High-throughput stability screening for detergent-solubilized membrane proteins

Abstract

Protein stability in detergent or membrane-like environments is the bottleneck for structural studies on integral membrane proteins (IMP). Irrespective of the method to study the structure of an IMP, detergent solubilization from the membrane is usually the first step in the workflow. Here, we establish a simple, high-throughput screening method to identify optimal detergent conditions for membrane protein stabilization. We apply differential scanning fluorimetry in combination with scattering upon thermal denaturation to study the unfolding of integral membrane proteins. Nine different prokaryotic and eukaryotic membrane proteins were used as test cases to benchmark our detergent screening method. Our results show that it is possible to measure the stability and solubility of IMPs by diluting them from their initial solubilization condition into different detergents. We were able to identify groups of detergents with characteristic stabilization and destabilization effects for selected targets. We further show that fos-choline and PEG family detergents may lead to membrane protein destabilization and unfolding. Finally, we determined thenmodynamic parameters that are important indicators of IMP stability. The described protocol allows the identification of conditions that are suitable for downstream handling of membrane proteins during purification.

Contribution of Intrinsic Fluorescence to the Design of a New 3D-Printed Implant for Releasing SDABS

“…The prolonged duration of contact between the sdAb and the components of the CRS could lead to protein degradation—for example, loss of tertiary structure—and eventually to aggregation [ 35 , 36 ]. Formulation screening using sdAb fluorescence and backscattering could be used to find formulations that promote sdAb stability and enhance the range of use of our CRS [ 37 , 38 , 39 , 40 ]. The solution’s composition, including pH, ionic strength, and choice of polymer, strongly altered sdAb thermal stability.…”

Changes in Membrane Protein Structural Biology

“…Where HTP-SEC is unavailable, filter plates can be used to crudely separate differently sized MPs, but these are less suitable for large MPs [101]. Alternatively, a simple extraction assay can be applied to assess folded state [102]. It is important to note that the most suitable condition is often not the condition that extracts the largest amount of MP.…”

“…It is important to note that the most suitable condition is often not the condition that extracts the largest amount of MP. A prime example is with Fos-Choline-based detergents that readily extract MPs, often in an inactive and mis-folded state [102]. This is particularly important when working with GFP tagged MPs expressed in eukaryotic expression systems such as yeast, insect cells and mammalian cells as GFP can remain fluorescent regardless of whether the MP is correctly folded [102].…”

“…The data from these detectors can be used to calculate the oligomeric state of an MP, the amount of detergent associated, and the amount of free detergent by The compositional stability of an MP in detergent is often assessed early in the purification process via FSEC. More recent approaches such as nanoDSF and in situ dynamic light scattering can also be used to access the effect of encapsulation reagents on the stability of an MP [102,170,171]. Negative stain is another method that can be used to assess the compositional stability of MPs but is limited by sample throughput but does have the advantage of using nanograms of sample.…”

Preparation of Recombinant Membrane Proteins from Pichia pastoris for Molecular Investigations

“…If no information is already available for the solubilization of the targeted IMP, the initial step usually consists in screening a panel of extraction conditions varying a number of parameters such as the choice and concentration of the detergent, the ionic strength, the membrane protein concentration and the addition of stabilizing compounds (Champeil et al 2016;Kotov et al 2019). The ideal combination is hardly predictable as it depends on the nature of the IMP, on its local lipid environment in the membrane where it stands, and on the physicochemical properties of the chosen detergent.…”