DENNIS POST-DOC/TECHNICIAN SUPPORT

这个子项目是许多研究子项目中的一个 由NIH/NCRR资助的中心赠款提供的资源。子项目及 研究者（PI）可能从另一个NIH来源获得了主要资金， 因此可以在其他CRISP条目中表示。所列机构为 研究中心，而研究中心不一定是研究者所在的机构。 该提案实现了AREA计划的目标：1）支持有价值的研究; 2）使本科生接触研究; 3）加强非研究密集型大学的研究环境。本研究的目的是 阐明聚羟基烷酸酯包合物的生物成因机制。电子显微镜的研究一直无法解决PHA包涵体的结构，这抑制了运动的包容物生物成因的凝聚力模型。采用原子力显微镜，我们已经确定有三层结构，外层是膜双层的厚度，中间的网络层， 下面的结晶层状层。遗传学研究表明，中间网络至少部分由PhaP组成，并且PhaP可能易位到周质。因此，似乎包裹体生物成因可能发生， 蛋白质和/或蛋白质向周质的移动，并通过细胞质膜出芽进入细胞质，促进细胞质膜作为包膜的获得。这项研究的目的是证明或反驳这一假设。本研究的具体目的是：1）明确证明周质 通过荧光定位和亚细胞级分的蛋白质印迹分析来定位PhaP，2）通过蛋白质组学分析证明包涵体包膜来源于细胞质膜，和3）表征瞬时和永久结合PhaP的蛋白质，希望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涵体生物发生的了解，使这个过程可以控制并用于医学应用。例如，可以设想，代替聚合物插入到包合物中，可以插入生物活性化合物，使包合物成为药物递送载体。

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. 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.