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Medical physics

Medical physics

Novel method reveals protein interactions in membranes

13 Oct 2017 Hannah Behrens 
Interaction sites of the membrane protein OmpF
Interaction sites of the membrane protein OmpF

Researchers from the Universities of Nottingham and Oxford have developed a new method to investigate membrane proteins, which are important drug targets but difficult to study. The approach allows scientists to map those protein interaction sites that are located in membranes. Neil Oldham and his colleagues developed a probe that is mixed with solubilized membrane proteins and, upon exposure to UV light, labels the accessible surface area of said proteins. From this accessible surface area, the inaccessible surfaces involved in interaction with other membrane proteins can be inferred.

Membrane proteins make up 20 to 30% of our genes and are important drug targets. However, they are particularly difficult to study because they have large hydrophobic surfaces that facilitate aggregation in water and other aqueous solutions, similar to the process by which milk curdles. To solubilize membrane proteins without observing aggregation, detergents are needed to cover the hydrophobic surfaces with their hydrophobic tails, while the hydrophilic heads mediate the interaction with water. The resulting structures are called micelles.

The missing puzzle piece

Co-author Carol Robinson pioneered the investigation of membrane proteins by mass spectrometry in her research group. While the methods she developed opened up many possibilities to study membrane proteins, the question of how proteins interact with each other in membranes remained intangible.

In this latest study, the team tackled the problem by using a probe that incorporates into micelles and can therefore label even the hydrophobic areas of proteins (Angew. Chem. Int. Ed.doi: 10.1002/anie.201708254). The membrane protein that Oldham and his team used to prove that this probe works was OmpF. This protein assembles into trimers in bacterial membranes, where it acts as a pore for nutrient uptake.

As the OmpF trimer is one of the few membrane protein complexes that can be successfully crystallized, the researchers could compare the interaction surfaces determined using the new probe with the interaction surfaces known from the crystal structure. These were found to be identical.

How does the probe get to the membrane?

Once the probe has labelled the accessible surface by binding to it, the researchers used mass spectrometry to determine which parts of the protein were labelled. The study showed that only the parts of the protein’s surface that are found in the membrane were labelled. This is despite the successful use of this probe to label soluble proteins in previous studies.

Oldham and his co-workers hypothesized that the probe might preferentially incorporate in micelles because it has – like the detergent molecules – a hydrophobic and a hydrophilic part. The authors were able to show that this was indeed the case and that in the presence of a detergent that forms micelles, soluble proteins could not be cross-linked.

Future promise

The new method, termed carbene footprinting, will fill in a methodological gap, allowing scientists to study interactions between proteins in membranes. This way, researchers will be able to learn more about the important class of membrane proteins involved in so many critical functions, such as cell signalling, transport, reception and metabolism. Hopefully, a better understanding of membrane proteins will help to expand the repertoire of drugs that can successfully target them.

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