Characterising protein/detergent complexes by triple-detection size-exclusion chromatography

Abstract

BackgroundIn vitro investigations of membrane proteins usually depend on detergents for protein solubilisation and stabilisation. The amount of detergent bound to a membrane protein is relevant to successful experiment design and data analysis but is often unknown. Triple-detection size-exclusion chromatography enables simultaneous separation of protein/detergent complexes and protein-free detergent micelles and determination of their molar masses in a straightforward and absolute manner. Size-exclusion chromatography is used to separate different species, while ultraviolet absorbance, static light scattering, and refractive index measurements allow molar mass determination of protein and detergent components.ResultsWe refined standard experimental and data-analysis procedures for challenging membrane-protein samples that elude routine approaches. The general procedures including preparatory steps, measurements, and data analysis for the characterisation of both routine and complex samples in difficult solvents such as concentrated denaturant solutions are demonstrated. The applicability of the protocol but also its limitations and possible solutions are discussed, and an extensive troubleshooting section is provided.ConclusionsWe established and validated a protocol for triple-detection size-exclusion chromatography that enables the inexperienced user to perform and analyse measurements of well-behaved protein/detergent complexes. More experienced users are provided with an example of a more sophisticated analysis procedure allowing mass determination under challenging separation conditions.

A Cholesterol Analog Induces an Oligomeric Reorganization of VDAC

“…This is due to several complications, with the main issue being the confinement of VDAC in the ''detergent micelle phase'' of the solution, drastically reducing the volume of the solution that VDAC molecules can possibly occupy. A more suitable model may be suggested by incorporating the volume of available detergent micelles into the model; however, this approach itself would be too simplistic because the AUC results presented in this work (Table 2) as well as information presented by others (32,44) have demonstrated that integral membrane proteins do not associate with the same mass of detergent as is in a single detergent micelle. In the case of VDAC, a further complication arises with different oligomers of VDAC associating with different quantities of detergent (Table 2).…”

Characterization of Proteins by Size-Exclusion Chromatography Coupled to Multi-Angle Light Scattering (SEC-MALS)

“…By far the most common applications are establishing whether a purified protein is monomeric or oligomeric and the degree of oligomerization, and assessing aggregates 3,10,11,17,31,36,37,38 . The ability to do so for detergentsolubilized membrane proteins that cannot be characterized by traditional means is especially prized, and detailed protocols for this have been published 31,39,40,41,42,43 . Other common applications include establishing the degree of post-translational modification and polydispersity of glycoprotein, lipoproteins and similar conjugates 4,31,44,45,46,47 ; the formation (or lack thereof) and absolute stoichiometry (as opposed to stoichiometric ratio) of heterocomplexes including protein-protein, protein-nucleic acid and protein-polysaccharide complexes 24,46,48,49,50,51,52 ; determining the monomer-dimer equilibrium dissociation constant 49,53,54 ; and evaluating protein conformation 55,56 .…”

Coincidence detection and bi-directional transmembrane signaling control a bacterial second messenger receptor

“…This approach enables assessment of the quaternary structure of detergent-solubilized, full-length LapD in response to c-di-GMP and LapG (Figure 1B and C, Figure 1—figure supplement 3). SEC-MALS is particularly advantageous for the study of membrane proteins because the respective scattering contributions of the protein and detergent components of the protein-detergent conjugate can be accurately deconvoluted to yield the molecular mass of the protein analyte (Gimpl et al, 2016) (Figure 1—figure supplement 3C). With this approach, purified apo-LapD was shown to be monodisperse and dimeric (Figure 1C, Figure 1—figure supplement 3C).…”