemigtiy 46 5072 SOg2 ln order to study such nonrandorn structures m a residue tspecific mdimer "e studied proteLtion factots against the Hil) exchtinge reaction for an mdividual NH by medns of NMR measurements m DMSOID.O niixtu]e as desLnbed in our papeireferredabeve 1 P 038 Structural change of P13K SH3 domain at acid-c

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... Yobhitakd Mdt-umura (1} MasNl Shenpo (1) Nebu} ukl OkEshio (2) Hiroghi Kihara (1H(1) Kanscu MLdi"U Un-tnto C2) Hokkaitlo RLcl Cross Btf od LLntrr) We have been LniestigitLng fDlding pioLesses of Fyii and grc SH3 domaln pietLins ("]th its mutant A45G) ThL} formLd an compact a helix nch intermediate on their toldmg pathways We have started studymg another SH3 domarn protem PBK SHa domain The recombinant of thits piotem -as expresbed in E eoh PI3K wds purihed is betoie [1] Masg gpcLtiomctry shows a peak at 9764 while rnoleculai weightot PI3K SH3 is le031 (tl) Seh 5(t&Jech Kvianset Gakt"/n (Jmv (2) Sch Btotosl Oment{d Sct dileck Ktnkt Vnu) We presume that protein foldmg pioceeds vvith Lhangm,., thui conforrnatiens ]n a non native state md then final]y mAke a Loopcrative transition towelrd the natiye gLruLturc IL ig crucia] ioi the understanding of folding pioLess to expleie non natrve structures located near the nat-e one Fo] that cnd vve have studied partially folded struLturLs in d]sulfidc deliciLnt iariants ot lysozyme So fcu we have teund that two disulfide variants 1ie on the boider bcLwcLn the nauve and ne" native stiuLturLs I'or examp]L 2SS[6 127 64 8e] which contatns two d]sulfide bi idges ol Cyg6 Cysl27 and Cyg6iL Lvs80 has a non nauve struLture in water but progressiyely reLoveis d nativL hkL tLrt]aty structuie v-tth the increase in thc concentratton of glycerol [Biopolymers 91(2009) 665 675] At present we study the nonrandom structures remaining in 1gg sd"ants ot 1}soLlrme Fai UV and nedr UV CD speLtra ot tour gpeucg ot ISS variants and OSS vin mt wuL mLftsurLd in thL absLnce and presence of glyLero] Only thL vanant of ]gS[6 127] ghowed a sigmficant diffeienLe m CD spcLtia rclative to other 1SS variants In the absencL ot glycerol [e ]po was bignifiLantly la]gc in the magmtude only m the far UV speLtrum of ISS[6 127] The near LV CD specuum of only 1SS[6 127] was siniilar Lo Lhal of 2SSL6 l27 64 80] in thL prLsLnLL of LonLcnllnled glyLLrol Thls means that the presenLe od thL dlsulClde b"dge Cys6 Cys127 is important fo] inducmg noniandem structures m lyso7yme IP042 Mstfi1]NLkJL.IS-AOKf:deft6fmsczaOlft:11 ChanLtenLation ol a propeptidL iLgmn in preuroguan)hn using de novo ctesigneci Disulfide H)brid Protem Masaki Okumura tl} HironoJ/Ko]ishi (2) Yuhei Ydmazdki {1} H:ioshi Yamaguchi (D Yup Hiddka (O) (fJ) Giaduatt vhoot of s"encL and t"hnoiosy KTtanseT Gak"rn Untv f2) Gtaductt[ schoot ttisuenLL and Lngine[ rtng Ntt"kt tsnty ) Urogumihn ig pioduced via a proteolytic pioecgsing ot a precursor protcin prouioguanylm The propeptide region in prouroguanylin functions as an mtra molecular LhapcionL leiding to the proper folcling ef the mature peptide uroguanylin [121 ln order to mvestigate the recogmtion mechdmbm of the propeptide regien twe peptides hybiid peptide l CrvDDCLhLALNVALTCJAL) and h}brid peptide 2 (NDCAELCCNVAATGCL) were de nevo desi.med bised on the companson of the pninary htructuieb between uroguanylm and heat stable enterotoxm (Slh) vLhiLh Lenta]ns three disulfide bo"ds and thL tv-o disulfide bonds aie locaLLd at relatively game posrtions as those of uroguanyhn The h)bnd pretetns En -hTLh the hybud peptides wLrL fused to thL pro rLgion ef urogua"ylin wcrL ilso picpaied ugmg an E coLi expression systern Folding reactions ot the hybrld peptldes and pToteins vLere menLtored by HPLC ind CD measuieme"ts ThL iLsultb indiLatL that Lhe pio peptide region tunctiong as the mua molecular chaperone for hybrtd peptide l but did not for hybrid peptide 2 Importantly hybrTd peptide 1 shovvcd a bindmg activily to its receptor protLm guanylyl cyLlasLC In order to fuither study the propLptidL mLdidtLd foldmg unteldmg proLLsscs of the h}biid proteing and prouToguanylm weie estrmated r31 Reductive unfoldrng reactiong of those proteins were carried out in O l M TrislHCI (pH 8 O) buffer containmg 40 mM DTT We will disLuss hew thL ex]daUvc ielding and the reductive unfoldmg of the hybTid proteLns tnd pTouiogud"y]in tre regulated by thepropeptidLiLgion ll]Y Hidaka etal Biochemistr} 37 S498(1998) [2]Y Hidaka aal T Bio] Chcm 275 2S155<2000)