Protein-protein interactions (PPis) are a key part of many biological processes, such as cellular uptake/signaling. Although these PPis are often to the benefit of the body, many diseases result from dysfunctional interactions. An example of this is Alzheimers Disease, where the aggregation of beta-amyloid fragments to form plaques. These plaques can then lead to neuron degeneration. By understanding how and where proteins interact with one another, it is possible to develop small molecule or peptide inhibitors of these interactions. Several methods, such as mass spectrometry and x-ray crystallography, exist to understand protein tertiary structure, as well as identify points of interaction between two proteins. However, many of these methods require specialized equipment, as well as trained individuals to both prepare samples and interpret them.<br/><br/>Fluorescamine is a fluoregenic dye that targets primary amines. When interacting with proteins, fluorescamine selectively tags lysine and arginine residues on the protein's surface. If two proteins are able to stably interact with one another, fluorescamine will be unable to bind to residues at that interaction interface. This translates to a reduction in fluorescence for the complex when compared to the proteins alone. Fluorescamine can be used as a means of rapidly screening PPis, in order to determine whether specific protein pairs interact. Since fluorescamine is a covalent tag, these proteins can then be digested and analyzed by mass spectrometry in order to identify the location of the interaction interface.<br/><br/>To assess fluorescamine's ability to be used in this fashion, several protein-protein interactions (protein A and IgG, actin and gc-globulin) were evaluated. In addition, the broad applicability of this assay to protein-biomolecule interactions was tested, through investigation of a protein-peptide pair (calmodulin and it's inhibitory peptide) or a protein-DNA pair (thrombin and two different thrombin-binding aptamers). For those interactions that were observed via reduction in fluorescence, mass spectrometry was conducted to confirm the location of the interaction. This method works best as an initial screen of interactions, in order to lower the amount of potential pairs to be analyzed by a more comprehensive analysis method.

Fluorescence-based screening of protein-protein interactions via fluorescamine incubation

ACS Spring 2022

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