Membrane Protein Modeling

我小组的总体研究目标是利用分子建模和生物信息学来分析膜蛋白的结构、功能和分子进化。膜蛋白是最重要的一类蛋白质。它们约占大多数基因组的30%，并参与许多生物过程。它们在生物医学研究中尤其重要，因为当前制药项目的大多数靶点都是膜蛋白。不幸的是，膜蛋白的结构很难通过实验来确定，而且大多数已经确定的结构都来自原核生物。我们通过开发分析序列的计算方法和开发膜蛋白的结构模型来填补这种结构空白。我们使用计算分析来做以下工作：1)解决晶体结构不能回答的问题。2)帮助理解同源蛋白之间的异同。3)将结构和序列信息与功能属性联系起来。4)协助实验研究的设计和解释。我们目前的项目包括开发钾离子通道及其相关通道的结构和门控机制模型。钾离子通道和相关蛋白构成了人类基因的第三大超家族。这些蛋白质几乎存在于所有细胞中，从细菌开始。这类膜蛋白包含几种不同的通道超家族，包括Na+、Ca2+、环核苷酸门控、TRP及其同源物、谷氨酸激活、Ca2+释放通道以及一些K+同向转运体和转运体。这些蛋白质中最小的是2TM K+通道，有四个相同的亚基；每一个都只有两个跨膜螺旋，M1和M2。位于M1和M2之间的“P”发夹段仅横跨跨膜区域的外半部。P段决定了信道的选择性。6TM K+通道更为复杂，每个α亚基有四个额外的跨膜片段S1-S4，它们在形成孔隙的S5-P-S6基序（类似于2TM通道的M1-P-M2基序）之前，并在电压门控通道中形成电压感应域。电压门控Ca2+和Na+通道只有一个α亚基；然而，它含有4个同源的6TM基序。我们的目标之一是建立每个主要的K+通道相关蛋白家族中至少一个成员的跨膜区域的结构模型。半个多世纪以来，膜生物物理学的一个主要目标是了解电压依赖性通道门的分子机制。我们正在开发这些机制的三维模型。诺贝尔化学奖授予Roderick MacKinnon，以表彰他在实验室中解决K+通道晶体结构的工作，认识到理解K+通道结构和功能机制的重要性。我们正在利用他们的晶体学数据来开发结晶的KvAP和Kv1.2通道蛋白的门控机制的结构模型。我们也在开发一些真核同源物的模型，这些同源物已经被广泛研究过，是重要的药物靶点。KvAP晶体结构提出了一个特别有趣的分子建模挑战。完整的KvAP通道蛋白的晶体结构，以及MacKinnon团队基于该结构建立的电压依赖性门控的桨形模型，很难与许多实验结果和膜蛋白能量学的基本原理相一致。我们怀疑这种结构的电压感应域（S1-S4）是严重扭曲的，但隔离电压感应域的第二晶体结构具有天然的开放构象。

The general research aims of my group are to use molecular modeling and bioinformatics to analyze structure, function, and molecular evolution of membrane proteins. Membrane proteins are one of the most important classes of proteins. They comprise about 30% of most genomes and are involved in many biological processes. They are especially important in biomedical research because most targets of current pharmaceutical projects are membrane proteins. Unfortunately, membrane protein structures are difficult to determine experimentally, and most that are determined come from prokaryotes. We fill some of this structural void by developing computational methods of analyzing sequences and developing structural models of membrane proteins. We use computational analyses to do the following:1)Address questions that are not answered by crystal structures. 2)Assist in understanding similarities and differences among homologous proteins.3)Relate structural and sequence information to functional properties.4)Assist in the design and interpretation of experimental studies.Our current projects involve developing models of the structure and gating mechanisms of potassium (K+) channels and their relatives.Potassium channels and related protein comprise the third largest superfamily of human genes. These proteins are found in almost all cells from bacteria on up. This category of membrane proteins contains several diverse superfamilies of channels including Na+, Ca2+, cyclic nucleotide-gated, TRP and its homologs, glutamate-activated, and Ca2+ release channels plus some K+ symporters and transporters. The smallest of these proteins are 2TM K+ channels that have four identical subunits; each of which has only two transmembrane helices, M1 and M2. A 'P' hairpin segment that spans only the outer half of the transmembrane region is located between M1 and M2. The P segment determines the selectivity of the channel. 6TM K+ channels are more complex, with each alpha subunit having four additional transmembrane segments, S1-S4, that precede the pore-forming S5-P-S6 motif (analogous to the M1-P-M2 motif of 2TM channels) and that forms a voltage-sensing domain in voltage-gated channels. Voltage-gated Ca2+ and Na+ channels have only one alpha subunit; however, it contains four homologous 6TM motifs. One of our goals is to develop structural models of the transmembrane region of at least one member of each major family of K+ channel related proteins. A major goal in membrane biophysics for over half a century has been to understand the molecular mechanisms by which voltage-dependent channels gate. We are developing three-dimensional models of these mechanisms. The importance of understanding the structure and functional mechanisms of K+ channels has been recognized by the awarding of the Nobel Prize in Chemistry to Roderick MacKinnon for the work in his laboratory in solving the crystal structures of K+ channels. We are utilizing their crystallographic data to develop structural models of the gating mechanisms of the crystallized KvAP and Kv1.2 channel proteins. We are also developing models of some of their eukaryotic homologs that have been studied extensively and that are important drug targets. The KvAP crystal structure presents a particularly interesting molecular modeling challenge. It is difficult to reconcile the crystal structure of the complete KvAP channel protein, and the paddle model of the voltage-dependent gating that MacKinnon's group developed based on this structure, with many experimental results and with basic principles of membrane protein energetics. We suspect that the voltage-sensing domain (S1-S4) of this structure is grossly distorted, but that a second crystal structure of an isolated voltage-sensing domain has a native open conformation.