Nobel strategy of structural biology for investigation of membrane proteins-ligands

1)大蛋白质核磁共振方法的发展：在之前的文章中，交叉饱和方法已经证明了我们可以识别接触。与化学位移摄动和氢-氘交换实验相比，更严格的方法研究了大型蛋白质复合物残基[j].结构，生物工程，7(3):220-223。然而，在交叉饱和法测定接触残留物的过程中，该方法存在着难以应用于具有一定分子量的蛋白质复合物的局限性。超过150k和/或弱结合，因为在该方法中应直接观察到由配合物产生的共振。在目前的研究中。为了克服这些限制，我们在核磁共振时间尺度上的自由和束缚态快速交换条件下进行了交叉饱和测量，并确定了蛋白a和完整的B结构域配合物的接触残基。IgG的分子量为164k，结合弱。通过转移交叉饱和（TCS）实验确定了agitoxin2 （AgTx2）与KcsA K+通道的结合位点。在TCS实验中受到显著影响的残基在AgTx2上形成了一个连续的表面，表面残基的取代降低了与KcsA K+通道的结合亲和力。基于AgTx2结合位点与KcsA K+通道的特性，我们提出了一个表面基元，可以在影响K+通道的孔阻断毒素上观察到。此外，我们还解释了K+通道对毒素特异性的结构基础。这里使用的TCS方法不仅适用于信道。它们与其他抑制剂和多种调节分子络合，并提供了它们在溶液中的界面的重要信息。

1)Development of NMR method for lager proteins :In the previous paper, it has been shown that the cross-saturation method enables us to identify the contact. residues of large protein complexes in a more rigorous manner than chemical shift perturbation and hydrogen-deuterium exchange experiments [Nat.Struct.Biol.7(3)220-223]. However, within the determination of the contact residues by the cross-saturation method, there are limitations that the method is difficult to apply to protein complexes with a molecular weight. over 150 K and/or with weak binding, since the resonances originating from the complexes should be directly observed in the method. In the present research. to overcome these limitations, we carried out the cross-saturation measurements under the condition of a fast exchange between free and bound states on the NMR time scale, and determined the contact residues of the complex of the B domain of protein A and intact. IgG which has a molecular weight of 164 K and shows weak binding.2)Interaction analyses between an ion channel and its pore blockerWe have determined the binding site on agitoxin2 (AgTx2) to the KcsA K+ channel by a transferred cross-saturation (TCS) experiment. The residues significantly affected in the TCS experiments formed a contiguous surface on AgTx2, and substitutions of the surface residues decreased the binding affinity to the KcsA K+ channel. Based on properties of the AgTx2 binding site with the KcsA K+ channel, we present a surface motif that is observed on pore-blocking toxins affecting the K+ channel. Furthermore, we also explain the structural basis of the specificity of the K+ channel to the toxins. The TCS method utilized here is applicable not only for the channels. which are complexed with other inhibitors but also with a variety of regulatory molecules, and provides important information about their interface in solution.