Dynamics and folding of a membrane protein and transcription activator

所有动物、植物和细菌细胞周围都有一层脂膜。嵌入在脂质中的蛋白质通道和受体精确地控制营养物质、废物、能量和信息进出细胞的流动，但人们对此知之甚少。我们的长期目标是开发方法来克服膜蛋白结构、折叠和动力学分析中的许多困难。最近，我们在研究细菌膜蛋白的折叠方面取得了进展，该蛋白是洗涤剂环境中的甘油促进剂。我们建议继续这项工作，应用一种新的方法，利用细胞提取物合成蛋白质，并研究蛋白质在脂膜环境中的折叠。无细胞合成开启了用同位素标记蛋白质以供核磁共振光谱分析的可能性，这是一种类似于磁共振成像(MRI)的方法。通过标记蛋白质，我们将研究蛋白质的详细原子动力学，这是理解其生物运输机制的关键。我们还将继续沿着类似的路线进行真菌抗菌肽阿拉米星的工作，这是一种模型膜蛋白。 所有慢病毒，包括人类免疫缺陷病毒(HIV)，都会产生一种称为转录反式激活因子(TAT)的调节蛋白，这是允许病毒自我复制的机制的重要组成部分。通过对一段蛋白质的详细动力学研究，我们发现TAT在生理条件下本质上是无序的，在其生物活性表达过程中经历了一个小的无序到有序的转变。我们现在建议使用核磁共振波谱来研究整个蛋白质的动力学，并延长研究动力学的时间范围，以便更好地表征未结合的蛋白质。我们还将继续研究该蛋白与另一种重要的细胞蛋白钙调蛋白和RNA的结合。由于TAT是调节病毒复制和病毒潜伏期的关键角色，了解TAT的动态和折叠可能为改进治疗提供基础。

A lipid membrane surrounds all animal, plant, and bacterial cells. Protein channels and receptors embedded in the lipid precisely control the flow of nutrients, waste products, energy, and information into and out of cells, but are poorly understood. Our long-term goal is to develop methods to overcome the many difficulties in membrane protein structure, folding and dynamics analysis. Recently, we have made progress in studying the folding of a bacterial membrane protein, the glycerol facilitator in detergent environments. We propose to continue this work by applying a new method for synthesizing the protein using cellular extracts and studying folding of the protein in a lipid membrane environment. Cell-free synthesis opens up the possibility of labelling the protein with isotopes for analysis by Nuclear Magnetic Resonance (NMR) spectroscopy, a method analogous to Magnetic Resonance Imaging (MRI). With labeled protein, we will study the detailed atomic dynamics of the protein that are key to understanding its biological transport mechanism. We will also continue our work along similar lines on the fungal antibiotic peptide alamethicin that is a model membrane protein. All lentiviruses, including the Human Immunodeficiency Virus (HIV), produce a regulatory protein called the Transactivator of transcription (Tat) that is an essential component of the machinery that permits the virus to replicate itself. By studying the detailed dynamics of a fragment of the protein we discovered that Tat is intrinsically disordered under physiological conditions and undergoes a small disorder-to-order transition during the expression of its biological activity. We now propose to study the dynamics of the entire protein using NMR spectroscopy and to extend the range of times over which dynamics will be studied in order to better characterize the unbound protein. We will also continue our studies characterizing the binding of the protein to another important cellular protein, calmodulin, and to RNA. Since Tat is a key player in the regulation of viral replication and viral latency understanding the dynamics and folding of Tat may provide the foundation for improved therapies.