Article

Determination of the molecular mass and dimensions of membrane proteins by size exclusion chromatography

The Medical Research Council, Dunn Human Nutrition Unit, Hills Road, Cambridge CB2 0XY, UK.
Methods (Impact Factor: 3.65). 11/2008; 46(2):62-72. DOI: 10.1016/j.ymeth.2008.10.020
Source: PubMed

ABSTRACT

Size exclusion chromatography is an established technique for the determination of hydrodynamic volumes of proteins or protein complexes. When applied to membrane proteins, the contribution of the detergent micelle, which is required to keep the protein soluble in the aqueous phase, needs to be determined to obtain accurate measurements for the protein. In a detergent series, in which the detergents differ only by the length of the alkyl chain, the contribution of the detergent micelle to the hydrodynamic volume is variable, whereas the contribution of the protein is constant. By using this approach, several parameters of membrane proteins can be estimated by extrapolation, such as the radius at the midpoint of the membrane, the average radius, the Stokes radius, and the excluded volume. The molecular mass of the protein can be determined by two independent measurements that arise from the behaviour of the free detergent micelle and protein-detergent micelle during size exclusion chromatography and the determination of the detergent-protein ratio. Determining the dimensions of protein-detergent micelles may facilitate membrane protein purification and crystallization by defining the accessibility of the protein surface.

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    • "Mass estimation by SEC is based on a comparison of the elution behaviour of the protein of interest with that of standard proteins, which are globular and water-soluble. Since detergent binding substantially alters a protein's elution behaviour [16] and, thus, its apparent size, analysing membrane proteins with SEC will provide, at best, a very crude size estimate. Nevertheless, with careful sample preparation, information on the coexistence of different oligomeric species can be deduced from SEC elution profiles even without knowledge of exact masses [5] . "
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