Conformation and Thermal Denaturation of Apocalmodulin: Role of Electrostatic Mutations

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• 作者：Irina Protasevich ; Bijan Ranjbar ; Vladimir Lobachov ; Alexander Makarov ; Robert Gilli ; Claudette Briand ; Daniel Lafitte ; and Jacques Haiech
• 刊名：Biochemistry
• 出版年：1997
• 出版时间：February 25, 1997
• 年：1997
• 卷：36
• 期：8
• 页码：2017 - 2024
• 全文大小：171K
• 年卷期：v.36,no.8(February 25, 1997)
• ISSN：1520-4995

文摘

Scanning microcalorimetry and circular dichroism were used tostudy conformational stateand heat denaturation of Ca²⁺-free synthetic calmodulin(SynCaM) and three charge reversal mutants.We produced evidence for the major role of the electrostaticpotential in the stability and flexibility ofSynCaM. The substitution of ¹¹⁸DEE¹²⁰by ¹¹⁸KKK¹²⁰ (SynCaM12A) does notinfluence the flexibilityof the protein; the replacement of ⁸²EEE⁸⁴by ⁸²KKK⁸⁴ (SynCaM8) decreases its level,while the combinationof these two mutations in SynCaM18A significantly increases theflexibility. The heat denaturation ofapoSynCaM and its mutants is well approximated by two two-statetransitions with the lower-temperaturetransition corresponding to C-terminal lobe melting and thehigher-temperature one to N-terminal lobemelting. The difference in transition temperatures for the twolobes decreases in SynCaM8 and increasesin SynCaM18A, suggesting a modification in the influence of one lobe tothe other. The electrostaticmutations change the parameters of thermal denaturation of SynCaM lobesin a similar way as pH conditionsaffect thermal transition parameters of multidomain proteins, leadingto a linear temperature dependenceof transition enthalpy. One domain of the N-terminal lobe inapoSynCaM18A is unfolded in the nativestate. Near-UV CD spectra point out the invariability of the localstructure of aromatic residues uponmutations, although the secondary structure undergoes strikingtransformations. Cacodylate ions stronglyand specifically alter the helical content of SynCaM. Our dataunambiguously demonstrate that the twolobes are not independent, and interactions between the lobes aremediated by the electrostatic potentialof the molecule.