CAJ | 학술논문

运用多种光谱法研究PCR定点突变获得的肌红蛋白突变体（D44K、D60K、K56D）与Cu（Ⅱ）的相互作用。结果表明：Cu（Ⅱ）对突变体的猝灭机理与野生型相同，均为静态猝灭，但结合常数、结合位点数、热力学常数、结合距离以及三维构象方面发生了一些变化。在相同温度下，蛋白与Cu（Ⅱ）的结合能力顺序为Mb（WT）＜Mb（D60K）＜ Mb（K56D）＜ Mb（D44K），结合力主要以静电和疏水相互作用为主；结合距离顺序为 Mb（WT）＜ Mb（K56D）＜Mb（D60K）＜Mb（D44K）。同时，借由多种光谱测定了Cu（Ⅱ）引起肌红蛋白及其突变体的构象变化情况，得出突变后蛋白更易与Cu（Ⅱ）发生相互作用，α-螺旋含量下降的幅度更大。总之，肌红蛋白表面44、60、56位带电氨基酸突变后对其构象和功能产生了一定影响。
운용다충광보법연구PCR정점돌변획득적기홍단백돌변체（D44K、D60K、K56D）여Cu（Ⅱ）적상호작용。결과표명：Cu（Ⅱ）대돌변체적졸멸궤리여야생형상동，균위정태졸멸，단결합상수、결합위점수、열역학상수、결합거리이급삼유구상방면발생료일사변화。재상동온도하，단백여Cu（Ⅱ）적결합능력순서위Mb（WT）＜Mb（D60K）＜ Mb（K56D）＜ Mb（D44K），결합력주요이정전화소수상호작용위주；결합거리순서위 Mb（WT）＜ Mb（K56D）＜Mb（D60K）＜Mb（D44K）。동시，차유다충광보측정료Cu（Ⅱ）인기기홍단백급기돌변체적구상변화정황，득출돌변후단백경역여Cu（Ⅱ）발생상호작용，α-라선함량하강적폭도경대。총지，기홍단백표면44、60、56위대전안기산돌변후대기구상화공능산생료일정영향。
The interaction between Cu(Ⅱ) and the mutant proteins (D44K ,D60K and K56D) which are attained by PCR site-directed mutagenesis is investigated by multi-spectroscopic techniques .The results show that the fluorescence of all the Mbs is quenched regularly with the addition of Cu (Ⅱ ) . The quenching belongs to the static fluorescence quenching .But the binding constants ,the numbers of the binding sites , the thermodynamic parameters , the binding distance and three-dimensional conformation of mutants are different from wild type .At the same temperature ,the sequence for binding strength is Mb(WT)< Mb(D60K)< Mb(K56D)< Mb(D44K) ,the electrostatic interaction and hydrophobic power play dominating roles in the course of binding .The binding distance sequence is Mb(WT)<Mb(K56D)<Mb(D60K)<Mb(D44K) .The effects of Cu(Ⅱ) on conformation of Mb and mutants are further analyzed by multi-spectroscopic techniques .T he experimental results indicate that the mutants of Mb become easier to interact with Cu (Ⅱ ) ,and α-helix content of the mutants decreases more after interaction with Cu (Ⅱ ) .In all ,mutation in surface-charged residue Asp44 , Asp60 and Lys56 has an effect on the conformation and function of myoglobin .