Foldase Catalyzed Protein Folding and Molecular Biology Workshops for K-12 Students

9631485 noiva蛋白二硫键异构酶(Pdi)是一种位于粗面内质网管腔内的“折叠酶”，它促进了分泌蛋白和膜蛋白的折叠、组装和翻译后修饰所需的几个反应。PDI分子上的一个识别未折叠和错误折叠的新生多肽的位点已经被发现，并被证明参与了PDI促进的折叠。在这项提案中，这个肽结合位点在PDI活性中的作用将进一步表征。寡核苷酸定点突变技术将被用来产生缺乏多肽结合位点或含有该位点单一氨基酸取代的PDI突变体。野生型和突变型PDI将在大肠杆菌和毕赤酵母表达系统中表达，以产生最高产量的酶。肽结合活性的丧失对PDI活性的影响将通过狗胰腺微粒体中可溶性酶的活性及其催化正确折叠蛋白质的能力来评估。初步证据表明，PDI很少或没有底物序列偏好，但假设识别多肽链上的6个氨基酸。对PDI折叠酶活性的底物偏好将通过结合使用定点突变、亲和层析和亲和平移技术来确定PDI结合多肽的长度和序列要求来进一步表征。钙在细胞内PDI功能中的作用也将被评估。在体外，钙对PDI促进的折叠或肽结合活性没有任何直接影响，但可能参与了PDI在粗面内质网腔的定位。这种钙介导的PDI定位将使用脉冲追逐标记、蛋白质印迹和共聚焦显微镜技术进一步表征。将为K-12学生和教师举办一系列关于实验生物学现代技术的暑期工作坊。工作坊将由一系列实验模块组成，介绍现代分子生物学实验室中使用的技术，并将根据初中生/高中生的水平量身定做。单元将根据学生的能力和先前的经验在工作坊之间互换。这些研讨会将使用由霍华德·休斯医学研究所资助的最近建成的分子生物学实验室教学设施。目前的技术允许科学家使用细菌培养来生产具有临床和制药重要性的重组人类蛋白质。然而，细菌缺乏有效折叠这些哺乳动物蛋白质所需的伴侣蛋白和折叠酶蛋白。在这个方案中，蛋白质二硫键异构酶(PDI)，一种帮助人类蛋白质折叠的折叠酶，将被研究。为了帮助蛋白质折叠，PDI必须识别新合成的蛋白质上的一个基序，该基序识别该蛋白质需要帮助才能正确折叠。在本提案中，将描述PDI与蛋白质结合在辅助折叠中的作用以及PDI识别的基序。此外，细胞内钙在将PDI引导到细胞内发生蛋白质折叠的区域中的作用也将被研究。这些信息将加强我们对PDI细胞功能的理解，并导致更好地利用PDI在细菌中提高重组人蛋白的产量。将为K-12学生和教师举办一系列关于实验生物学现代技术的暑期工作坊。这些讲习班将为来自美洲原住民保留地和教育资源有限的农村社区的学生提供机会，使他们能够接触到用于实验生物学的最新技术。将鼓励学生学习分子水平的生物技术，如电泳法、聚合酶链式反应和克隆重组DNA。S在研讨会上提供的实验模块将加强各自学校的科学，展示令人兴奋的新概念，并有望激励学生在生物学和生物医学科学领域寻求更多机会。***

9631485 Noiva Protein disulfide isomerase (PDI) is a "foldase" located in the lumen of the rough endoplasmic reticulum where it facilitates several of the reactions required for the folding, assembly, and posttranslational modification of secretory and membrane proteins. A site on the PDI molecule which recognizes unfolded and misfolded nascent polypeptides has been identified and demonstrated to be involved in PDI-facilitated folding. In this proposal, the role of this peptide binding site in PDI activity will be further characterized. The technique of oligonucleotide-directed site mutagenesis will be used to generate mutants of PDI lacking the peptide binding site or containing single amino acid substitutions in that site. Wild type and mutant PDI will be expressed in both E. coli and Pichia pastoris expression systems to generate the highest yield of enzyme. The effect of loss of peptide binding activity on PDI activity will be assessed on both the activity of the soluble enzyme and its ability to catalyze properly folded proteins in dog pancreas microsomes. Preliminary evidence indicates that PDI has little or no substrate sequence preference, but is hypothesized to recognize a stretch of 6 amino acids in the polypeptide chain. Substrate preferences for the PDI foldase activity will be further characterized by determining length and sequence requirements for peptide binding by PDI using a combination of site-directed mutagenesis, affinity chromatography, and affinity panning techniques. The role of calcium in PDI function in the cell will also be assessed. Calcium does not have any direct effect on PDI-facilitated folding or peptide binding activity in vitro, but may be involved in localization of PDI to the lumen of the rough endoplasmic reticulum. This calcium-mediated localization of PDI will be further characterized using the techniques of pulse-chase labeling, western blotting, and confocal microscopy. A series of summer workshops in Modern Techniques in Experimental Biology for K-12 students and teachers will be developed. Workshops will be composed of a series of experimental modules presenting techniques used in the modern molecular biology laboratory and will be tailored to the level of junior/senior high school students. Modules will be interchanged between workshops depending on the abilities and prior experiences of the students. The workshops will use a recently completed Molecular Biology Laboratory teaching facility funded by the Howard Hughes Medical Institute. %%% Current technology allows scientists to use bacterial cultures to produce recombinant human proteins of clinical and pharmaceutical importance. However, bacteria lack chaperone and foldase proteins required to fold these mammalian proteins efficiently. In this proposal, protein disulfide isomerase (PDI), one of the foldases which assists protein folding in humans, will be studied. In order to assist proteins in folding, PDI must recognize a motif on the newly synthesized protein which identifies that protein as requiring assistance for proper folding. In this proposal, the role of protein binding by PDI in assisted folding and the motif that is recognized by PDI will be characterized. Furthermore, the role of intracellular calcium in directing PDI to the area in cells where protein folding occurs will also be studied. This information will strengthen our understanding of the cellular function of PDI and lead to the better use of PDI in improving yields in the production of recombinant human proteins in bacteria. A series of summer workshops in Modern Techniques in Experimental Biology for K-12 students and teachers will be developed. These workshops will provide opportunities for students from Native American reservations and rural communities with limited educational resources to have access to current technology used in experimental biology. Students will be encouraged to learn biological techniques at the molecular level such as electrophoresis, PCR, and cloning recombinant DNA. Experimental module s offered at the workshop will reinforce science at their own schools, present new and exciting concepts and hopefully motivate students to pursue further opportunities in Biology and the Biomedical Sciences. ***