Physics Chat
A label free method to measure the dynamics of membranes to determine their biophysical properties and the effect of protein insertion
Speaker: Freya Turley (W Langbein)
Date: Friday 29 April 2022
Time: 15:00
Venue: Zoom
Study of the dynamics of membranes and their components is important as they often modulate the function of proteins. These dynamics have been studied using fluorescence techniques which suffer from artefacts originating from fluorophores and have limited speed and observation time due to photobleaching [1]. With more than 60% of therapeutic targets being membrane proteins, developing a method for quantification of biophysical properties of the membrane and its response upon protein insertion will aid understanding these systems [2]. We are developing interferometric gated off-axis reflectometry (iGOR) to fill this need for a dynamic, sensitive imaging technique without fluorescent labelling. It reduces the chance of altering the system and improves the probing of non-lateral dynamics. This technique uses off-axis digital holography to measure the complex field of reflected signals. This can be done in real time, providing several hundred frames per second, allowing observation of the dynamic phase and amplitude of the reflection, encoding the axial position and membrane thickness. To measure the membrane dynamics of suspended lipid bilayers the sample is ideally a weakly curved surface within the observed volume of 20#m length in all dimensions. To provide such samples, we have developed a protocol using electroformation to produce giant unilamellar vesicles (GUVs) of sufficient size, void of internal or attached lipid structures and minimal lipid debris in the surrounding [3]. This is necessary to avoid the complexity of the scattering from such structures being superimposed to the membrane reflection. These samples are being optimised for stability to allow longer measurements upon insertion of pore-forming proteins. Inserting Cry6Aa1 (a Bacillus thuringiensis toxin [4]) into GUVs will be analysed using iGOR to determine the biophysical properties of the membrane in response to protein insertion such as the bending modulus and possibly raft formation arising from phase separation.
References 1. A.L. Robson et al., Front. Pharmacol. 9, 80 (2018). 2. J. Carroll et al., Eur J Biochem 214, 771-778 (1993). 3. D. Drabik et al., Chemistry and Physics of Lipids 212, 88-95 (2018). 4. A. W. Ravna et al., Transporters as Targets for Drugs, 4, 15-51 (2018)
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