Development of dynamical structural analysis for the active site of multi functional proteins

在本研究中，我们发展了核磁共振的方法来分析多功能蛋白的活性位点的动态结构。需要解决的问题是：1)我们如何选择性地获得多功能蛋白活性位点的结构信息？2)我们如何分析溶液中蛋白质的动力学结构？利用稳定同位素标记的氨基酸蛋白和分析酰胺基团的弛豫时间是解决这一问题的方法。从本研究中得到以下结果。anti-dansyl Fv片段的研究1)利用双标记法和序列特异性共振法确定了来自anti-dansyl Fv片段主链酰胺基的信号的归属。2)根据自旋标记半抗原结合实验结果，我们得出Fv片段的抗原结合位点由高变环H1、H3和VH结构域n端组成。3)根据NOE数据，我们得出负责抗原结合的残基为Y96H、Y104H、F27H和V2H。4)抗原结合作用通过Fv片段的界面从VH结构域传递到VL结构域。5)在没有抗原的情况下，负责抗原结合的超变环呈现多种构象。构象之间的交换速率受抗原结合的影响。6)从无抗原和有抗原的抗原结合位点结构比较，Fv片段的抗原结合机制为“诱导契合”。抗丹酚Fab片段的研究用分子量为25K的Fv片段建立的方法也适用于分子量为50K的Fab片段。

In the present study, we develop the NMR method in order to analyze the dynamical structure of the active site of multi-functional proteins. The questions to resolve are1) How do we selectively obtain the information about the structure of the active site of the multi-functional protein?2) How do we analyze the dynamical structure of the protein in solution?The answere are the use of protein which are amino-acid specifically labeled withe stable isotope and the analysis of the relaxation time of the amide group.The following results are obtained from the present study.Study by using of anti-dansvl Fv fragment1) The assignments of the signals originating from the amide group of the main chain of anti-dansyl Fv fragment are established by using the double labeling method along with the sequence-specific resonance assingment.2) On the basis of the results obtained from the experiment of the binding of the spin labeled hapten, we conclude that the antigen-binding site of the Fv fragment is composed of H1, H3 of the hypervariable loops and N-terminal in VH domain.3) On the basis of the NOE data, we conclude that the residues which are responsible for the antigen binding are Y96H,Y104H,F27H and V2H.4) The effect of the antigen binding is transmitted from VH domain to VL domain through the interface of the Fv fragment.5) In the absence of the antigen, the hyper variable loop which is responsible for the antigen binding take multi-conformations. The exchange rate among the conformations is affected by the antigen binding.6) From the comparison of the structure of the antigen binding site between in the absence and presence of the antigen, the mechanism of the antigen binding for the Fv fragment is 'induced fit'.Study by using of anti-dansyl Fab fragmentThe method established by using the Fv fragment with the molecular weight of 25K is able to apply to the Fab fragment with the molecular weight of 50K.