- Email: [email protected]
P-A4-02 Folding energetics of a heterodimeric leucine zipper
A4 Folding and stability P-A44 1 EFFECT OF ALIPHATIC ALCOHOLS ON POLY (LLYSINE) ARUNKUlbiAR AI, KUMAR TKS, YU C.
P-A442 FOLUING ENElQ2ETKS OF A HETERODiMlHbjfC LEUCINE ZIPPER JELESAROV I, BOSSHARD HR Dept. Biochem., Univ. of Zurich (CH)
Dept. of Chem., National Tsing Hua Univ, Hsinchu, Taiwan. R.O.C.
Purpose: We report on the energeticsof the coupled folding and associationof engineered heterodimeric coiled coils (leucine zippers) consisting of one acidic and one basic polypeptide chain.
Purpose: TFX (2.2,2-T~ifluoroethanol) has been shown to specificallyinduce helix in those fragments of proteins/peptides which have a propensity to form helix. Using homopolymers such as poly (L-lysine) we study the helix inducing ability of alcohols above the pKa of lysine(11.5). Methods: The study was carried out using the technique
of
circular
dichroism.
Methods: The thermodynamic parametersof folding of severalLeu-to-Ala mutants were analysedby ITC in the temperature region of maximum stability and by van? Hoff analysisof temperature-inducedunfolding monitored by CD spectroscopy.
All
measurements were carried out in buffered solutionsmatinkined to a pH of 11.5. Results: We find that all the alcoholsincreases the helical content in poly (L-lysine) at pH 11.5. The increasein helical content is about twice as that found in poly (L-lysine) at pH 11.5 in the aqueousbuffer solution. Conclusion: From the results of our study we conclude that there is a deftite but indirect relationship between helical induction in homopolymers and the dielectric constant of the solvent used.
Results: AHfold,A!$,,ldand AC,,normalized per mol of residue were typical for cooperatively folding units. However, discrepancieswere noticed between van? Hoff and calorimetric enthalpies. Conclusions: 1, The energetic factors driving folding and associationof two polypeptide chains are balancedvery similarly to the folding of a single polypeptide chain. 2. Over an extended temperature range, the assumtionof a common denatured state implicit in the van? Hoff analysismay not always be valid.
P-A443 MET HKh$fW.@U’I OF FUX%%SC$+Ex PAULrnSt IEvmENcE Fat s-f&z&SW AND CONTROL INTmHmiEIrocKET.
P-A444 SPEcTORSCOPlC MONITQRING OF GELAm AND MODEL FOLWEPTIDES. KELLER RCA,’ DE WOLF FA.’
OF WBUNl’T INTB%AWtONS
‘ATO-DLO,
AGUSTINHO SCM., TINT0 M&L. PERUSSI J.R, TOMJNAGA T.T., IMASATO H, TABAK M. hstituto de Qulmka de k$&oCarIos-LISP, C.P. 780, 13560-970, SPOCarIos, SP (B&I)
Wageningen
(NL)
Purpose: In order to rationalize the wosking mechanismof gelatins in gelation, the important conformations need to be recognized and quantified. Sincequantitatively reliable reference CD-spectra are not available, we constructed a library of relevant spectra.
Cbromatograpby of met Hb of G. in SephadexGZOO at pH 7.9 gives the intact oIigouxz At pH 9.0 compkte dissociition into rrimers and monois rccomplished. Gptiil absorption rpeotrs of the intact oIigomer (&a&m I), trbner @aotioa II) and monomer @action III) were monitored as a Smetion of pH using the qex constraint aaalysis (CCA) programm developed by Fasomn et al. For the whole protein two pKs of 7.7 and 9.5 were observed corresponding to an inever$ibIa transition from aqua met acid Emmto his-imkkole hemiciuomeI and a rwcrsibletraasition&emhemiduomeI to hepkbrome II which is a +ies with we&ned and/or dlsaupted bond of iron to i&&44 N ofgnx&ual bistidine. The pKs are d&-eat Ear tbe ‘%&ions I, II and III denoting di@brazes in the beme ~ooket induced by subunit intera&aa BiimidaaoIe hemiobrome 1 was essily Wan&nod &to the oyanomot tkm for which the woakoniag of the bond at the proxhnal side was quite difkdt. IO waclwion the fbmation of hemicbromesat alkaline. pH oouId be a.znodu&m to oontro1 subonit interaotiens in this oIigomer tluougb obanga in the home pocket.
Methods: Circular dichroism is used to monitor the structural elements present and fluorescence to quantify the polymeric state of the polypeptides. Results: In gelatin essentially three types of secondary struchtres can be distinguished: random coil and the extended polyProlinel1helix (as a single or triple helix). Denatured gelatin can be used as generalreference for random coil. The prolyl chr6mophore was shown to have an effect on both the shaoe and w&ions of the CD-spectra of the poiyProlinei1 helix (or 3, helix). Conclusions: Referencespectrafor both random coil and 3,-helices were found. Discerning multiple and single helices is possible using temu. dewndent CD. the presenceof either intraor ikerniolecular interactions is addressedusing
Support: CNPq, CAPES and FINEP.
fluorescence spectroscopy.
51
P-A442 FOLUING ENElQ2ETKS OF A HETERODiMlHbjfC LEUCINE ZIPPER JELESAROV I, BOSSHARD HR Dept. Biochem., Univ. of Zurich (CH)
Dept. of Chem., National Tsing Hua Univ, Hsinchu, Taiwan. R.O.C.
Purpose: We report on the energeticsof the coupled folding and associationof engineered heterodimeric coiled coils (leucine zippers) consisting of one acidic and one basic polypeptide chain.
Purpose: TFX (2.2,2-T~ifluoroethanol) has been shown to specificallyinduce helix in those fragments of proteins/peptides which have a propensity to form helix. Using homopolymers such as poly (L-lysine) we study the helix inducing ability of alcohols above the pKa of lysine(11.5). Methods: The study was carried out using the technique
of
circular
dichroism.
Methods: The thermodynamic parametersof folding of severalLeu-to-Ala mutants were analysedby ITC in the temperature region of maximum stability and by van? Hoff analysisof temperature-inducedunfolding monitored by CD spectroscopy.
All
measurements were carried out in buffered solutionsmatinkined to a pH of 11.5. Results: We find that all the alcoholsincreases the helical content in poly (L-lysine) at pH 11.5. The increasein helical content is about twice as that found in poly (L-lysine) at pH 11.5 in the aqueousbuffer solution. Conclusion: From the results of our study we conclude that there is a deftite but indirect relationship between helical induction in homopolymers and the dielectric constant of the solvent used.
Results: AHfold,A!$,,ldand AC,,normalized per mol of residue were typical for cooperatively folding units. However, discrepancieswere noticed between van? Hoff and calorimetric enthalpies. Conclusions: 1, The energetic factors driving folding and associationof two polypeptide chains are balancedvery similarly to the folding of a single polypeptide chain. 2. Over an extended temperature range, the assumtionof a common denatured state implicit in the van? Hoff analysismay not always be valid.
P-A443 MET HKh$fW.@U’I OF FUX%%SC$+Ex PAULrnSt IEvmENcE Fat s-f&z&SW AND CONTROL INTmHmiEIrocKET.
P-A444 SPEcTORSCOPlC MONITQRING OF GELAm AND MODEL FOLWEPTIDES. KELLER RCA,’ DE WOLF FA.’
OF WBUNl’T INTB%AWtONS
‘ATO-DLO,
AGUSTINHO SCM., TINT0 M&L. PERUSSI J.R, TOMJNAGA T.T., IMASATO H, TABAK M. hstituto de Qulmka de k$&oCarIos-LISP, C.P. 780, 13560-970, SPOCarIos, SP (B&I)
Wageningen
(NL)
Purpose: In order to rationalize the wosking mechanismof gelatins in gelation, the important conformations need to be recognized and quantified. Sincequantitatively reliable reference CD-spectra are not available, we constructed a library of relevant spectra.
Cbromatograpby of met Hb of G. in SephadexGZOO at pH 7.9 gives the intact oIigouxz At pH 9.0 compkte dissociition into rrimers and monois rccomplished. Gptiil absorption rpeotrs of the intact oIigomer (&a&m I), trbner @aotioa II) and monomer @action III) were monitored as a Smetion of pH using the qex constraint aaalysis (CCA) programm developed by Fasomn et al. For the whole protein two pKs of 7.7 and 9.5 were observed corresponding to an inever$ibIa transition from aqua met acid Emmto his-imkkole hemiciuomeI and a rwcrsibletraasition&emhemiduomeI to hepkbrome II which is a +ies with we&ned and/or dlsaupted bond of iron to i&&44 N ofgnx&ual bistidine. The pKs are d&-eat Ear tbe ‘%&ions I, II and III denoting di@brazes in the beme ~ooket induced by subunit intera&aa BiimidaaoIe hemiobrome 1 was essily Wan&nod &to the oyanomot tkm for which the woakoniag of the bond at the proxhnal side was quite difkdt. IO waclwion the fbmation of hemicbromesat alkaline. pH oouId be a.znodu&m to oontro1 subonit interaotiens in this oIigomer tluougb obanga in the home pocket.
Methods: Circular dichroism is used to monitor the structural elements present and fluorescence to quantify the polymeric state of the polypeptides. Results: In gelatin essentially three types of secondary struchtres can be distinguished: random coil and the extended polyProlinel1helix (as a single or triple helix). Denatured gelatin can be used as generalreference for random coil. The prolyl chr6mophore was shown to have an effect on both the shaoe and w&ions of the CD-spectra of the poiyProlinei1 helix (or 3, helix). Conclusions: Referencespectrafor both random coil and 3,-helices were found. Discerning multiple and single helices is possible using temu. dewndent CD. the presenceof either intraor ikerniolecular interactions is addressedusing
Support: CNPq, CAPES and FINEP.
fluorescence spectroscopy.
51