Web Date: September 26, 2011
Cheap, Simple Test Spots Protein-Protein Interactions
A graphene oxide-based assay could provide chemists with an inexpensive means to detect protein-protein interactions (Anal. Chem., DOI: 10.1021/ac200617k).
To discover peptide-based drug candidates, researchers often monitor how a disease-related protein interacts with libraries of small peptides. The biggest challenge is developing an easily measurable signal for when the proteins bind to peptides, says Chun-Hua Lu of Fuzhou University, in China. Fluorescence resonance energy transfer (FRET) spectroscopy, which monitors the distance between fluorescent molecules attached to the proteins, is commonly used, but to generate a signal, it often requires a protein to change shape upon binding. To study proteins that don’t shape shift, Lu and his colleagues developed a more general approach.
To the end of a peptide, the researchers attach pyrene and measure its fluorescence with a spectrometer. Then, they mix the tagged peptide with graphene oxide, which pyrene binds to. Graphene oxide, made from the same inexpensive graphite at the core of most pencils, quenches the fluorescent signal from the pyrene-bound peptide when pyrene stacks onto its flat surface. Finally, the researchers add the protein of interest. If it binds to the peptide, the tagged peptide leaves the graphene oxide and the fluorescent signal returns.
The team tested the assay with a well-studied system: a peptide that is a hallmark of HIV infection along with a human antibody that binds it. They found that as little as 200 pM of the antibody rekindled pyrene’s glow.
To test their method further, the researchers next applied the antibody in samples of human saliva and serum, which contain molecules that could disrupt the protein-peptide interaction. Even with the additional chemicals present, the assay had detection limits of 2 nM in saliva and 5 nM in a solution made from human serum. These limits match those of existing methods, Lu says.
To test their method with other protein-peptide pairs, the researchers successfully detected binding between different peptides and a second HIV antibody, as well as with a protein called α-bungarotoxin from snake venom.
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