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，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发挥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在核酸（如DNA）中编码。最近的努力，如人类基因组计划，提供了越来越多的核酸序列，从这些序列可以得到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，以及它在生物体中的功能。然而，越来越明显的是，了解许多蛋白质的生物分子功能将需要我们考虑多种结构。特别是，有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，伴随蛋白质功能。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非常重要。例如，在努力创造抗癌剂的过程中，通常的策略通常是靶向快速分裂的细胞。然而，最近已经设计出通过与特定蛋白质的特定构象结合而起作用的新药，这阻止了它们与它们通常相互作用的分子结合，从而阻断了它们的功能。这条研究路线似乎是非常有前途的，并依赖于知识的替代蛋白质构象。构象变化的计算机模拟可能会在上述努力中发挥重要作用，提供很难通过实验获得的机制信息。原则上，我们可以采用蛋白质中原子间力的模型，然后在计算机上求解牛顿运动方程，通过一系列小步骤及时推进蛋白质的状态。这种方法的主要问题是需要大量的步骤，即使计算机技术最近取得了进展，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，其可以通过进行额外的近似来弥合时间尺度差距。目前的建议是采用这种最近开发的方法来表征涉及四种特别感兴趣的蛋白质的构象变化。在每种情况下，终点构象是已知的，或已建议，从实验数据，和计算机模拟可以用来计算的干预路径。这些模拟还将提供相应机制的速率常数，并可用于分析将战略定位的氨基酸突变成替代形式的效果。在这项研究中，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。例如，腺苷酸激酶（Adk）蛋白质执行重要的生物学功能，但在不同生物体之间其精确的氨基酸序列略有不同。与来自“正常”细菌的Adk相比，来自适应在80摄氏度温度下生活的生物体的Adk蛋白在20摄氏度下表现出降低的活性。最近的实验表明，这种差异是由较慢的构象动力学。计算机模拟将被用来测试这一假设，并得出所涉及的途径的细节，为未来的实验方向提出建议。

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.