THE FOLDING OF AN ENZYME .1. THEORY OF PROTEIN ENGINEERING ANALYSIS OF STABILITY AND PATHWAY OF PROTEIN FOLDING
THE FOLDING OF AN ENZYME .1. THEORY OF PROTEIN ENGINEERING ANALYSIS OF STABILITY AND PATHWAY OF PROTEIN FOLDING
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DOI：
10.1016/0022-2836(92)90561-w
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发表时间：
1992-04-05
影响因子：
5.6
通讯作者：
SERRANO, L
中科院分区：
文献类型：
--
作者：
FERSHT, AR;MATOUSCHEK, A;SERRANO, L
概述了蛋白质折叠的稳定性和途径问题的简单蛋白质工程方法的理论、假设和局限性。它是分析非共价键合（包括酶催化）中结构-活性关系的一般程序，将实验可获得的数据与非共价键合的变化联系起来。对突变蛋白质的展开和重折叠的动力学和平衡测量可用于绘制过渡态和折叠中间体的结构形成。例如，测量突变时解折叠的活化能和解折叠的自由能的变化的比率以给出参数φ。有两个φ的极值，在实践中经常发现，可以用简单的方式解释。φ = 0的值意味着突变位点处的结构在过渡态中与在折叠态中一样折叠。相反，φ = 1表明突变位点的结构在过渡态和未折叠结构中一样未折叠。φ的分数值更难解释，需要更复杂的方法。最合适的突变涉及侧链的截短以去除优选地与蛋白质的其余部分很少相互作用并且不与掩埋电荷配对的部分。对于这种类型的突变发现的φ的分数值可能意味着存在部分非共价键形成或状态的混合。该方法的主要假设是：（1）突变不会改变折叠的途径;（2）突变不会显著改变折叠态的结构;（3）突变不会扰乱未折叠态的结构;（4）在反应能量的过程中，靶基团不会与新的伴侣发生新的相互作用。假设（2）和（3）对于φ = 0或1（最常见的值）的简单情况不一定是必需的，因为结构破坏的影响可以抵消。假设（4）可以通过双突变循环过程来检验，可以对双突变循环过程进行分析，以在复杂的背景下分离出仅仅一对相互作用的影响。这一分析为芽孢杆菌RNA酶的稳定性和折叠途径的相关研究提供了正式的基础，可以看出该理论在实践中非常有效。
The theory, assumptions and limitations are outlined for a simple protein engineering approach to the problem of the stability and pathway of protein folding. It is a general procedure for analysing structure-activity relationships in non-covalent bonding, including enzyme catalysis, that relates experimentally accessible data to changes in non-covalent bonding. Kinetic and equilibrium measurements on the unfolding and refolding of mutant proteins can be used to map the formation of structure in transition states and folding intermediates. For example, the ratio of the changes in the activation energy of unfolding and the free energy of unfolding on mutation is measured to give a parameter φ. There are two extreme values of φ that are often found in practice and may be interpreted in a simple manner. A value of φ = 0 implies that the structure at the site of mutation is as folded in the transition state as it is in the folded state. Conversely, φ = 1 shows that the structure at the site of mutation is as unfolded in the transition state as it is in the unfolded structure. Fractional values of φ are more difficult to interpret and require a more sophisticated approach. The most suitable mutations involve truncation of side-chains to remove moieties that preferably make few interactions with the rest of the protein and do not pair with buried charges. Fractional values of φ found for this type of mutation may imply that there is partial non-covalent bond formation or a mixture of states. The major assumptions of the method are: (1) mutation does not alter the pathway of folding; (2) mutation does not significantly change the structure of the folded state; (3) mutation does not perturb the structure of the unfolded state; and (4) the target groups do not make new interactions with new partners during the course of reaction energy. Assumptions (2) and (3) are not necessarily essential for the simple cases of φ = 0 or 1, the most common values, since effects of disruption of structure can cancel out. Assumption (4) may be checked by the doublemutant cycle procedure, which may be analysed to isolate the effects of just a pair of interactions against a complicated background. This analysis provides the formal basis of the accompanying studies on the stability and pathway of folding of barnase, where it is seen that the theory holds very well in practice.