Grant Duration
1/24 – 12/25

Overview: This project sought to define the structural and functional mechanisms of the human dysferlin, with particular emphasis on domains involved in membrane repair and the structural effects of pathogenic variants. The grant supported advances in protein expression, purification, and structural characterization, with complementary computational efforts to explore membrane interactions.

Aim 1: Test multiple designs for nanodysferlin_v2.0 using nanodysferlin_v1.0 as a starting point

This aim, conducted in collaboration with Dr. Peter Keyel (Texas Tech University), focused on the design and functional testing of new nanodysferlin constructs. Six engineered versions of “nanodysferlin_v2.0” were generated, each lacking specific C2 domains but retaining the C2-FerA domain, based on the hypothesis that the FerA four-helix bundle provides a conserved functional role among ferlins. Functional assays in Dr. Keyel’s toxin repair assay displayed variable protective activity among constructs: the ABF construct (C2A–C2B–C2FerA–DysF–C2F–TM) exhibited the greatest membrane protection, whereas the BCG construct (C2B–C2C–C2FerA–DysF–C2G–TM) showed the least. These results suggest unique contributions of domain composition to dysferlin’s repair capacity and present a framework for future mechanistic tests of domain function. While our truncated dysferlin constructs and previous constructs showed efficacious membrane repair activity, it is unclear whether nano-dysferlin could fully rescue wild-type dysferlin activity.

Aim 2: Structural and biophysical validation of dysferlin domains

This aim centered on high-resolution structural analysis of key dysferlin domains, with particular progress on the C2G domain, including clinically relevant variants (D1837E, D1837N, Y1839F, and D1914N). Structural data revealed new insights into domain stability, membrane-interaction interfaces, and the molecular consequences of disease-associated mutations. A manuscript describing the results is in preparation.

Salary support from the award enabled Dr. Matthew Dominguez to lead the experimental component of this aim, encompassing expression, purification, and structural characterization of full-length dysferlin for cryo-electron microscopy (cryoEM). Dr. Dominguez successfully optimized expression in HEK293 cells, developed reproducible purification protocols, and produced high-purity full-length and truncated constructs. Purified samples were submitted to the Stanford Synchrotron Radiation Lightsource (SLAC) for cryoEM grid preparation and data collection. Due to limited access to external cryoEM facilities—restricted to twice yearly—optimization of grid quality and imaging parameters was protracted. Although Dr. Dominguez’s departure from the lab and concurrent cryoEM publications by other groups shifted project priorities, the expression and purification systems developed are still a significant outcome, supporting ongoing structural studies.

Parallel molecular dynamics (MD) simulations have been initiated to assess dysferlin–membrane interactions and their possible roles in modulating membrane curvature. Early simulations indicated that dysferlin domains can reorganize lipids and alter curvature, consistent with emerging structural observations. Completion of extended production simulations will require expanded computing resources in future phases to capture long-timescale remodeling dynamics.