Investigation of the Transport of Nitric Oxide by Myoglobin Using Sol-Gel Glass Encapsulation

尽管对一氧化氮（NO）的生理学研究越来越深入，但关于一氧化氮在生物系统中的寿命和传递的一些基本和基本问题仍然未知。本研究旨在验证肌红蛋白（Mb）促进NO向平滑肌细胞方向扩散的假设，并可开发一种溶胶-凝胶包封方法对其进行检测。这是基于P.I.对Mb输送氧的独特特性及其相关生理条件的新发现。目的是确定Mb是否在与生理条件相似的特定条件下运输NO，重点关注脱氧肌红蛋白（deoxyMb）通过亚硝基肌红蛋白的形成运输NO，而不是NO与氧基肌红蛋白的相互作用。deoxyMb在促进运输中的作用涉及在deoxyMb存在的情况下，NO以快速的速度从一个位置移动到另一个位置，O2的运输也是如此。与结合和解离不同，研究NO的运输需要考察脱氧分子对NO的空间运动。正如O2输运所证明的那样，溶胶-凝胶包封方法不仅提供了一种简单有效的确定空间运动的手段，而且其实验条件可以很容易地调整为生理的。这项工作可能会确定Mb（可能还有其他血红蛋白）在控制和指导NO相关生物效应方面的另一个基本和重要作用，从根本上改变了NO生理学的看法，即指导而不是随机。由于NO化学在许多生物过程中的重要性，Mb在NO运输中发挥重要作用的证据将产生直接、深刻和广泛的影响。一氧化氮（NO）最近被发现是一种内源性产生的分子，具有多种生物活性，例如在心血管系统，大脑和免疫系统中。作为一种非极性的小双原子物质，NO可以从其生物合成位点自由扩散。由于NO的生物靶标（例如，鸟苷酸环化酶）相对于NO的生物合成位于不同的和明确的位置，如果允许NO自由扩散，只有少量的生物合成的NO会到达它的靶标或受体，导致难以置信的生物合成努力的浪费（NO的生物合成是一个多步骤的过程，需要使用NADPH）。这与自然的简约性明显的不一致表明NO的扩散不可能是自由的，而是朝着目标的方向被促进。本研究旨在通过一种新的检测方法，即溶胶-凝胶包封法，探讨一氧化氮在血红蛋白肌红蛋白中的扩散机制。这项研究的结果可能会导致对肌红蛋白和生物运输的作用有更深入的了解。*** 9604680 Fane病毒蛋白和核酸正确组装成具有生物活性的病毒粒子涉及许多不同的大分子相互作用。主要目的是阐明这些关键的相互作用，并定义负责大分子的结构域。遗传学、生物化学和结构方法（x射线晶体学）的结合将被用来实现这些目标。像分子伴侣一样，支架蛋白指导其他蛋白质获得适当的三维构象。在这种类比的背景下，原衣壳的原子结构提供了一种与底物复合的伴侣样蛋白质的描述。先前的研究结果表明，微病毒科内部支架蛋白（gpB）与分子伴侣蛋白具有许多相同的特性。先前的结果还表明，内部支架蛋白要么具有固有的灵活性，要么以非特异性的方式与底物相互作用，可能通过界面。确定含有外源支架蛋白的杂化原壳的原子结构，将直接解决这个问题。目前的X174原衣壳结构不含在纯化过程中丢失的内部支架蛋白。在支持的第一年，将探索替代的遗传和纯化策略来稳定这种蛋白质。目前的方案仍将用于收集额外的数据，以完善外部支架蛋白结构。同样在第一年，产生α原囊体所需的遗传和纯化策略将被开发出来。与m·g·罗斯曼博士合作进行的结构工作将在整个支助期间继续进行。最近发现的外部支架蛋白gpD的原子结构表明，它与分子伴侣蛋白有许多共同的特征。因此，像分子伴侣一样，gpD可以以许多不同的方式与其他蛋白质结合。虽然一些结构域以不同的方式与外壳蛋白接触，但其中一个结构域可能决定蛋白质对特定病毒外壳蛋白的底物特异性。基于质粒的交叉互补系统已扩展到该基因。与内部支架蛋白在一级结构上存在很大差异不同，外部支架蛋白具有75%的序列同一性。然而，（X174）蛋白不能有效地指导其他微病毒科病毒粒子的组装。初级结构的比较表明，不同的残基定位于蛋白质的NH2末端，在X174原子结构中形成一个大的螺旋。这些观察结果提示了一个模型，其中支架对特定病毒外壳蛋白的特异性驻留在该区域。这一假设将用嵌合多肽进行验证。在第一年的支持中，将引入构建嵌合外部支架蛋白所需的限制性位点。（X174）基因将被重新克隆，其他微病毒科D基因的克隆将被生成。嵌合基因和基因产物的表征将在第二年开始。这些分析的结果也可能为重组蛋白的设计提供见解。所有的病毒都必须通过多种蛋白质的相互作用来组装自己。病毒组装通常依赖于被称为支架蛋白的蛋白质。类似于建筑中使用的支架，支架蛋白存在于病毒组装中间体中，但不存在于成熟病毒中。两种不同的支架蛋白，外部和内部，是组装微病毒科病毒所需的。有了这些病毒，我们就能够提纯病毒中间产物，其中仍然包括外部支架蛋白。通过检查外部支架蛋白的原子结构，并对内部和外部蛋白进行遗传分析，我们确定了支架蛋白的不同区域可能具有特定的可识别的功能。完善这些蛋白质的原子结构，并通过构建混合微病毒科支架蛋白来测试我们关于其各种功能的假设是本研究的主要目标。这些分析的结果将为病毒组装和重组蛋白的设计提供进一步的见解。

9603899 Fukuto In spite of the intensive studies on the physiology of nitric oxide(NO), some basic and fundamental questions regarding the lifetime and delivery of NO in biological systems remain unknown. This study is designed to test the hypothesis that myoglobin(Mb) facilitates diffusion of NO in the direction of smooth muscle cells, and a sol-gel encapsulation methodology can be developed for its detection. This is based on new findings by the P.I. on the unique characteristics of O2 transport by Mb, and its associated physiological conditions. The objective is to determine whether Mb transports NO under selected conditions similar to physiological ones, with focus on the transport of NO by deoxymyoglobin (deoxyMb) via nitrosylmyoglobin formation, but not the interaction of NO with oxymyoglobin. The proposed role of deoxyMb in facilitating transport involves the spatial movement of NO from one location to another in the presence of deoxyMb at a rapid rate, as is O2 transport. Unlike the binding and dissociation, the investigation of the transport of NO requires the examination of spatial movement of NO by deoxyMb molecules. As demonstrated with the O2 transport, the sol-gel encapsulation methodology not only provides a simple and effective means for determining spatial movement, but also its experimental conditions can be readily adjusted to physiological. This work may define another fundamental and vital role for Mb (and possibly other hemoproteins) in controlling and directing the biological effects associated with NO, fundamentally changing the way in which NO physiology is viewed, i.e. directed instead of random. Because of the importance of NO chemistry in numerous biological processes, evidence for Mb to play a major role in NO transport will have an immediate, profound, and broad impact. Nitric oxide (NO) has recently been found to be an endogenously generated molecule with a diverse array of biological activities, such as in the cardiovascular, the brain and the immune systems . As a non-polar, small diatomic species, NO can freely diffuse from its site of biosynthesis. Since the biological targets for NO (for example, the enzyme guanylate cyclase) are located in distinct and defined locations relative to NO biosynthesis, if NO were allowed to freely diffuse, only a small amount of the biosynthesized NO would ever reach it's target or receptor, resulting in an incredible waste of biosynthetic effort (NO biosynthesis is a multi-step process which requires the use of NADPH). This apparent inconsistency with the parsimony of Nature suggests that NO diffusion is unlikely to be free, but rather is facilitated towards the direction of its targets. This proposal aims to address the mechanism of facilitated diffusion for NO by the hemeprotein myoglobin by a new detection method, using sol-gel encapsulation. The results derived from this study may lead to a greater understanding of the role of myoglobin and of biological transport. *** 9604680 Fane The proper assembly of viral proteins and nucleic acids into a biologically active virion involves numerous and diverse macromolecular interactions. The main objectives are to elucidate these critical interactions and to define the structural domains of the responsible macromolecules. A combination of genetic, biochemical and structural approaches (X-ray crystallography) will be employed to accomplish the objectives. Like molecular chaperones, scaffolding proteins direct other proteins in achieving their proper three-dimensional conformations. Within the context of this analogy, the atomic structure of a procapsid offers a depiction of a chaperone-like protein complexed with its substrate. Prior results suggest that the Microviridae internal scaffolding proteins, gpB, share many properties with molecular chaperones. Prior results also indicate that the internal scaffolding proteins either possess inherent flexibility or interact with their substrates in nonspecific manners, perhaps via interfaces. Determining th e atomic structures of hybrid procapsids, containing foreign scaffolding proteins, will directly address this question. The present (X174 procapsid structure does not contain the internal scaffolding protein which is lost during purification. During the first year of support, alternate genetic and purification strategies will be explored to stabilize this protein. The current protocols will still be employed to gather additional data to refine the external scaffolding protein structure. Also within the first year, the genetic and purification strategies needed to generate alpha procapsids will be developed. Continuation of the structural work, done in collaboration with Dr. M. G. Rossmann, will proceed throughout the support period. The recently solved atomic structure of the external scaffolding protein, gpD, suggests that it also shares many features with molecular chaperones. Thus gpD, like a molecular chaperone, can bind to other proteins in many different ways. While some domains make contact with the coat protein in varied manners, one of these domains may determine the protein's substrate specificity for a particular viral coat protein. the plasmid-based cross complementation system has been extended to this gene. Unlike the internal scaffolding proteins which exhibit a great deal of divergence in primary structure, the external scaffolding proteins share 75% sequence identity. The (X174 protein, however, is unable to productively direct the assembly of other Microviridae virions. A comparison of the primary structure reveals that the divergent residues are localized to the NH2 -termini of the proteins which forms a large (-helix in the (X174 atomic structure. These observations suggest a model in which the scaffolding's specificity for a particular viral coat protein resides in this region. This hypothesis will be tested with chimeric polypeptides. During the first year of support, restriction sites needed to construct chimeric external scaffolding proteins will be intro duced. The (X174 gene will be recloned and clones of other Microviridae D genes will be generated. Characterization of the chimeric genes and gene products will commence in the second year. The results of these analyses may also provide insights into the design of recombinant proteins. All viruses must assemble themselves by means of multiple protein interactions. viral assembly is often dependent on proteins known as scaffolding proteins. Analogous to scaffoldings used in the construction of buildings, scaffolding proteins are found in virus assembly intermediates but not in the mature viruses. Two different scaffolding proteins, external and internal, are required for the assembly of the Microviridae family of viruses. With These viruses we are able to purify viral intermediates which still include the external scaffolding protein. By examining the atomic structure of the external scaffolding protein and performing genetic analyses with both the internal and external proteins, we have determined that different regions of the scaffolding proteins may have specific and identifiable functions. Refining the atomic structure of these proteins and testing our hypotheses regarding their various functions by constructing hybrid Microviridae scaffolding proteins are the main objectives of the proposed work. The results of these analyses will provide further insights into virus assembly and the design of recombinant proteins.