Jendeberg L, Persson B, Andersson R, Karlsson R, Uhlén M, Nilsson B
Department of Biochemistry and Biotechnology, Royal Institute of Technology, Stockholm, Sweden.
J Mol Recognit. 1995 Jul-Aug;8(4):270-8. doi: 10.1002/jmr.300080405.
A real-time biospecific interaction analysis (BIA) was performed to study the specific interaction between the Fc portion of human immunoglobulin G1 (Fc1) and a one domain analogue (designated Z) of staphylococcal protein A, in monovalent (Z) and divalent (ZZ) forms, and five different single amino acid substituted Z variants (L17D, N28A, F30A, I31A, K35A). Experimental BIA data were used to calculate association rate constants (kon), dissociation rate constants (koff) and affinity constants (Kaff). The divalent form (ZZ) showed a higher affinity for Fc1 mainly because of a slower off rate. Out of the five mutant Z proteins, four (L17D, N28A, I31A, K35A) showed a decreased affinity to Fc1 compared to the parent Z molecule. Surprisingly, two (L17D, I31A) of these four had the major effect of a decreased binding energy as a lowered kon while the other two (N28A, K35A) mutant proteins showed an increased koff as the major kinetic difference from Z in their binding to Fc1. For five of the six different Z variants, as well as for the ZZ molecule, calculated Kaff and calculated differences in binding free energies relative to the parent Z molecule (delta delta G), are in good agreement with the corresponding values obtained in a competitive displacement assay using radioactively labeled Z as a tracer (Cedergren et al., (1993) Prot. Eng. 6, 441-448). However, the I31A variant, with a measured kon that was more than three orders of magnitude lower than that of Z in the BIA assay, showed a significantly weaker affinity to Fc1 when calculated from the BIA data compared to the competitive displacement assay. The discrepancy between these two methods for Z(I31A) is discussed as well as possible explanations for the unexpected large effect of lowered kon for two of the mutant Z proteins.
进行了实时生物特异性相互作用分析（BIA），以研究人免疫球蛋白G1（Fc1）的Fc部分与单价（Z）和二价（ZZ）形式的葡萄球菌蛋白A的单结构域类似物（命名为Z）以及五个不同的单氨基酸取代Z变体（L17D、N28A、F30A、I31A、K35A）之间的特异性相互作用。实验性BIA数据用于计算缔合速率常数（kon）、解离速率常数（koff）和亲和常数（Kaff）。二价形式（ZZ）对Fc1表现出更高的亲和力，主要是因为解离速率较慢。在五个突变Z蛋白中，四个（L17D、N28A、I31A、K35A）与亲本Z分子相比，对Fc1的亲和力降低。令人惊讶的是，这四个中的两个（L17D、I31A）具有降低结合能的主要作用，表现为kon降低，而另外两个（N28A、K35A）突变蛋白在与Fc1结合时，表现出koff增加，这是与Z的主要动力学差异。对于六个不同Z变体中的五个以及ZZ分子，计算得到的Kaff和相对于亲本Z分子的结合自由能计算差异（δδG）与使用放射性标记Z作为示踪剂的竞争性置换试验（Cedergren等人，（1993年）《蛋白质工程》6，441 - 448）中获得的相应值高度一致。然而，在BIA测定中，I31A变体的kon测量值比Z低三个多数量级，与竞争性置换试验相比，根据BIA数据计算时，其对Fc1的亲和力明显较弱。讨论了这两种方法对Z（I31A）的差异以及对两个突变Z蛋白kon降低的意外大效应的可能解释。
