Here, we employed the Hill equation, used broadly to characterize cooperativity in protein-ligand binding, to describe the dimerization of transmembrane (TM) helices in hydrophobic environments. The Hill analysis of wild-type fibroblast growth factor receptor 3 (FGFR3) TM domain dimerization gives a Hill coefficient of approximately 1 for lipid bilayers but only approximately 0.2 for sodium dodecyl sulfate (SDS) micelles. We propose that this finding is indicative of heterogeneity in FGFR3 TM dimer structure and stability in SDS micelles. We further speculate that (1) the Hill equation can be used as a tool to assess the existence of multiple structural states of TM dimers in different hydrophobic environments and (2) the structural heterogeneity, detectable by Hill analysis, may be the underlying reason for the broad peaks and the low resolution NMR studies of peptides in detergents. Full Article

One of the most extensively studied receptor tyrosine kinases is EGFR/ErbB1. Although our knowledge of the role of the extracellular domains and ligands in ErbB1 activation has increased dramatically based on solved domain structures, the exact mechanism of signal transduction across the membrane remains unknown. The transmembrane domains are expected to play an important role in the dimerization process, but the contribution of ErbB1 TM domain to dimer stability is not known, with published results contradicting one another. We address this controversy by showing that ErbB1 TM domain dimerizes in lipid bilayers and by calculating its contribution to stability as -2.5 kcal/mol. The stability calculations use two different methods based on Förster resonance energy transfer, which give the same result. The ErbB1 TM domain contribution to stability exceeds the change in receptor tyrosine kinases dimerization propensities that can convert normal signaling processes into pathogenic processes, and is thus likely important for biological function. Full Article

Receptor tyrosine kinases (RTKs) are the second largest family of membrane receptors and play a key role in the regulation of vital cellular processes, such as control of cell growth, differentiation, metabolism, and migration. The production of whole-length RTKs in large quantities for biophysical or structural characterization, however, is a challenge. In this study, a cell engineering strategy using the anti-apoptotic Bcl-2 family protein, Bcl-x(L), was tested as a potential method for increasing stable expression levels of a recombinant RTK membrane protein in Chinese hamster ovary (CHO) cells. Wild-type and CHO cells stably overexpressing heterologous Bcl-x(L) were transformed with the gene for a model RTK membrane protein, ErbB2, on a plasmid also containing the Zeocin resistance gene. While CHO cells exhibited a gradual decrease in expression with passaging, CHO-bcl-x(L) cells offered an increased and sustained level of ErbB2 expression following continuous passaging over more than 33 days in culture. The increased ErbB2 expression in CHO-bcl-x(L) cells was evident both in stable transfected pools and in clonal isolates, and demonstrated both in Western blot analysis and flow cytometry. Furthermore, the sustained high-level protein expression in CHO-bcl-x(L) cells does not alter the correct membrane localization of the ErbB2 protein. Our results demonstrate that cellular engineering, specifically anti-apoptosis engineering, can provide increased and stable ErbB2 membrane protein expression in mammalian cells. This approach may also be useful for other membrane proteins in which large quantities are needed for biophysical and structural studies. Full Article

This review focuses on the methods that are available to study transmembrane (TM) helix dimerization in membrane-like environments (either bacterial membranes or lipid bilayers, as mimics of the eukaryotic cellular membrane), with an emphasis on the utility of surface-supported bilayers in such studies. Full Article

Membrane proteins fold, assemble and function within their native fluid lipid environment. Structural studies of fluid lipid bilayers are thus critically important for understanding processes in membranes. Here, we propose a simple approach to visualize the hydrocarbon core using neutron diffraction and deuterated lipids that are commercially available. This method should have broad utility in structural studies of the bilayer response to protein insertion and folding in membranes. Full Article