USE OF LIPIDIC NANODISCS FOR STRUCTURE/FUNCTION STUDIES ON MEMBRANE PROTEINS

这个子项目是许多利用资源的研究子项目之一 由NIH/NCRR资助的中心赠款提供。次级项目的主要支助 而子项目的主要调查员可能是由其他来源提供的， 包括其它NIH来源。 为子项目列出的总成本可能 表示子项目使用的中心基础设施的估计数量， 而不是由NCRR赠款提供给子项目或子项目工作人员的直接资金。 膜蛋白贡献20  30%的已知蛋白质。然而，它们对研究其结构和功能提出了挑战。X射线晶体学提供了原子分辨率的详细结构信息，但通常膜蛋白必须在去污剂中结晶，去污剂不能忠实地代表脂膜的天然环境，使蛋白质不稳定，结晶尝试涉及太多的劳动，并且结晶某些蛋白质太困难或不可能。因此，其他方法和途径被广泛用于研究膜蛋白，特别是在天然脂质环境中。纳米盘是尺寸可控的磷脂双层的小块，在过去十年中被开发为洗涤剂的替代品[1，2]，并已成功应用于研究膜蛋白[3]。脉冲偶极光谱（PDS）结合氮氧自由基自旋标记已发展成为一种非常有用的技术，以揭示脂质重构膜蛋白的功能机制[4，5]。 该方法基于测量引入蛋白质中所需位置的半胱氨酸特异性顺磁性标记之间的距离。 尽管PDS成功应用于重构成脂质囊泡的蛋白质，但由于蛋白质占据的样品体积相对较小，PDS确实存在灵敏度问题。这种有限的体积增加了局部蛋白质浓度，并且由于二维空间分布改变了信号，使得难以分离信号的信息部分。结果，需要更长的时间来进行信号平均，并且通常不能测量中等长的距离。最近，对重组成纳米盘的细菌膜转运蛋白进行了PDS研究，并报告了数据质量的大幅改善[6]。鉴于ACERT对膜蛋白的研究给予了高度重视，我们认为开发常规使用的纳米盘对ACERT的核心研究和合作至关重要的膜蛋白的结构和功能的研究。通过将这种方法与提高ACERT PDS仪器的灵敏度相结合，我们预计PDS数据的质量将得到非常显着的改善，并大幅减少数据平均时间，这是提高这一方向效率的重要因素。 [1]的文件。T. H. Bayburt，Y. V. Ginkova和S. G. Sligar，Nano Lett. （2002），2，853-856; [2]. I. G.杰尼索夫，Y. V. Ginkova，A. A. Lazarides和S. G. Sligar，J. Am.（2004），126，3477-87; [3]。T. H. Bayburt，Y. V. Ginkova和S. G.斯利加，弧。生物化学生物物理学（2006），450，215-222; [4] E. R. Georgieva，T. F. Ramlall，P. P. Borbat，J. H. Freed，D. Eliezer，J. Am.（2008），130，12856-12857; [5] P. P. Borbat，K. Surendhran，M. Bortolus，P. Zou，J. H.弗里德，H。S. Mchaourab，Plos Biol.（2007），5，221-2219; [6]. P. Zou和H. S. Mchaourab，Biophys. J.（2010），6，L18-L20.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Membrane proteins contribute 20  30% of all known proteins. However, they present a challenge to the study of their structure and function. X-ray crystallography provides detailed structural information at atomic resolution, but usually membrane proteins has to be crystallized in detergents, which do not faithfully represent the native environment of lipid membranes, destabilize proteins, too much labor is involved in making crystallization attempts, and it is just too difficult or impossible to crystallize certain proteins. Therefore, other methods and approaches are in wide use to study membrane proteins, being in particular true when it comes to the natural lipid environment. Nanodiscs, which are small patches of phospholipid bilayer of controlled size, were developed in the last decade as an alternative to detergents [1, 2] and have been successfully applied to study membrane proteins [3]. Pulsed dipolar spectroscopy (PDS) combined with nitroxide spin-labeling has grown into a highly useful technique to reveal functional mechanisms of lipid-reconstituted membrane proteins [4, 5]. The method is based on measuring distances between cysteine-specific paramagnetic labels introduced into desired position(s) in a protein. Despite its successful application to proteins reconstituted into lipid vesicles, PDS does experiences sensitivity problems due to a relatively small volume of the sample occupied by the protein. This limited volume increases local protein concentration and by the virtue of two-dimensional spatial distribution modifies the signal, making it difficult to isolate the informative part of the signal. In result, longer time for signal averaging is necessary and often moderately long distances cannot be measured. Recently, a PDS study was performed on a bacterial membrane transporter reconstituted into nanodiscs, and large improvement of data quality was reported [6]. Given the fact that ACERT set high priority to the studies on membrane proteins, we consider the development of routine use of lipidic nanodiscs for the studies on structure and function of membrane proteins of utmost importance to ACERT's core research and collaboration. By combining this method with increasing the sensitivity of ACERT PDS instrumentation, we expect very significant improvement of the quality of PDS data and substantial reduction of data averaging time, which are important contributing factors for increasing the efficiency in this direction. [1]. T. H. Bayburt, Y. V. Ginkova, and S. G. Sligar, Nano Lett. (2002), 2, 853-856; [2]. I. G. Denisov, Y. V. Ginkova, A. A. Lazarides, and S. G. Sligar, J. Am. Chem. Soc. (2004), 126, 3477-87; [3]. T. H. Bayburt, Y. V. Ginkova, and S. G. Sligar, Arc. Biochem. Biophys. (2006), 450, 215-222; [4] E. R. Georgieva, T. F. Ramlall, P. P. Borbat, J. H. Freed, D. Eliezer, J. Am. Chem. Soc. (2008), 130, 12856-12857; [5] P. P. Borbat, K. Surendhran, M. Bortolus, P. Zou, J. H. Freed, H. S. Mchaourab, Plos Biol.(2007), 5, 221-2219; [6]. P. Zou and H. S. Mchaourab, Biophys. J.(2010), 6, L18-L20.