EFFLUX-MEDIATED RESISTANCE TO TETRACYCLINES

描述（改编自申请人的摘要）：外排已经成为 越来越多地被认为是对抗生素和其他 自从研究人员发现四环素外排蛋白泰特以来， 大约20年前。今天，许多泰特外排蛋白（与原发性 序列），其具有两半，每半六个α-螺旋 跨膜结构域，由细胞质环分隔，并指定交换 四环素/阳离子复合物的质子。主动外排是两种 四环素耐药的主要机制，减少了临床 这是一个重要的抗生素家族。在校长的研究 研究人员的实验室专注于生物化学和分子基础， 由Tn 10指定的43 kD B类泰特蛋白流出。蛋白质被 在洗涤剂中纯化为多组氨酸融合物。近日有 成功地进行了2D分析。本建议旨在确定 通过结晶方法分析TetA蛋白的三级结构， 结合2D和3D的努力，通过半胱氨酸交联的定点 半胱氨酸突变残基，并通过基因分析抑制突变。中 第二个目标，他们将追求的发现，牵连域间循环， 泰特蛋白的功能，使用环和半胱氨酸的诱变 交联研究，以检查环如何与其他结构域的相互作用， 泰特蛋白。第三个目标将继续他们对放射性标记的研究。 光亲和性四环素类似物，以确定底物结合区域， 泰特蛋白。这种方法将得到使用铁作为 四环素流出的阳离子和产生肽的芬顿反应 在蛋白质中底物结合位点的键断裂。这些研究追求 进一步阐明了药物外排蛋白泰特反向转运的分子基础。 对泰特蛋白理解的进展将增强我们对以下方面的认识： 转运蛋白主要促进剂家族中的其他类似蛋白质 这将在感染性疾病的药物治疗领域产生广泛的影响， 疾病和癌症。

DESCRIPTION (Adapted from the Applicant's Abstract): Efflux has become increasingly recognized as a mechanism of resistance to antibiotics and other drugs since the investigators' discovery of the tetracycline efflux protein Tet some 20 years ago. Today, a number of Tet efflux proteins (related by primary sequence) have been described which have two halves, each six alpha-helical transmembrane domains, separated by a cytoplasmic loop and specify an exchange of a tetracycline/cation complex for a proton. Active efflux is one of two major mechanisms of tetracycline resistance which have curtailed the clinical effectiveness of this important family of antibiotics. Studies in the Principal Investigator's laboratory focus on the biochemical and molecular basis of efflux by the 43kD class B Tet protein specified by Tn10. The protein has been purified in detergent as a polyhistidine fusion. Recently, it has been successfully crystallized for 2D analysis. This proposal seeks to determine the tertiary structure of the TetA protein through crystallization methods, combining 2D and 3D efforts, by cysteine cross-linking of site-directed cysteine mutant residues, and by genetic analysis of suppressor mutations. In a second aim, they shall pursue findings that implicate the interdomain loop in the function of the Tet protein, using mutagenesis of the loop and cysteine cross-linking studies to examine how the loop interacts with other domains of the Tet protein. A third aim will continue their work on radiolabeled photoaffinity tetracycline analogs to identify the substrate-binding region in the Tet protein. This approach will be complemented by studies that use iron as the cation for tetracycline efflux and the Fenton reaction to produce peptide bond cleavage at substrate binding sites in the protein. These studies pursue further the molecular basis for antiport by the drug efflux protein Tet. Advances in understanding of the Tet protein will enhance our knowledge of other, similar proteins in the major facilitator family of transport proteins which should have broad impact in the area of drug treatment of infectious disease and cancer.