Periodic Reporting for period 1 - Fas_Life-Death (A Biophysical Investigation of the Duality in Fas Receptor)
Berichtszeitraum: 2021-04-01 bis 2023-03-31
Signaling receptors in the plasma membrane control information exchange within and across cells. Among the different transmembrane signaling receptors, the Fas protein is receiving growing attention, mainly because of its ‘dual character’ in promoting as well as preventing cell growth in both cancer and normal cells. Here, we engineer a bottom-up approach to investigate the physical mechanism behind the duality in Fas receptor-ligand complexes that control the cell functions.
Multi-domain signaling receptors, including Fas are known to undergo molecular conformational changes to facilitate the downstream recruitment of signaling molecules. Such a conformational switch is usually driven by the change in chemical state (ligand, phosphorylation, glycosylation, etc). Fas_Life-Death combines biochemical (for in-vitro protein reconstitution), synthetic biology (for site-specific fluorescence labeling), and optical methods (single-molecule FRET, single particle tracking) to explore new conformations, and molecular mechanisms of Fas-mediated clustering in membranes. Investigating the influence of structural and molecular dynamics of transmembrane proteins on the physiological activity of cells have both scientific and pharmaceutical significance. These could provide comprehensive insights to transmembrane communication and identify points of intervention for drug design.
Multi-domain signaling receptors, including Fas are known to undergo molecular conformational changes to facilitate the downstream recruitment of signaling molecules. Such a conformational switch is usually driven by the change in chemical state (ligand, phosphorylation, glycosylation, etc). Fas_Life-Death combines biochemical (for in-vitro protein reconstitution), synthetic biology (for site-specific fluorescence labeling), and optical methods (single-molecule FRET, single particle tracking) to explore new conformations, and molecular mechanisms of Fas-mediated clustering in membranes. Investigating the influence of structural and molecular dynamics of transmembrane proteins on the physiological activity of cells have both scientific and pharmaceutical significance. These could provide comprehensive insights to transmembrane communication and identify points of intervention for drug design.
• We have successfully obtained detergent solubilized functional, full-length Fas constructs with tags for purification and fluorescence labeling, thereby having a first ever protocol for such a transmembrane signalling receptor.
• Using ligand-binding assays together with various biochemical and synthetic biology approaches we optimized the fluorescent labeling of full-length Fas receptor for in-vitro optical spectroscopy.
• We established in-vitro assays using peptidisc that facilitates research on spatio-temporal dynamics of full-length Fas signaling receptor using single-molecule FRET.
• We demonstrated monomeric and dimeric insertion of Fas receptors in the absence of Fas ligand. Such constructs prove a novel platform for single-molecule investigation of Fas nanoscale conformations.
• In parallel, we developed a single-molecule technique on a model protein, the ABC transporter BmrA, that exhibits two conformations - apo or post-hydrolytic, depending on ATP. Future investigation of Fas will benefit from these in-house protocols that facilitate studying the effects of transmembrane domains & ligand activation in oligomeric states of membrane proteins.
• Overall, Fas_Life-Death allowed the coordinator to start an independent research career by establishing a new line of research program focusing on biophysics of transmembrane signaling receptors.
• Using ligand-binding assays together with various biochemical and synthetic biology approaches we optimized the fluorescent labeling of full-length Fas receptor for in-vitro optical spectroscopy.
• We established in-vitro assays using peptidisc that facilitates research on spatio-temporal dynamics of full-length Fas signaling receptor using single-molecule FRET.
• We demonstrated monomeric and dimeric insertion of Fas receptors in the absence of Fas ligand. Such constructs prove a novel platform for single-molecule investigation of Fas nanoscale conformations.
• In parallel, we developed a single-molecule technique on a model protein, the ABC transporter BmrA, that exhibits two conformations - apo or post-hydrolytic, depending on ATP. Future investigation of Fas will benefit from these in-house protocols that facilitate studying the effects of transmembrane domains & ligand activation in oligomeric states of membrane proteins.
• Overall, Fas_Life-Death allowed the coordinator to start an independent research career by establishing a new line of research program focusing on biophysics of transmembrane signaling receptors.
Thanks to the MSCA grant, we have been successful to in-vitro encapsulate the functional and full-length signaling receptor, Fas. During this project, we engineered a bottom-up approach to the study of protein conformations and diffusion dynamics using single-molecule fluorescence spectroscopies combined with the state-of-the-art synthetic biology approach. Given that Fas have implications in numerous cancers, and autoimmune disease, we anticipate that this new research direction seeded during this MSCA will provide new knowledge on how protein molecular dynamics and cooperativity with other receptors regulate cell functions.