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

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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.