CAREER: Molecular Assemblies of Multidomain Signaling Protein on Membrane Surface

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Many proteins that carry out critical life processes are modular in structure, i.e., composed of multiple independent domains, and the intra- and intermolecular interactions among these domains determines function. Often, changes in these structural organization that leads to activation occurs at the cellular membrane, which remains experimentally inaccessible. This project will develop a technology that can elucidate the molecular interactions on membrane surfaces using Bruton’s tyrosine kinase (Btk) as a model system. The project will develop experimental tools for elucidating structural details for biomolecular assemblies on membranes, as well as the understanding of Btk and other similar multidomain proteins. Integrated with this research strategy, the education plan seeks to instill the concepts of microscopy and single-molecule imaging in the public, in collaboration with the Colorado State University Spur Campus. The plan is based on the idea that aesthetic appreciation for scientific imageries can facilitate the understanding and affinity towards science. Artistic actives that combine natural concepts will be incorporated into creative activities in which artistic techniques illustrate the concepts.Bruton’s tyrosine kinase (Btk), a key signaling protein in both innate and adaptive immunity, is a multidomain protein that maintains an autoinhibited state at rest but undergoes a dramatic structural change upon membrane recruitment where it becomes active. However, it is very difficult to study membrane-specific structural changes and molecular interaction such as oligomerization or clustering that can play a critical role in the functional outcome of cellular signaling. In this project, synthetic membrane reconstitution will be combined with quantitative spectroscopic methods to systematically probe the membrane-specific supramolecular assemblies of Btk, with the following specific objectives: (1) determine the multimerization state of the full-length Btk on membranes, and the specific molecular interactions responsible for the multimerization, (2) evaluate the molecular assembly’s impact on Btk’s activation and catalytic activity, and (3) identify the material state of the macroscopic Btk lipid binding domain clusters. Overall, the project will advance our knowledge in cellular signaling, particularly those with emergent properties specific to cellular membranes.This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.