Novel Molecular Probes Detect Protein-Protein Interaction

Investigators from MIT are reporting in the online June 27 issue of J. Am. Chem. Soc. a new method to tag intracellular (or in vitro) proteins to allow the study of their interactions.
Here’s what the authors note in the study:

One protein partner is fused to Escherichia coli biotin ligase (BirA), while the other protein partner is fused to BirA’s “acceptor peptide” (AP) substrate. If the two proteins interact, BirA will catalyze site-specific biotinylation of AP, which can be detected by streptavidin staining. To minimize nonspecific signals, we engineered the AP sequence to reduce its intrinsic affinity for BirA. The rapamycin-controlled interaction between FKBP and FRB proteins could be detected in vitro and in cells with a signal to background ratio as high as 28. We also extended the method to imaging of the phosphorylation-dependent interaction between Cdc25C phosphatase and 14-3-3ε phosphoserine/threonine binding protein. Protein−protein interaction detection by proximity biotinylation has the advantages of low background, high sensitivity, small AP tag size, and good spatial resolution in cells.

The following is from a statement issued by MIT:

The new technique allows researchers to tag proteins with probes that link together like puzzle pieces if the proteins interact inside a cell. The probes are derived from an enzyme and its peptide substrate. If the probe-linked proteins interact, the enzyme and substrate also interact, which can be easily detected.
To create the probes, the researchers used the enzyme biotin ligase and its target, a 12-amino-acid peptide.
Their work is conceptually related to an approach that uses GFPs (green fluorescent proteins), which glow when activated, as probes. Half of each GFP molecule is attached to the proteins of interest, and when the proteins interact, the GFP halves fuse and glow. However, this technique results in many false positives, because the GFP halves seek each other out and bind even when the proteins they are attached to are not interacting, said Ting.
The new probes could be used to study nearly any protein-protein interaction, Ting [Alice Ting, MIT Pfizer-Laubach Career Development Assistant Professor of Chemistry] said. The researchers tested their probes on two signaling proteins involved in suppression of the immune system, and on two proteins that play a role in cell division. They are currently using the probe to image the interaction of proteins involved in synapse growth in live neurons.

Press release: New probe may help untangle cells’ signaling pathways …
Abstract: Protein-Protein Interaction Detection in Vitro and in Cells by Proximity Biotinylation J. Am. Chem. Soc., ASAP Article, 10.1021/ja801445p