Abstract
Detergents are widely used for the isolation and solubilization of membrane proteins to support crystallization and structure determination. Detergents are amphiphilic molecules that form micelles once the characteristic critical micelle concentration (CMC) is achieved and can solubilize membrane proteins by the formation of micelles around them. The results are presented of a study of micelle formation observed by in situ dynamic light-scattering (DLS) analyses performed on selected detergent solutions using a newly designed advanced hardware device. DLS was initially applied in situ to detergent samples with a total volume of approximately 2 µl. When measured with DLS, pure detergents show a monodisperse radial distribution in water at concentrations exceeding the CMC. A series of all-trans n-alkyl-β-D-maltopyranosides, from n-hexyl to n-tetradecyl, were used in the investigations. The results obtained verify that the application of DLS in situ is capable of distinguishing differences in the hydrodynamic radii of micelles formed by detergents differing in length by only a single CH2 group in their aliphatic tails. Subsequently, DLS was applied to investigate the distribution of hydrodynamic radii of membrane proteins and selected water-insoluble proteins in presence of detergent micelles. The results confirm that stable protein-detergent complexes were prepared for (i) bacteriorhodopsin and (ii) FetA in complex with a ligand as examples of transmembrane proteins. A fusion of maltose-binding protein and the Duck hepatitis B virus X protein was added to this investigation as an example of a non-membrane-associated protein with low water solubility. The increased solubility of this protein in the presence of detergent could be monitored, as well as the progress of proteolytic cleavage to separate the fusion partners. This study demonstrates the potential of in situ DLS to optimize solutions of protein-detergent complexes for crystallization applications.
Highlights
Detergents are bifunctional molecules with amphipathic properties
This study demonstrates the potential of in situ dynamic light-scattering (DLS) to optimize solutions of protein– detergent complexes for crystallization applications
The results indicate that DLS is a suitable diagnostic technique for the identification and characterization of protein–detergent complex (PDC)
Summary
Detergents are bifunctional molecules with amphipathic properties. Because of these amphipathic properties, pure detergent monomers show peculiar solubility properties in water such that once a minimum concentration (the critical micelle concentration or CMC; Birdi, 1997) is achieved, all further additions of detergent molecules go into the formation of micelles. A detergent must be used at concentrations above its CMC in order to act as an effective solubilizing agent of hydrophobic molecules such as membrane proteins (Arnold & Linke, 2008). A significant obstacle in membrane-protein research and, in particular, in membrane-protein crystallization is the need to doi:10.1107/S2053230X14027149 75 research communications identify a suitable detergent and concentration that, in combination with buffer and additives, will maintain the stability and biological functionality of a given protein as a protein–detergent complex (PDC) during solubilization and crystallization (Oliver et al, 2013). It is expected that intact detergent-solubilized membrane proteins will appear as PDCs with slightly but detectably different radii to those of pure micelles and, more importantly, they will show a dominant single-peak radial distribution when measured using DLS. A poorly soluble or insoluble protein mixture can be identified by DLS, as can be failures to achieve a PDC, whether owing to the presence of a misfolded protein or an unsuitable detergent
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a call
