Sunday, May 18, 2025
5/18/2025
Clonable Nanoparticles - PROJECT SUMMARY The objective of this proposal is to address the contrast problem in cellular electron microscopy. The cloneable fluorophore, Green Fluorescent Protein (GFP), and related fluorescent proteins complement small molecule stains and dyes to essentially solve the contrast problem in optical imaging. For cellular electron microscopy, however, contrast options are limited and there are no widely used cloneable contrast agents. Cloneable contrast in electron microscope imaging of biology can arise from a `cloneable inorganic nanoparticle (cNP).' A cNP is an NP made by a protein. The protein determines the properties of the nanoparticle such as elemental composition and shape. Because protein sequence, structure and function are encoded in DNA, the properties of the nanoparticle are also encoded in DNA. Modifications to DNA encoding a cNP may modify the resulting cNP. Our cNPs are based on inorganic ion oxidoreductase enzymes. Such enzymes select for and reduce inorganic biocoordination complexes, creating metal(loid) nanoprecipitates. Additional proteins/peptides (fused to the enzyme) act as ligands, influencing size, morphology, et cetera of the nanoparticle. DNA encoding the cNP can be concatenated to DNA encoding any protein of interest. Resulting protein chimeras contain an integral inorganic nanoparticle. The nanoparticle contrast allows the protein of interest to be identified against cellular background in electron micrographs. We have developed a cloneable selenium nanoparticle (cSeNP). We demonstrated cSeNP molecular labeling of FtsZ filaments in E. coli. The goal of this proposal period is to continue evaluation of this cSeNP and produce additional distinguishable cNPs. The proposal proceeds in 3 aims. Aim 1 is to evaluate the cSeNP in Drosophila, as a multicellular model organism, more complex than E. coli. Aim 1 also proposes to label ribosomal protein L1 in Caulobacter with the cSeNP and other cNPs. Because ribosomes can be unambiguously identified in electron cryo-tomograms, they provide an internal-standard for evaluation of cNPs. We will determine efficiency of cNP constructs by assessing what percentage of ribosomes have an associated cNP label. This assessment identifies shortcomings in cNPs, informing their subsequent refinement. Ideally, cNPs will emerge that label quantitatively. Aims 2 and 3 engineer different aspects of cNPs. Aim 2 uses protein design tools to minimize cNP protein size. Aim 2 also proposes directed evolution of cNP enzymes to produce cNPs of novel elemental composition. Aim 3 is to expand the palette of biomolecules that ligate cNPs, influencing their resulting size and morphology. Aim 3 will identify proteins within the protein coronas that form around cNPs, followed by identification of any serendipitous nanoparticle binding domains. Aim 3 also proposes to isolate nanoparticle binding peptide ligands through ribosome display approaches. Overall, this proposal is to develop and evaluate a pipeline of many size/shape/morphology distinguishable cNPs, facilitating multiplexed protein imaging in cellular electron microscopy.
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