A Förster resonance energy transfer-based sensor of steric pressure generated by protein crowding on membrane surfaces

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dc.contributor.advisorStachowiak, Jeanne Casstevens
dc.contributor.committeeMemberYeh, Hsin-Chih
dc.contributor.committeeMemberRen, Pengyu
dc.contributor.committeeMemberSenning, Eric
dc.creatorHouser, Justin Ryan
dc.date.accessioned2022-12-05T22:25:49Z
dc.date.available2022-12-05T22:25:49Z
dc.date.created2022-08
dc.date.issued2022-08-12
dc.date.submittedAugust 2022
dc.date.updated2022-12-05T22:25:50Z
dc.description.abstractBiological membranes are densely crowded with proteins. Rapid diffusion and collision of membrane-bound proteins generates substantial steric pressure that is capable of shaping membrane surfaces. Despite efforts to model this steric pressure through analytical and computational models, a direct measurement of steric pressure at membrane surfaces is still lacking. Towards addressing this gap, we have developed a Förster resonance energy transfer (FRET) based sensor to directly measure steric pressure generation at membrane surfaces. Using this FRET sensor, we measured the steric pressure generated by crowding the N-terminal structured domain, ENTH, of the endocytic adaptor protein epsin1 on synthetic liposomes. Here, we found the measured steric pressure from our FRET sensor, by crowding ENTH domains on the membrane surface, is in good agreement with crowded particle theory. Further, we found that ENTH’s ability to break up larger vesicles into smaller ones correlates with steric pressure rather than the chemistry used to attach ENTH to the membrane. However, while this work investigates the effects of crowding globular proteins on the membrane, many proteins involved in membrane remodeling are intrinsically disordered. Having large hydrodynamic radii, disordered domains could contribute substantially to membrane crowding through steric effects. However, it is unclear to what extent they are capable of generating steric pressure, owing to their conformational flexibility. Toward resolving this uncertainty, we used our recently developed FRET sensor to measure steric pressure generated by intrinsically disordered proteins at membrane surfaces during dynamic membrane remodeling events. Our data indicate that disordered domains generate significant steric pressure through entropic and electrostatic mechanisms, suggesting that they may constitute a critical, yet previously neglected class of membrane remodeling proteins.
dc.description.departmentBiomedical Engineering
dc.format.mimetypeapplication/pdf
dc.identifier.urihttps://hdl.handle.net/2152/116921
dc.identifier.urihttp://dx.doi.org/10.26153/tsw/43816
dc.language.isoen
dc.subjectFRET
dc.titleA Förster resonance energy transfer-based sensor of steric pressure generated by protein crowding on membrane surfaces
dc.typeThesis
dc.type.materialtext
thesis.degree.departmentBiomedical Engineering
thesis.degree.disciplineBiomedical Engineering
thesis.degree.grantorThe University of Texas at Austin
thesis.degree.levelDoctoral
thesis.degree.nameDoctor of Philosophy

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