Transmembrane helix dimerization in lipid bilayers and mammalian membranes

Lateral interactions of membrane proteins play important roles in signal transduction across the cell membrane. A quantitative understanding of the interaction strengths is crucial for describing signaling networks and predicting cellular responses to environmental stimuli. However, such knowledge is lacking due to many experimental limitations. We developed and applied quantitative Forster Resonance Energy Transfer (FRET) methods to characterize the thermodynamics of membrane protein interactions in cell membrane mimetic model systems.;Emission-excitation FRET (EmEx-FRET) method was developed to measure the interaction strengths of transmembrane helix in lipid bilayers. The method was described and verified with published data on fibroblast growth factor receptor 3 (FGFR3) transmembrane domain. Then, the Gly382Asp pathogenic mutant of FGFR3 transmembrane domain was studied and the dimerization free energy was determined as -2.78+/-0.04 kcal/mol. This result was very similar with the wild type. Thus, the pathogenic mutation did not affect the dimer stability. Furthermore, the dimerization free energy of epidermal growth factor receptor 1 (EGFR1) transmembrane domains was determined and the results showed that their interactions contribute a few kcal/mole to the overall stability of the EGFR1 dimer. This suggested that the transmembrane domains played an important structural role in signal transduction.;Quantitative imaging FRET (QI-FRET) method was developed to measure the interaction strengths of transmembrane proteins in mammalian membranes. Glycophorin A (GpA) transmembrane domain was studied and a dimerization free energy of -3.9 +/- 0.2 kcal/mole was measured. This first quantitative free energy measurement in a mammalian membrane showed that the crowded environment weakens the strength of GpA interactions. Furthermore, the contribution of the extracellular domain to FGFR3 dimerization was measured. The result showed that the overall contribution of the extracellular domain was repulsive and the magnitude is about 1 kal/mol. This observation highlights the fine balance in receptor tyrosine kinase domain interactions that regulate signaling.;In summary, novel FRET methods were developed and applied to yield binding curves and equilibrium constants describing membrane protein interactions, and they give us the power to predict the distribution of proteins in their monomeric and associated states, as a function of protein expression, and ultimately predict biological activity.