To characterize the thermal stability of 3-isopropylmalate dehydrogenase (IPMDH) from an extreme thermophile, Thermus thermophilus, urea-induced unfolding of the enzyme and of its mesophilic counterpart from Escherichia coli was investigated at various temperatures. The unfolding curves were analyzed with a three-state model for E.coli IPMDH and with a two-state model for T.thermophilus IPMDH, to obtain the free energy change DeltaG degrees of each unfolding process. Other thermodynamic parameters, enthalpy change DeltaH, entropy change DeltaS and heat capacity change DeltaC(p), were derived from the temperature dependence of DeltaG degrees. The main feature of the thermophilic enzyme was its lower dependence of DeltaG degrees on temperature resulting from a low DeltaC(p). The thermophilic IPMDH had a significantly lower DeltaC(p), 1.73 kcal/mol.K, than that of E.coli IPMDH (20.7 kcal/mol.K). The low DeltaC(p) of T.thermophilus IPMDH could not be predicted from its change in solvent-accessible surface area DeltaASA. The results suggested that there is a large structural difference between the unfolded state of T.thermophilus and that of E.coli IPMDH. Another responsible factor for the higher thermal stability of T.thermophilus IPMDH was the increase in the most stable temperature T(s). The DeltaG degrees maximum of T.thermophilus IPMDH was much smaller than that of E.coli IPMDH. The present results clearly demonstrated that a higher melting temperature T(m) is not necessarily accompanied by a higher DeltaG degrees maximum. Study holds ProTherm entries: 12762, 12763, 12764 Extra Details: Tween 20(0.01%) was added in the experiment. N - I heat capacity change; 3-isopropylmalate dehydrogenase; protein unfolding;,thermal stability; thermophilic protein
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:44 p.m.
|Number of data points||9|
|Proteins||3-isopropylmalate dehydrogenase ; 3-isopropylmalate dehydrogenase ; 3-isopropylmalate dehydrogenase ; 3-isopropylmalate dehydrogenase|
|Assays/Quantities/Protocols||Experimental Assay: dCp ; Experimental Assay: Tm ; Experimental Assay: dHvH|
|Libraries||Mutations for sequence MSKNYHIAVLPGDGIGPEVMTQALKVLDAVRNRFAMRITTSHYDVGGAAIDNHGQPLPPATVEGCEQADAVLFGSVGGPKWEHLPPDQQPERGALLPLRKHFKLFSNLRPAKLYQGLEAFCPLRADIAANGFDILCVRELTGGIYFGQPKGREGSGQYEKAFDTEVYHRFEIERIARIAFESARKRRHKVTSIDKANVLQSSILWREIVNEIATEYPDVELAHMYIDNATMQLIKDPSQFDVLLCSNLFGDILSDECAMITGSMGMLPSASLNEQGFGLYEPAGGSAPDIAGKNIANPIAQILSLALLLRYSLDADDAACAIERAINRALEEGIRTGDLARGAAAVSTDEMGDIIARYVAEGV ; Mutations for sequence MKVAVLPGDGIGPEVTEAALKVLRALDEAEGLGLAYEVFPFGGAAIDAFGEPFPEPTRKGVEEAEAVLLGSVGGPKWDGLPRKIRPETGLLSLRKSQDLFANLRPAKVFPGLERLSPLKEEIARGVDVLIVRELTGGIYFGEPRGMSEAEAWNTERYSKPEVERVARVAFEAARKRRKHVVSVDKANVLEVGEFWRKTVEEVGRGYPDVALEHQYVDAMAMHLVRSPARFDVVVTGNIFGDILSDLASVLPGSLGLLPSASLGRGTPVFEPVHGSAPDIAGKGIANPTAAILSAAMMLEHAFGLVELARKVEDAVAKALLETPPPDLGGSAGTEAFTATVLRHLA|