Tamara L. Kinzer-Ursem - US grants

Project Summary/Abstract 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.

Purdue University Molecular Biophysics Training Program PROJECT SUMMARY In 2015, the Zika virus outbreak emerged as an international public health crisis. In less than a year, scientists at Purdue University published the structure of the mature Zika virus capsid, providing immunologists and drug discovery experts with a sophisticated molecular understanding of how to rationally develop selective anti-viral agents. This remarkably fast transition from the discovery of a novel disease to an atomic model was made possible by a cutting-edge electron microscopy facility, a deep understanding of the theory and application of biophysics, and a diverse team of researchers spearheaded by a well-trained predoctoral student. Successful molecular biophysics training programs will instill upon its trainees many of the same qualities that made this example so effective. To this end, the mission of the Purdue University Molecular Biophysics Training program is to bring together 27 preceptors from six different departments to train an outstanding cohort of graduate students in the underlying theory and practice of cutting-edge biophysical techniques. The program's chief objectives are: (i) to provide enhanced training in the application of molecular biophysics in a rigorous and reproducible way to modern problems in human health and disease, (ii) to foster effective and inclusive teamwork, and (iii) to provide career development opportunities tailored to the goals of individual trainees. To achieve these objectives, selected trainees (6 in each year of the award, typically beginning in their 2nd year of study and supported for up to 2 years) will take a new two-semester gateway class that merges theory with team- based project design and implementation, participate in and help implement an interdepartmental biophysics seminar series that will showcase student-invited external speakers and trainee research on campus, and plan, develop, and implement Purdue's annual biophysics symposium called the Hitchhiker's Guide to the Biomolecular Galaxy. Examples of key activities that support the professional development of these trainees are exercises in teamwork built into program coursework and symposium planning, active development and implementation of detailed individual development plans reviewed and revised annually in collaboration with the mentor, personalized teaching and/or internship opportunities, a grant-writing class tailored to biophysical topics, training in the responsible conduct of research, and participation in the recruitment and retention of underrepresented and/or disabled students. By leveraging Purdue's expertise in Biology Education and self- assessment, the training program and its individual activities will be evaluated annually and refined to ensure that the program is meeting its objectives and the needs of the scientific community.