Calcium Carbonate Formation by Genetically Engineered Inorganic Binding Peptides

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Calcium Carbonate Formation by Genetically Engineered Inorganic Binding Peptides

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Title: Calcium Carbonate Formation by Genetically Engineered Inorganic Binding Peptides
Author: Gresswell, Carolyn Gayle
Abstract: Understanding how organisms are capable of forming (synthesize, crystallize, and organize) solid minerals into complex architectures has been a fundamental question of biomimetic materials chemistry and biominealization for decades. This study utilizes short peptides selected using a cell surface display library for the specific polymorphs of calcium carbonate, i.e., aragonite and calcite, to identify two sets of sequences which can then be used to examine their effects in the formation, crystal structure, morphology of the CaCO3 minerals. A procedure of counter selection, along with fluorescence microscopy (FM)characterization, was adapted to insure that the sequences on the cells were specific to their respective substrate, i.e., aragonite or calcite. From the resulting two sets of sequences selected, five distinct strong binders were identified with a variety of biochemical characteristics and synthesized for further study. Protein derived peptides, using the known sequences of the proteins that are associated with calcite or aragonite, were also designed using a bioinformatics-based similarity analysis of the two sets of binders. In particular, an aragonite binding protein segment, AP7, a protein found in nacre, was chosen for this design and the resulting effects of the designed peptides and the AP7 were examined. Specifically, the binding affinities of the selected and the protein derived peptides off the cells were then tested using FM; these studies resulted in different binding characteristics of the synthesized and cellular bound peptides . Two of the peptides that displayed strong binding on the cells bound to neither of the CaCO3 substrates and both the high and low similarity protein-derived peptides bound to both polymorphs. However, two of the peptides were found to only bind to their respective polymorph showing; these results are significant in that with this study it is demonstrated that the designed peptides based on experimental library-based selection and sequence identification, can be designed to have recognition capability to a given crystal structure, specifically, in this case, of calcium carbonate. Calcite mineralization with the peptides produced vaterite when several of the peptides were used in the synthesis process, many having unique morphologies studied using scanning electron microscopy (SEM). The amount of vaterite crystal percentage in these biomineralized mixtures was calculated and it was found to be closely related to peptide concentration for the aragonite-binding peptides. In the aragonite mineralization experiments, a separate solid phase, namely, calcium nitrate hydrate, was produced for one of the peptides along with the other polymorphs of calcite carbonate (ie., aragonite, calcite and vaterite) in the solution in the form of a flat film. These biomineralization results are examined in the light of the effects of peptide sequences and their related solid-binding characteristics
Description: Thesis (Master's)--University of Washington, 2012
Author requested restriction: No embargo

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