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Analytical Chemistry

Intact Membrane Proteins Analyzed

Micelles protect complexes from solution to vacuum

by Celia Henry Arnaud
June 16, 2008 | APPEARED IN VOLUME 86, ISSUE 24

Credit: Courtesy of Carol Robinson
Credit: Courtesy of Carol Robinson

BRITISH RESEARCHERS REPORT that they have analyzed intact membrane protein complexes with mass spectrometry. MS analysis of membrane proteins has been difficult because the proteins are often insoluble in the buffers used for electrospray ionization (ESI), a widely used technique for MS analysis of large proteins, and because subunits of the complexes readily dissociate.

Carol V. Robinson, a chemistry professor at the University of Cambridge, and coworkers both there and at the University of Bristol avoid these problems by using nonionic detergent micelles. The micelles protect the membrane protein complex so that all of its transmembrane and cytoplasmic components reach the mass spectrometer (Science, DOI: 10.1126/science.1159292). In the spectrometer, the detergent dissociates and releases the intact complex.

"We will be able to look at membrane and soluble subunits together in the same experiment," Robinson says. "This is really a first because in general the conditions required for both types of subunit are markedly different and cannot normally be captured within the same set of experimental conditions."

Robinson and her colleagues demonstrate the method with a transmembrane complex from Escherichia coli called BtuC2D2, which imports vitamin B-12 into the cell. The protein has two transmembrane subunits (BtuC) and two cytoplasmic nucleotide-binding subunits (BtuD). The researchers used the detergent n-dodecyl-β-D-maltoside (DDM) to hold the complex together in an active state.

The micelles allow the scientists to use harsher conditions in the ESI unit and the mass analyzer than they usually do, Robinson says. In the resulting mass spectrum, they identify isolated DDM clusters, an assembly of more than 100 DDM molecules associated with the intact complex, and a dissociated, unfolded BtuD subunit. Normally, the same experimental conditions would lead to complete dissociation of the complex. In addition, the researchers analyzed a complex of BtuC2D2 bound to adenosine triphosphate and Mg2+.

"Without the micelles, we only see the soluble part of the complex, the cytoplasmic subunits," Robinson says. In that situation, the transmembrane portions are lost. "With the micelles, we are able to protect interactions between the different types of subunits," she says.

The work represents a "significant development for membrane protein analysis," says Joseph A. Loo, professor of chemistry and biochemistry at UCLA. Robinson's group "has demonstrated not only that membrane proteins can be detected but also that noncovalent complexes with their full complement of ligands can be measured. Using this method, I can envision that ESI-MS will be the frontline method to characterize membrane proteins prior to using high-resolution structural methods, such as X-ray crystallography and NMR," Loo says.

Robinson's group has also been using the method to analyze other membrane protein complexes, but its full applicability remains to be seen. "Many more different types of complexes will need to be measured to get a better feel for the generality of the method," Loo says.



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