Abstract
Digitonin has long been used as a mild detergent for extracting proteins from membranes for structure and function studies. As supplied commercially, digitonin is inhomogeneous and requires lengthy pre-treatment for reliable downstream use. Glyco-diosgenin (GDN) is a recently introduced synthetic surfactant with features that mimic digitonin. It is available in homogeneously pure form. GDN is proving to be a useful detergent, particularly in the area of single-particle cryo-electron microscopic studies of membrane integral proteins. With a view to using it as a detergent for crystallization trials by the in meso or lipid cubic phase method, it was important to establish the carrying capacity of the cubic mesophase for GDN. This was quantified in the current study using small-angle X-ray scattering for mesophase identification and phase microstructure characterization as a function of temperature and GDN concentration. The data show that the lipid cubic phase formed by hydrated monoolein tolerates GDN to concentrations orders of magnitude in excess of those used for membrane protein studies. Thus, having GDN in a typical membrane protein preparation should not deter use of the in meso method for crystallogenesis.
Highlights
The foxglove plant, Digitalis purpurea, is the source of useful natural products that include digitalin and digitonin
Samples were prepared in the way they would be for membrane protein crystallization trials by mixing molten monoolein with aqueous solution in a weight ratio of 3/2
The aqueous solution contained GDN over a range of concentrations up to 100 mM [11.65%(w/v)]. This is orders of magnitude above (i) the critical micelle concentration (CMC) of GDN at 18 mM [0.0021%(w/v); Chae et al, 2012] and (ii) the concentration that is likely to be used with membrane proteins for cryo-electron microscopy (cryoEM) or crystallization studies (Zhang et al, 2017; Krishna Kumar et al, 2019; Walter et al, 2019; Xu et al, 2019)
Summary
The foxglove plant, Digitalis purpurea, is the source of useful natural products that include digitalin and digitonin. Digitonin is a steroidal saponin used extensively as a mild non-ionic detergent (Fig. 1) (Bridges, 1977). Digitonin finds application as a cholesterol precipitating agent (Haust et al, 1966). It has long been used in membrane protein structure and function studies, where it serves to provide a hospitable micellar environment for fragile membrane integral proteins and complexes (Weigel et al, 1983). As supplied commercially, digitonin is expensive, of variable quality, and requires lengthy and often repeated pre-treatments that include boiling and a week-long incubation followed by filtration and lyophilization (Bridges, 1977). Delivering membrane proteins of consistently high quality and stability using digitonin has generally been considered a challenge
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