Dynamic structural science: exploring energy landscapes in complex enzyme systems

蛋白质是动态分子。蛋白质分子内的运动可以是局部的（例如键振动，主链和侧链运动），发生在相对较快的时间尺度上，或大规模的运动（区域运动，构象变化和缓慢的呼吸模式），通常发生在毫秒到秒的时间尺度上。局部快速运动可以影响酶活性位点的化学性质；更大规模的运动将活性位点聚集在一起，或促进远距离通信，例如氧化还原酶中的远距离电子转移，通过信号级联或蛋白质分子折叠的信息传递。这些大规模的运动产生了能量景观的概念——这是一个自由能表面，可以容纳所有的蛋白质大分子的构象。构象状态在这一景观中的分布可能受到干扰，例如配体或药物结合、序列的自然变异（多态性）或伴侣蛋白结合。我们对这些景观的空间分布和时间探索的知识充其量是有限的，主要归因于缺乏捕获这些信息的一般结构和生物物理工具。“刚性”蛋白质模块的三维结构很容易使用传统方法（晶体学，核磁共振波谱）获得。然而，在复杂的蛋白质系统中，多个模块如何通信是无法使用这些技术访问的。在这个应用中，我们的目标是开发强大的实验方法，使用最先进的光谱，动力学和计算方法，使研究人员能够研究景观的空间和时间特性及其通过小分子/蛋白质结合的重塑。我们的目标是利用哺乳动物一氧化氮合酶来开发这些方法，氧化还原酶是由多个功能域构建的，在酶催化反应过程中，这些功能域的化学作用与主要的动力学漂移相耦合。我们的目标是在整个景观中定义多个构象状态的结构，定义它们相互转化的时间常数，并评估这些结构转变在酶的催化循环中的功能重要性。通过提供一氧化氮合酶功能动力学的原子级空间和时间分辨率信息，我们设想开发选择性抑制剂干扰与功能相关的动态过程的新机会。这将重振对这些酶的异构体特异性抑制剂的研究，并为其他动态系统的类似分析提供通用工具，从中可以研究功能和治疗干预。

Proteins are dynamic molecules. Motions within a protein molecule can be localised (e.g. bond vibrations, backbone and side chain motions) occuring on relatively fast timescales, or large scale motions (domain motions, conformational changes and slow breathing modes) that typically occur on the millisecond to second timescale. Localised fast motions can influence the chemistry in enzyme active sites; larger scale motions bring active sites together, or facilitate long range communication, for example in the transfer of electrons over large distances in redox enzymes, information transfer through signalling cascades or the folding of protein molecules. These large scale motions give rise to the concept of energy landscapes - that is the free energy surface that accommodates all conformations of the protein macromolecule that are populated. The distribution of conformational states across this landscape can be perturbed, for example by ligand or drug binding, natural variation in sequence (polymorphisms) or partner protein binding. Our knowledge of the spatial distribution and temporal exploration of these landscapes is at best limited, attributed in the main to the lack of general structural and biophysical tools to capture this information. The three dimensional structures of 'rigid' protein modules are readily accessed using conventional approaches (crystallography, NMR spectroscopy). How multiple modules communicate in complex protein systems however is not accessible using these techniques. In this application we aim to develop robust experimental methods using state-of-the-art spectroscopic, kinetic and computational methods that enable investigators to study the spatial and temporal properties of landscapes and their remodelling by small molecule/protein binding. We aim to develop these methods using mammalian nitric oxide synthases, redox enzymes that are constructed from multiple functional domain the chemistry of which is coupled to major dynamical excursions during the course of the enzyme catalysed reaction. We aim to define the structures of multiple conformational states across the landscape, define the timeconstants for their interconversion and assess the functional importance of these structural transitions in the catalytic cycle of the enzyme. By providing atomic level spatial and time resolved information on the functional dynamics in nitric oxide synthase enzymes we envisage that new opportunities will accrue to develop selective inhibitors that interfere with dynamical processes linked to function. This will reinvigorate the search for isoform specific inhibitors of these enzymes, and also provide general tools for similar analysis of other dynamic systems from which function and therapeutic intervention can be studied.