SummaryWe have investigated self-association propensities of aqueous unfolded (U AQ ) forms of eight outer membrane proteins, OmpA, OmpW, OmpX, PagP, OmpT, OmpLa, FadL and Omp85. We found that high urea concentrations maintain all of these OMPs as monomers and that OmpA and OmpX remain monomeric upon dilution to 1M urea. A pH screen showed that basic pH supports the least amount of U AQ OMP self-association, consistent with earlier studies showing that basic pH was optimal for better folding efficiencies. The addition of KCl increased U AQ OMP self-association, although the magnitudes of the responses were varied. These studies showed that urea can be used to tune the amount of U AQ OMP self-association and indicate that the presence of some urea may be useful in optimizing folding conditions because it diminishes aggregation.

The EGF receptor is a classical receptor-tyrosine kinase. In the absence of ligand, the receptor adopts a closed conformation in which the dimerization arm of subdomain II interacts with the tethering arm in subdomain IV. Following the binding of EGF, the receptor opens to form a symmetric, back-toback dimer. Although it is clear that the dimerization arm of subdomain II is central to the formation of receptor dimers, the role of the tethering arm of subdomain IV (residues 561-585) in this configuration is not known. Here we use 125 I-EGF binding studies to assess the functional role of the tethering arm in the EGF receptor dimer. Mutation of the three major residues that contribute to tethering (D563A,H566A,K585A-EGF receptor) did not significantly alter either the ligand binding properties or the signaling properties of the EGF receptor. By contrast, breaking the Cys 558 -Cys 567 disulfide bond through double alanine replacements or deleting the loop entirely led to a decrease in the negative cooperativity in EGF binding and was associated with small changes in downstream signaling. Deletion of the Cys 571 -Cys 593 disulfide bond abrogated cooperativity, resulting in a high affinity receptor and increased sensitivity of downstream signaling pathways to EGF. Releasing the Cys 571 -Cys 593 disulfide bond resulted in extreme negative cooperativity, ligand-independent kinase activity, and impaired downstream signaling. These data demonstrate that the tethering arm plays an important role in supporting cooperativity in ligand binding. Because cooperativity implies subunitsubunit interactions, these results also suggest that the tethering arm contributes to intersubunit interactions within the EGF receptor dimer.The EGF receptor is a receptor-tyrosine kinase composed of an extracellular ligand binding domain, a single ␣-helical transmembrane domain, and a cytoplasmic tyrosine kinase domain (1). In the absence of ligand, the EGF receptor exists as a monomer, although substantial evidence suggests that it is also present as an inactive predimer (2-5). Binding of EGF induces dimerization of the receptor and leads to the stimulation of its intracellular tyrosine kinase activity (6).The primary target of this tyrosine kinase is the receptor itself, with phosphorylation occurring in trans on the C-terminal tail of the receptor (7). These phosphotyrosine residues serve as sites for the binding of Src homology 2 and PTB domain-containing proteins that promote the assembly of the signaling complexes that mediate the intracellular effects of EGF (8 -11).The extracellular domain of the EGF receptor is composed of four subdomains referred to as subdomains I-IV (12, 13). Subdomains I and III are homologous and together form the site at which EGF is bound. Subdomains II and IV are also homologous and are regions of high cysteine content. In the absence of ligand, the extracellular domain is held in a closed conformation by interactions between the dimerization arm in subdomain II and the tethering arm in subdomain IV (14). Upon b...

The physiological functions of a tissue in the body are carried out by its complement of expressed genes. Genes that execute a particular function should be more specifically expressed in tissues that perform the function. Given this premise, we mined public microarray expression data to build a database of genes ranked by their specificity of expression in multiple organs. The database permitted the accurate identification of genes and functions known to be specific to individual organs. Next, we used the database to predict transcriptional regulators of brown adipose tissue (BAT) and validated two candidate genes. Based upon hypotheses regarding pathways shared between combinations of BAT or white adipose tissue (WAT) and other organs, we identified genes that met threshold criteria for specific or counterspecific expression in each tissue. By contrasting WAT to the heart and BAT, the two most mitochondria-rich tissues in the body, we discovered a novel function for the transcription factor ESRRG in the induction of BAT genes in white adipocytes. Because the heart and other estrogen-related receptor gamma (ESRRG)-rich tissues do not express BAT markers, we hypothesized that an adipocyte co-regulator acts with ESRRG. By comparing WAT and BAT to the heart, brain, kidney and skeletal muscle, we discovered that an isoform of the transcription factor sterol regulatory element binding transcription factor 1 (SREBF1) induces BAT markers in C2C12 myocytes in the presence of ESRRG. The results demonstrate a straightforward bioinformatic strategy to associate genes with functions. The database upon which the strategy is based is provided so that investigators can perform their own screens.