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首页> 外文期刊>Proceedings of the National Academy of Sciences of the United States of America >Nmr Studies Of A Channel Protein Without Membranes: Structure And Dynamics Of Water-solubilized Kcsa
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Nmr Studies Of A Channel Protein Without Membranes: Structure And Dynamics Of Water-solubilized Kcsa

机译：没有膜的通道蛋白的Nmr研究：水溶性Kcsa的结构和动力学

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摘要

Structural studies of polytopic membrane proteins are often hampered by the vagaries of these proteins in membrane mimetic environments and by the difficulties in handling them with conventional techniques. Designing and creating water-soluble analogues with preserved native structures offer an attractive alternative. We report here solution NMR studies of WSK3, a water-soluble analogue of the potassium channel KcsA. The WSK3 NMR structure (PDB ID code 2K1E) resembles the KcsA crystal structures, validating the approach. By more stringent comparison criteria, however, the introduction of several charged residues aimed at improving water solubility seems to have led to the possible formations of a few salt bridges and hydrogen bonds not present in the native structure, resulting in slight differences in the structure of WSK3 relative to KcsA. NMR dynamics measurements show that WSK3 is highly flexible in the absence of a lipid environment. Reduced spectral density mapping and model-free analyses reveal dynamic characteristics consistent with an isotro-pically tumbling tetramer experiencing slow (nanosecond) motions with unusually low local ordering. An altered hydrogen-bond network near the selectivity filter and the pore helix, and the intrinsically dynamic nature of the selectivity filter, support the notion that this region is crucial for slow inactivation. Our results have implications not only for the design of water-soluble analogues of membrane proteins but also for our understanding of the basic determinants of intrinsic protein structure and dynamics.
机译：在膜模拟环境中，这些蛋白质的种类繁多，并且难以通过常规技术处理它们，因此常常会妨碍多位膜蛋白的结构研究。设计和制造具有保留的天然结构的水溶性类似物提供了一种有吸引力的选择。我们在这里报告WSK3（钾通道KcsA的水溶性类似物）的溶液NMR研究。 WSK3 NMR结构（PDB ID码2K1E）类似于KcsA晶体结构，验证了该方法。然而，通过更严格的比较标准，引入一些旨在改善水溶性的带电荷残基似乎导致可能形成一些天然结构中不存在的盐键和氢键，从而导致结构上的细微差别。相对于KcsA的WSK3。 NMR动力学测量表明，在没有脂质环境的情况下，WSK3具有很高的柔韧性。降低的光谱密度图和无模型分析显示出动态特性，与等速旋转四聚体经历缓慢（纳秒）运动且具有异常低的局部有序性。选择性过滤器和孔螺旋附近的氢键网络发生了变化，以及选择性过滤器的内在动力学性质，支持了这一区域对于缓慢灭活至关重要的观点。我们的结果不仅对膜蛋白的水溶性类似物的设计有影响，而且对我们对内在蛋白结构和动力学的基本决定因素的理解也有影响。

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