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

1)大分子蛋白质NMR方法的发展：在以前的论文中，已经表明交叉饱和方法使我们能够识别接触。以比化学位移扰动和氢-氘交换实验更严格的方式测定大蛋白质复合物的残基[Nat.Struct.Biol.7（3）220 - 223]。然而，在通过交叉饱和法测定接触残基的范围内，存在该方法难以应用于具有分子量的蛋白质复合物的局限性。超过150 K和/或具有弱结合，因为在该方法中应该直接观察到源自络合物的共振。在目前的研究中。为了克服这些限制，我们在NMR时间尺度上自由态和结合态之间快速交换的条件下进行了交叉饱和测量，并确定了蛋白A的B结构域和完整的复合物的接触残基。IgG，分子量为164K，显示弱结合。2）离子通道与其孔阻断剂的相互作用分析我们通过转移交叉饱和（transferred cross-saturation，TCS）实验确定了agitoxin 2（AgTx2）与KcsA K+通道的结合位点。在TCS实验中显著受影响的残基在AgTx2上形成连续表面，并且表面残基的取代降低了与KcsA K+通道的结合亲和力。基于与KcsA K+通道的AgTx2结合位点的性质，我们提出了一个表面基序，该基序在影响K+通道的孔阻断毒素上观察到。此外，我们还解释了K+通道对毒素的特异性的结构基础。这里使用的TCS方法不仅适用于信道。其与其它抑制剂复合，但也与多种调节分子复合，并提供关于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.