Developing Mechanisms from Protein Structures

在一组研究中，我们一直在研究蛋白质中最保守的部分，它们只占总结构的一小部分，通常是6-20个残基。同时，我们也在研究各种分子性质，如堆积，与序列保守区的相关性。了解序列保守的结构物理基础将使我们能够仅从序列中对蛋白质核心做出一些关键的预测。我们研究了几种蛋白质，最近的溶菌酶和a-乳清蛋白，在那里我们发现了一个连接所有二级结构元素的残基核，可以作为关键的核。此外，我们正在开发几种新的线程化方法，以全面考虑核心和序列保护。我们还一直在研究蛋白质折叠中间体的荧光，以确定分子内距离，以便将这些与天然结构距离进行比较。在到目前为止的情况下，熔融球体中间体中的距离与自然状态的距离相似。 计算生物学的另一个目标是了解分子机制。用传统的分子动力学处理的蛋白质结构并不能提供关于大规模运动的信息。我们正在用一种新的粗粒度模型研究蛋白质动力学，每个残基只有一个点。这种新方法代表了一种从结构推断功能行为的简单方法。它基于高斯网络模型考虑已知蛋白质结构的波动。这一过程已被证明令人满意地采样了蛋白质天然构象周围的残基波动分布，并与晶体温度因素和氢交换数据产生了良好的一致性，适用于各种蛋白质和核酸结构。由于这种方法简单，所以结果直观而引人注目。这种方法产生了一系列运动模式，通常是铰链弯曲运动，甚至包括最慢、最全局的运动。在最近的一次方法开发中，我们将计算从标量扩展到了矢量，因此我们现在能够跟踪平移变形。这为理解超大型甚至超分子结构的总体功能动力学开辟了新的和令人兴奋的前景。在另一项计算改进中，计算所需的时间显著减少了几个数量级。我们最近用这种方法进行的研究包括：1)逆转录酶，在逆转录酶中，我们展示了手指/拇指结合部位的运动与核糖核酸酶H位之间的反相关性如何导致核酸链在一系列释放-拉动-转弯运动中通过酶进行逐步处理的机制；2)t-RNA自由并与其同源合成酶结合(两者独立或共同显示相似的运动)；3)GroEL-Groes蛋白质伴侣系统，这是一个非常大的系统(8000个残基)，显示空腔如何以多种不同的方式被压缩，以及可用的结合内部结合表面如何通过这些运动发生变化；以及4)微管蛋白，其中二聚化对于增强运动的协同性至关重要，二聚体运动包括亚基之间的摆动，沿二聚体长轴的伸长和压缩，以及两个单体在相反方向上的扭曲。最近的应用包括纤维形式的微管蛋白，以及其他几种核酸结合蛋白。预期的应用包括研究多种蛋白质的结合和构象转变。 我们现在可以将这些计算扩展到极大的结构(100万个氨基酸)。功能运动，例如加工，通常只依赖于形状，而不依赖于结构的所有细节。通常，这些可以是两个结构域之间的铰链，整个结构的伸展，亚基之间的旋转。重要的发现是，在这些大规模运动中，结合位点上的一些局部环打开和关闭，而不是独立的。这对序列保护具有重要的意义。整个蛋白质结构对于促进这些明显的局部运动至关重要！ 新的快速结构测定。通过将计算的结构库与使用其连接通过质谱学鉴定的交联结构的表面距离测量相结合。 通过磷酸化来调节蛋白质。级联效应与构象的变化有关。我们开始对为什么磷酸化引起构象改变或不引起构象改变有一些理解。 药物发现部。还应用了用于计算蛋白质运动的相同数学形式(奇异值分解)来分析细胞系筛选数据。有可能将122种药物分成25个不同的组，并以高度系统化的方式对细胞系本身进行分类。这60个细胞系分成21个组，其中肾癌、白血病和卵巢癌的组别最强。 Z01公元前08370-17

In one group of studies we have been studying the very most conserved parts of proteins, which comprise a small fraction of the total structure, typically 6 - 20 residues. At the same time we are also investigating the correlations of various molecular properties, such as packing, with the sequence conserved regions. Understanding the physical basis in structure for sequence conservation would enable us to make some critical predictions of protein cores from sequences alone. We studied several proteins, most recently lysozyme and a-lactalbumin, where we find a nucleus of residues that connect among all of the secondary structure elements and could act as a critical nucleus. In addition we are developing several new approaches to threading to consider cores and sequence conservation in a comprehensive way. We also have been studying protein folding intermediates by fluorescence to determine intramolecular distances in order to compare these with the native structure distances. In cases to date, the distances in the molten globule intermediates are similar to the native state distances. Another goal of computational biology is to understand molecular mechanisms. Protein structures treated with conventional molecular dynamics have not been so informative about large scale motions. We are investigating protein dynamics with a new coarse-grained model having only one point per residue. This new approach represents a simple way to infer functional behavior from structures. It considers fluctuations about known protein structures based on a Gaussian network model. This procedure has been shown to sample satisfactorily the distribution of residue fluctuations around the native conformation in proteins, and to yield excellent agreement with crystallographic temperature factors and hydrogen exchange data, for a broad variety of proteins and nucleic acid structures. Since this method is simple, results are intuitive and compelling. The approach yields a series of modes of motion, typically hinge bending motions, including even the slowest, most global motions. In a recent method development we have extended the calculations from scalar to vector, so that we are now able to follow translational deformations. This opens new and exciting prospects for comprehending the total functional dynamics of extremely large, even supra-molecular structures. In another computational improvement, the time required for calculations has been significantly reduced by several orders of magnitude. Our recent studies with this approach have included: 1) reverse transcriptase in which we showed how the anti-correlations between the motions of the fingers/thumb binding site and the ribonuclease H site could lead to a step-wise processing mechanism for the progression of the nucleic acid chain through the enzyme in a release-pull-turn series of motions; 2) t-RNA free and bound to its cognate synthetase (both show similar motions, independently and together); 3) the GroEl-GroES protein chaperone system which is an extremely large system (8000 residues), to show how the cavity is compressed in many different ways and how the available binding interior biding surface changes through these motions; and 4) tubulin where the dimerization is critical for enhancing the cooperativity of motions and the dimer motions include a wobble between the subunits, elongation and compression along the long axis of the dimer, and twisting of the two monomers in directions opposite to one another. Recent applications have included tubulin in its fibrillar form, as well as several other nucleic acid binding proteins. Anticipated applications include studies of binding and conformational transitions for a broad variety of proteins. We now can extend these calculations to extremely large structures (1 million amino acids). Functional motions, e.g. processing, usually depend only on shape and not on all details of structure. Typically these can be hinges between two domains, stretching of whole structure, rotations between subunits. The important finding is that some local loops over binding sites open and close during some of these large scale motions, not independently. This has important implications for sequence conservation. The entire protein structure is critical for facilitating these apparently local motions! New Fast Structure Determination. By combining computed structure libraries with surface distance measurements using crosslinked structures whose links are identified by mass spectroscopy. Phosphorylation to Regulate Proteins. Cascade effects relate to changes in conformation. We are beginning to develop some understanding of why phosphorylation either causes conformational change or it does not. Drug Discovery. An application of the same mathematical formalism (singular value decomposition) utilized for calculating the motions of proteins has also been made to analyze the cell-line screening data. It was possible to cluster the 122 agents into 25 distinct groups, as well as to classify the cell lines themselves, in a highly systematic way. The 60 cell lines cluster into 21 groups, with the strongest groupings found for renal, leukemia and ovarian cancer. Z01 BC 08370-17