Department of Biochemistry and Cell Biology
Kevin R. MacKenzie
integral membrane proteins
Signal transduction by the T cell receptor (TCR) provides a number of opportunities for investigating the sequence dependence of helix-helix associations. Interactions between the transmembrane a-helices (TMs) of components of the TCR and other proteins involved in downstream aspects of signaling are important for TCR function. Because these proteins span the membrane with single helices, the approach will be to start by examining pairwise interactions between isolated TMs. Direct studies of larger complexes may also prove possible; if these systems are experimentally intractable it will be necessary to analyze multiple sets of pairwise interactions.
Questions about membrane protein folding and stability can be addressed using computational, biophysical, biochemical or molecular biological methods, and combinations of these approaches can be particularly effective. The glycophorin A transmembrane domain has been studied using many different approaches; for a list of papers describing these experiments click here.
Nuclear magnetic resonance spectroscopy (NMR) is the primary biophysical tool applied in the lab; the samples for these experiments are isotopically labeled membrane proteins or peptides solubilized in detergent micelles. Solution NMR can be used to determine the structures of these proteins, to monitor the fast-time scale motions of the polypeptide backbone, and to measure the rates at which the backbone amide protons exchange with solvent (D2O).
Fourier-transform infrared (FTIR) or circular dichroism (CD) spectroscopy can be used to study the secondary structure (and orientation, using ATR-FTIR) of membrane proteins in lipid bilayer environments; these measurements complement the solution NMR studies.
Measurements of the dissociation free energies of transmembrane helix-helix complexes can be made using a ultracentrifugation or fluorescence resonance energy transfer. Small angle x-ray scattering can measure the stoichiometry and the orientation (parallel or antiparallel) of the helices. Data from gel electrophoresis experiments or in vivo assays are generally treated qualitatively. The applicability of these different methods (and the development of new methods) depends strongly on the suitability of the particular sample.