Small membrane proteins may appear larger than life on blue native electrophoretic gels. The migration of purified Aac3 (left) and free detergent micelles (right) in samples prepared with detergent at ~30-fold the critical micelle concentration. Image courtesy of Edmund Kunji.
When data don’t make sense, it’s time to take a closer look and figure out what’s going on. A team led by Edmund Kunji at the Medical Research Council in the U.K. did just that in a recent paper in the Journal of Biological Chemistry: They established that the anomalous migration of a protein in blue native electrophoretic gels was leading researchers to think it worked as a dimer when in reality, it is a monomer.
The protein is a mitochondrial carrier, which allows molecules such as ATP and ADP exit and enter the organelle. “We were struggling to understand the discrepancy between many biophysical data showing that mitochondrial carriers were monomeric – crystal structures, ultracentrifugation data, and size exclusion chromatography – and the large number of claims in the literature for a dimeric form when blue native electrophoresis was applied,” explains Kunji.
To analyze membrane proteins by gel electrophoresis, researchers use detergents to extract it from its native organelle. The detergent molecules, as well as the lipid from the membranes, form a belt around the protein, says Kunji. The investigators had done earlier work with size-exclusion chromatography to show the big influence that the detergent/lipid micelle could have on the protein’s migration in the gel filtration column. Depending on the lipid and detergent, the belt can be two to four times the mass of the protein.
“We knew that this belt makes a large contribution to the overall mass and size of the complex,” says Kunji. They wondered if the same belt phenomenon applied to the technique of blue native gel electrophoresis.
The investigators decided to study the yeast ADP/ATP carrier Aac3 because it is a good representative of mitochondrial carriers. It had a large apparent molecular mass in blue native electrophoretic gels but in 2003, its crystal structure showed the basic fold to be monomeric. (Other investigators also confirmed this with the bovine counterpart of Aac3.)
“This was a shock to us and to the field, as everybody had been expecting a dimer,” says Kunji. “Plausible models for the mechanism had been proposed based on the dimer, but these structures showed they were incorrect”
So what was causing Aac3 to look larger in blue native gels? Paul Crichton, one of the investigators, looked into the influence of lipids, detergents and the blue Coomassie stain on the protein’s migration in blue native electrophoretic gels. After testing out each component, the investigators concluded that all of these components increased the apparent mass of the protein.
Kunji says he and his colleagues now think that other studies that used blue native gel electrophoresis for the sizing of small membrane proteins may have misinterpreted the mass data from the gels. In some other cases, the apparent larger mass may have led researchers to mistakenly “assume associations of these proteins with other proteins into large protein complexes,” says Kunji. “But these conclusions might be unsafe, as they could be simply due to effects of lipids and detergents on migration.”
