Conformational changes in proteins: rates and mechanisms from discrete path sampling

蛋白质是由20种不同的氨基酸组成的链，它们在细胞中合成后必须折叠成具有生物活性的形式。它们在所有生物体中起着重要的结构和催化作用。蛋白质发挥功能所需的所有信息最终都必须编码在核酸（如DNA）中。最近的努力，如人类基因组计划，正在提供越来越多的核酸序列，从中可以推导出给定生物体中每种蛋白质的氨基酸序列。一旦知道了蛋白质的氨基酸序列，我们就可以试图确定它折叠成的三维结构，以及它在生物体中发挥的功能。然而，越来越明显的是，理解许多蛋白质的生物分子功能将需要我们考虑不止一种结构。特别是，越来越多的实验证据表明，明确定义的结构变化，通常称为构象变化，伴随着蛋白质的功能。因此，为了深入了解这些蛋白质是如何工作的，我们必须发现所涉及的途径，以及它们被穿越的速度。这项研究的潜在益处在药物设计等领域具有重要意义。例如，创造抗癌药物的通常策略通常是针对快速分裂的细胞。然而，最近已经设计出新的药物，通过与特定蛋白质的特定构象结合起作用，这阻止了它们与通常相互作用的分子结合，从而阻碍了它们的功能。这条研究路线似乎非常有前途，并且依赖于对替代蛋白质构象的了解。计算机模拟构象变化可能在上述努力中发挥重要作用，提供难以通过实验获得的机理信息。原则上，我们可以采用蛋白质中原子间作用力的模型，然后在计算机上求解牛顿运动方程，通过一系列的小步骤在时间上推进蛋白质的状态。这种方法的主要问题是需要大量的步骤，即使最近计算机技术的进步，也不可能以这种方式在所需的微到毫秒的时间尺度上跟踪蛋白质动力学。然而，替代的模拟方法是可用的，它可以通过进行额外的近似来弥补时间尺度的差距。目前的建议是采用这种最近发展的方法来表征四种特别感兴趣的蛋白质的构象变化。在每种情况下，终点构象都是已知的，或者已经从实验数据中提出，计算机模拟可以用来计算中间路径。这些模拟还将为相应的机制提供速率常数，并可用于分析策略性位置的氨基酸突变成其他形式的影响。本研究将处理四个特定的例子，以涵盖一系列重要的蛋白质功能，包括催化，生物合成和信号传导。例如，腺苷酸激酶（Adk）蛋白具有重要的生物学功能，但在不同的生物体之间，其精确的氨基酸序列略有不同。与“正常”细菌中的Adk相比，适应80摄氏度温度的生物体中的Adk蛋白在20摄氏度时表现出较低的活性。最近的实验表明，这种差异是由较慢的构象动力学引起的。计算机模拟将用于验证这一假设，并得出所涉及的途径的细节，以建议未来的实验方向。

Proteins are chains constructed from twenty different amino acids, which must fold up into a biologically active form after they are synthesised in cells. They play essential structural and catalytic roles in all living organisms. All the information required for a protein to function must ultimately be encoded in nucleic acids such as DNA. Recent efforts, such as the human genome project, are providing an increasing number of nucleic acid sequences, from which the amino acid sequences of every protein in a given organism can be derived. Once the amino acid sequence of a protein is known we can seek to determine the three-dimensional structure that it folds into, and the function that it performs in the organism. However, it has become increasingly apparent that understanding the biomolecular function of many proteins will require us to consider more than one structure. In particular, there is a growing body of experimental evidence suggesting that well-defined structural changes, usually called conformational changes, accompany protein function. Hence, to gain insight into how such proteins work, we must discover the pathways involved, and how quickly they are traversed. The potential benefits of this research are of great importance in fields such as drug design. For example, the usual strategy in efforts to create anti-cancer agents has generally been to target rapidly dividing cells. Recently, however, new drugs have been designed that work by binding to specific conformations of particular proteins, which prevents them from binding the molecules that they usually interact with, thus blocking their function. This line of research appears to be very promising, and depends upon knowledge of alternative protein conformations. Computer simulation of conformational changes could potentially play an important role in the above effort by supplying mechanistic information that is hard to obtain experimentally. In principle, we could adopt a model for the interatomic forces in the protein, and then solve Newton's equations of motion on a computer to advance the state of the protein in time using a series of small steps. The main problem with this approach is that a huge number of steps are needed, and even with recent advances in computer technology, it is not yet possible to follow the protein dynamics on the required micro- to millisecond time scale in this way. However, alternative simulation methods are available, which can bridge the time scale gap by making additional approximations. The present proposal is to employ a recently developed approach of this sort to characterise the conformational changes involved in four proteins of particular interest. In each case, the end-point conformations are known, or have been suggested, from experimental data, and computer simulations could be used to calculate the intervening paths. These simulations would also provide rate constants for the corresponding mechanisms, and could be used to analyse the effect of mutating strategically located amino acids into alternative forms. Four particular examples would be treated in this study to cover a range of important protein function, including catalysis, biosynthesis and signalling. For example, adenylate kinase (Adk) proteins perform a vital biological function, but differ slightly in their precise amino acid sequence between different organisms. The Adk protein from an organism adapted to live at a temperature of 80 centigrade exhibits reduced activity at 20 centigrade when compared with Adk from 'normal' bacteria. Recent experiments suggest that this difference is caused by slower conformational dynamics. Computer simulation would be used to test this hypothesis, and to derive details of the pathways involved to suggest future directions for experiment.