Date of Award
Doctor of Philosophy (PhD)
Membrane curvature provides a means to control spatial organization and activity of cells. It is regulated by plenty of peripherally binding membrane proteins, including BAR domain proteins. Two important sub-families of BAR domain containing proteins are NBAR and FBAR domain proteins. However, the current understanding of BAR domain protein membrane curvature (MC) sensing and generation is insufficient. My thesis intends to contribute to alleviating this situation.
We first performed curvature sorting and generation experiments of an NBAR domain protein: endophilin. We found that the endophilin NBAR domain (ENBAR) behaved as a curvature sensor or generator at different concentrations. We studied lateral diffusion of ENBAR and found its diffusion coefficients depending on its membrane density. We developed an analytical model to explain our experimental results. Our theory predicts that the membrane curvature serves as a switch that is modulated by a thermodynamic phase transition.
Then we studied the influence of membrane insertion helices on NBAR domain protein MC sensing and membrane dissociation kinetics by comparing ENBAR WT protein with helices deletion mutants. We found that the two helices had essential contributions for the NBAR domain curvature sorting. The WT and mutant membrane dissociation time were positively correlated with their densities on the membrane. Irreversible binding fractions for both variants were observed. These phenomena suggest higher order structure formation of these variants on the membrane.
Furthermore, we investigated the autoinhibition mechanism of full length endophilin via its H0 variants and the SH3 domain, by fluorescence experiments. We obtained evidence of the interaction between H0 helix and SH3 domain in solution and determined their binding affinities. These results experimentally support an H0/ SH3 domain mediated autoinhibition mechanism.
Finally we explored the regulation of a possible autoinhibition of the tubulation ability of syndapin, an FBAR domain protein. We compared the curvature sensing, generation and initiation abilities of syndapin FBAR, full length and full length with proline rich ligand. We found that the syndapin FBAR curvature initiation ability was larger compared to the full length protein, which was likely due to the differences in their curvature sensing abilities.
Zhu, Chen, "Membrane Curvature Sorting And Generation By The Bar Domain Proteins Endophilin And Syndapin" (2014). Publicly Accessible Penn Dissertations. 1527.