Monitoring Dimerization of GpA using FRET

Abstract

The long-term goal of this project is to design complex membrane proteins that have been functionalized with appropriate cofactors and can serve as components of artificial bioenergetic systems. We seek to achieve this by engineering specific interactions between the helical interfaces of transmembrane proteins. This will allow us to create functional nanostructures that contain the appropriate components in particular orientations. The association of transmembrane helical proteins to homodimers is driven by a handful of sequence motifs, the most common of which is GXXXG. We used this sequence to introduce additional interactions so that the formation of heterodimers occurred.We designed a buried salt bridge in the transmembrane domain of a well known dimeric membrane protein, GpA. We mutated Thr 87, which is not part of the dimerization interface, to diaminopropionic acid (Dap) on one of the helices and to aspartic acid on the other. Dap and aspartic acid interact electrostatically only in a narrow pH window. We characterized the pHdependent association of the peptides when incorporated into micelles using fluorescence resonance energy transfer (FRET). This allowed us to establish the pH profile for heterodimer formation and to measure the strength of the interaction.