ANALYSIS OF POLYHYDROXYALKANOATE INCLUSION BIOGENESIS

这个子项目是利用资源的许多研究子项目之一。 由NIH/NCRR资助的中心拨款提供。对子项目的主要支持 子项目的首席调查员可能是由其他来源提供的， 包括美国国立卫生研究院的其他来源。为子项目列出的总成本可能 表示该子项目使用的中心基础设施的估计数量， 不是由NCRR赠款提供给次级项目或次级项目工作人员的直接资金。 这项建议实现了以下领域的计划目标：1)支持有价值的研究；2)让本科生接触研究；3)加强非研究密集型大学的研究环境。这项研究的目标是 目的：阐明聚羟基烷酸包合物的生物形成机制。电子显微镜研究一直无法解析PHA包裹体的结构，这阻碍了包裹体生物发生的内聚模型的发展。利用原子力显微镜，我们确定了它有三层结构，外层是膜双层的厚度，中间网络层，以及 下面是一层水晶层。遗传学研究表明，中间网络至少部分由Phap组成，Phap很可能被移位到周质。因此，似乎包裹体生物发生可以通过以下方式发生 蛋白质和/或蛋白质向周质移动，并通过细胞质膜发芽进入细胞质，促进细胞质膜作为包膜的获得。本研究的目的是证明或反驳这一假设。研究的具体目的是：1)明确证明周质 通过亚细胞组分的荧光定位和Western印迹分析对Phap进行定位，2)蛋白质组学分析证明包涵体包膜来自细胞膜，3)鉴定与Phap瞬时和永久结合的蛋白质，以期阐明包涵体生物发生的机制。最终，这项研究的目标是扩大我们对包裹体生物发生的知识，使这一过程可以控制并用于医疗应用。例如，可以设想，不是将聚合物插入包合物，而是可以插入生物活性化合物，使包合物进入药物输送载体。

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. This proposal accomplishes the AREA program objectives of: 1) supporting meritorious research; 2) exposing undergraduates to research; and 3) strengthening the research environment in non-research intensive universities. The goal of this research is to elucidate the mechanism of polyhydroxyalkanoate inclusion biogenesis. Electron microscopy studies have been unable to resolve the structure of PHA inclusions and this has inhibited movement toward a cohesive model of inclusion biogenesis. Employing atomic force microscopy, we have determined that there are three layers of structure, an outer envelope that is the thickness of a membrane bilayer, a middle network layer, and an underlying crystalline lamellar layer. Genetic studies have indicated that the middle network is comprised at least partially of PhaP and that PhaP is likely to be translocated to the periplasm. Thus, it would appear that inclusion biogenesis may occur by movement of protein and/or proteins to the periplasm and budding through the cytoplasmic membrane into the cytoplasm, facilitating the acquisition of the cytoplasmic membrane as an envelope. The goal of this research is to prove or disprove this supposition. The specific aims of the research are: 1) definitively prove periplasmic localization of PhaP via fluorescence localization and Western blot analyses of subcellular fractions, 2) demonstrate that the inclusion envelope is derived from the cytoplasmic membrane by proteomic analysis, and 3) characterize proteins that bind transiently and permanently to PhaP in hopes of elucidating the mechanism of inclusion biogenesis. Ultimately, the goal of the research is to enlarge our knowledge of inclusion biogenesis to the point that this process can be controlled and utilized for medical applications. For instance, it could be envisioned that instead of polymer being inserted into the inclusion, bioactive compounds could be inserted, making the inclusion into a drug delivery vehicle.