Biophysical Society Conference | Tahoe 2022

Molecular Biophysics of Membranes

Poster Abstracts

42-POS Board 11 ULTRA-DARK NANODISCS AS A MEMBRANE MIMETIC FOR ASSESSING MEMBRANE PROTEIN STABILITY BY NANO DIFFERENTIAL SCANNING FLUORIMETRY (NANODSF) Jazlyn Selvasingh 1 ; Jake Mckinney 1 ; Jens Meiler 1,2 ; Kaitlyn Ledwitch 1 ; 1 Vanderbilt University, Nashville, TN, USA 2 Leipzig University, Leipzig, Germany Membrane proteins are particularly challenging targets to study because they associate with unique, asymmetric lipid environments and therefore, require the presence of a lipid membrane mimetic for in vitro studies. Here, we developed a new method that leverages nano Differential Scanning Fluorimetry (nanoDSF) coupled with an engineered non-fluorescent ‘ultra-dark’ nanodisc (replaces both tryptophan and tyrosine residues with non-fluorescent sidechains) to measure membrane protein thermostability. In this work, we demonstrate the utility of this approach using the disulfide bond formation protein B (DsbB) as a model membrane protein system. This new technology platform allows for the evaluation of membrane protein stability in the presence of lipids without signal interference from the lipid-loaded nanodisc mimetic. NanoDSF experiments for DsbB measured in ultra-dark nanodiscs resulted in a thermal unfolding curve with two distinct inflection points corresponding to a melting temperature of 70.5 and 77.5 °C respectively. We compared this to DsbB in a panel of detergent micelles, which all resulted in a single inflection point. DsbB in DDM (n-dodecyl- β -D-maltoside) micelles showed the highest melting temperature of 55.5 °C. This new technology platform facilitates high-throughput evaluation of membrane protein thermostability in the context of a lipid-based membrane mimetic. The application of this method is broadly applicable to determining the change in stability associated with a range of membrane proteins in complex lipid environments.

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