Molecular Characterization of Key Proteins Directing Otoconial Mineralization

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DESCRIPTION (provided by applicant): Otoconia are biomineral particles of microscopic size essential for perception of gravity and maintenance of balance. Millions of older Americans are affected in their mobility, quality of life, and in their health by progressive demineralization of otoconia. Currently, no effective means to prevent or counteract this process are available. Because of prohibitive anatomical and biological constraints, otoconial research is lagging far behind other systems, such as bone and teeth. We have overcome the major obstacles to rigorous otoconial research by generating recombinant versions of the principal matrix proteins, Otoconin 90 and 22, as well as Otolin 1. The proteins were evaluated in vitro for their effects upon calcite crystal growth parameters, including nucleation, growth rates and morphology, and combined with biophysical solution state studies, which provided essential, but limited structural and mechanistic insights. Our results to date combined with those of other researchers suggest three key concepts to be pursued: 1) Tertiary structure matters because it juxtaposes distant residues to create a stereochemical relationship between protein and mineralizing species that could not come about from a linear chain or a random coil (i.e. primary structure). 2) Otoconia are mesocrystals and therefore it is necessary to understand their role in stabilizing nanoparticles (that may be amorphous) and/or mediating aggregation. 3) The fibrillar Otolin 1 core of otoconia indicates a role for an insoluble organic matrix in directing the initial stages of mineralization. Thus we have now arrived at a stage that demands more advanced tools capable of precise quantitative control over growth conditions and evaluation of growth kinetics and underlying mechanisms at a molecular level. For this purpose we joined forces with J. DeYoreo, a leading expert in crystal growth science who has established the most powerful molecular scale technology. This team will perform the advanced molecular level studies for which we will provide the necessary high quality normal and mutated protein and constructs. These will be validated exhaustively by biochemical and biophysical studies, complemented by standard crystal growth determinations, prior to submitting them to molecular level studies. The joint team is expected to arrive at essential mechanistic insights, enabling us to design and implement measures to prevent, slow down or ameliorate otoconial degeneration.