In normal learning and memory, dynamic changes in the strength of synaptic connections (called synaptic plasticity) are brought about through exquisite coordination of neurotransmitter release, protein synthesis, protein localization and cytoskeletal reorganization. The timing, magnitude, and location of these processes are determined by protein binding and enzyme activation events within protein signaling networks. In many neurological disorders the spatial and temporal regulations of these protein interactions are disrupted. Thus, in order to effectively design treatments for these complex disorders, detailed information about the spatial organization of protein signaling molecules is absolutely necessary. Current experimental paradigms of qualitative studies with knock-down, overexpression or mutation of particular proteins in mutant animals or in cell lines alone are not adequate to advance our knowledge to the necessary level of mechanistic detail. To address this gap, we are simultaneously developing: 1) a protein labeling technique that is site-specifically and covalently tags a protein with click chemistry functionality and 2) a novel non-fouling, click chemistry- functionalized transmission electron microscopy (TEM) grid coating. The grid coating will enable selective covalent capture of the tagged protein alone and in complex with its interacting proteins onto TEM grids for cryo-EM imaging. This allows to fine control over the reaction, wash, and incubation conditions that the proteins are subjected to, thus allowing control over i) the state of activation of the protein of interest, ii) the surface deposition of the protein, and iii) the binding of the protein with its associated proteins. In addition the TEM grid coatings are non-fouling and thus minimize non-specific binding interactions that would otherwise obscure protein complex identification. Direct imaging of the complexes will be performed using cryo-EM; this maintains proteins in their naturally hydrated state and allows for large protein complexes to be imaged at high resolution. Single particle analysis will be performed to reconstruct the complexes to sub-nanometer resolution.
(Relevance) Dynamic changes in the strength of neuronal connections underlie learning and memory function, and when disrupted, constitute the molecular basis of many neurological disorders and psychiatric diseases. In this project we will develop a novel technique with which high-resolution images of neuronal protein complexes can be reliably and reproducibly obtained. These techniques will allow for future investigations of the structure of protein complexes under conditions that mimick both normal and disease conditions. Thus our finding will provide new insights into how to mediate disruptions in protein complexes that contribute to cognitive dysfunction.
|Fraseur, Julia G; Kinzer-Ursem, Tamara L (2018) Next generation calmodulin affinity purification: Clickable calmodulin facilitates improved protein purification. PLoS One 13:e0197120|
|Kinzer-Ursem, Tamara (2017) Relieving the Pressure on Tissue Development. Biophys J 113:360-361|
|Benjamin, Christopher J; Wright, Kyle J; Bolton, Scott C et al. (2016) Selective Capture of Histidine-tagged Proteins from Cell Lysates Using TEM grids Modified with NTA-Graphene Oxide. Sci Rep 6:32500|