Methods to measure TMD-TMD interaction
TMD-TMD interactions can be investigated with a range of biochemical methods including gel-shift assays, chemical crosslinking, density gradient centrifugation and gel filtration chromatography. Because working with membrane proteins leads to unique challenges, answering a research question often leads to the development or improvement of newer assays.
ToxR-based assays in the E. coli inner membrane
Measuring these interactions in lipid bilayers is possible by using a genetic device previously developed in the Langosch lab, the ToxR assay. This system can be used for the analysis of individual sequences as well as for library screening. It makes use of a chimeric protein consisting of the cytoplasmic ToxR transcription activator domain connected via a TMD of interest to the periplasmic maltose-binding protein (MalE) domain. Upon TMD-TMD interaction in the inner membrane of appropriately engineered E. coli cells, the ToxR moiety activates a ToxR-driven promoter that is coupled to various reporter proteins. Expression of chloramphenicol acetyltransferase (CAT) results in chloramphenicol resistance, thus enabling selection of self-assembling TMD sequences in vivo. Relative TMD-TMD affinity is determined by quantification of ?-galactosidase (?-gal) activity after dimerization-induced lacZ expression. In combination with scanning mutagenesis, the ToxR system is useful for uncovering critical amino acids and motifs in the specific assembly of TMDs - the structural “code” of membrane-spanning interaction domains.
Förster Resonance Energy Transfer (FRET) assays
FRET can occur when two fluorophores are in close proximity, and is therefore a flexible tool to investigate whether a protein exists as a monomer or dimer. TMDs can be labeled with a fluorescent protein such as a GFP or RFP variant, or organic fluorophores. When TMD-TMD interactions bring the two fluorophores together, the increase in FRET can be detected though a change in fluorescence emission or polarization. FRET-based assays are therefore powerful tools to investigate the lipid dependence of TMD-TMD interactions, distinguish between dimers/trimers/higher-oligomers, and also measure the physicochemical parameters of the interaction.
Protein-Fragment Complementation Assays
For protein-fragment complementation assays (PCA) an enzyme regains activity only after dimerization. A number of proteins are available for PCA, which have been artificially split (e.g. GFP, adenylat cyclase, TEV protease, beta-lactamase, DHFR) into N- and C-terminal fragments which can be expressed in cells as fusion proteins with the protein of interest. For example, TMDs thought to interact could each be fused to a domains of a split protein. If the TMDs undergo dimerization, the enzyme will regain activity and lead to the production of a detectable substrate. In in-vivo assays, the enzyme activity can be detected through an increased expression of reporter genes or increased cellular growth. PCA methods can therefore allow the measurement of affinity between existing TMDs. In combination with random mutagenesis, PCA can also allow the selection of novel TMDs with high affinity.