Materials and MethodsAs in the study of the previously determined MscL structure (S1), multiple homologs of MscS [encoded by the yggb gene (S2)] were identified by BLAST searching of the NCBI genome database. Ten homologs from prokaryotes and Archaea were identified and subsequently cloned. The channels were subcloned into expression vectors (pET system, Novagen) and expression screening was carried out. Cells expressing sufficient channels to be identified by Western blotting were subjected to extensive detergent screening utilizing ~50 detergents (Anatrace, Sigma, Aldrich) where both the ability of the channels to be extracted out of the membrane and the ability to remain as a homo-oligomer were determined. Subsequent large-scale expressions, extraction and purification produced sufficient amounts of protein for three channels (E. coli, B. subtilis and C. tepidum) for crystallization trials. Each of these channels was produced recombinantly (vector pet28b, Novagen) in 50-liter fermenter growths in a modified Terrific Broth media containing 1% glucose and 0.4% glycerol. Protein expression was initiated by the addition of 2% lactose and 2 mM IPTG for 2-4 hours, resulting in ~1.5 kg of wet cells. To obtain phase information, selenomethionine-derivatized protein was purified from cells grown in a modified M9 media containing 50 mg/l selenomethionine, and the remaining amino acids at 40 µg/l. Extraction of the E. coli MscS was carried out using sonication and solubilization with 1% Foscholine-14. Ni-affinity chromatography, anion exchange, and size exclusion chromatography in the presence of 0.05% Foscholine-14 were used to purify the protein to homogeneity. The apparent molecular mass of the protein, as indicated by size-exclusion chromatography, was in excess of 200 kD, similar to that reported for recombinant MscS by Sukharev (S3). Crystals were obtained with 10-15 mg/ml MscS by hanging drop vapor diffusion with 100 mM pH 7.2 Hepes buffer, 150 mM Na-formate, 8% glycerol, and 16% PEG-3350 as the precipitant. Crystals grew to ~200 µm in each dimension, and were assigned to space group P4 3 2 1 2 (a = b = 184.7 Å, c = 260.7 Å) with one MscS oligomer in the asymmetric unit (corresponding to ~71% solvent content). Only residues in the extramembrane (water-soluble) regions of MscS participated in lattice contacts.

The chemosensory pathway of bacterial chemotaxis has become a paradigm for the two-component superfamily of receptor-regulated phosphorylation pathways. This simple pathway illustrates many of the fundamental principles and unanswered questions in the field of signaling biology. A molecular description of pathway function has progressed rapidly because it is accessible to diverse structural, biochemical, and genetic approaches. As a result, structures are emerging for most of the pathway elements, biochemical studies are elucidating the mechanisms of key signaling events, and genetic methods are revealing the intermolecular interactions that transmit information between components. Recent advances include (a) the first molecular picture of a conformational transmembrane signal in a cell surface receptor, (b) four new structures of kinase domains and adaptation enzymes, and (c) significant new insights into the mechanisms of receptor-mediated kinase regulation, receptor adaptation, and the phospho-activation of signaling proteins. Overall, the chemosensory pathway and the propulsion system it regulates provide an ideal system in which to probe molecular principles underlying complex cellular signaling and behavior.

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The transmembrane, homodimeric aspartate receptor of Escherichia coli and Salmonella typhimurium controls the chemotactic response to aspartate, an attractant, by regulating the activity of a cytoplasmic histidine kinase. The cytoplasmic domain of the receptor plays a central role in both kinase regulation and sensory adaptation, although its structure and regulatory mechanisms are unknown. The present study utilizes cysteine and disulfide scanning to probe residues Leu-250 through Gln-309, a region that contains the first of two adaptive methylation segments within the cytoplasmic domain. Following the introduction of consecutive cysteine residues by scanning mutagenesis, the measurement of sulfhydryl chemical reactivities reveals an ␣-helical pattern of exposed and buried positions spanning residues 270 -309. This detected helix, termed the "first methylation helix," is strongly amphiphilic; its exposed face is highly anionic and possesses three methylation sites, while its buried face is hydrophobic. In vivo and in vitro assays of receptor function indicate that inhibitory cysteine substitutions are most prevalent on the buried face of the first methylation helix, demonstrating that this face is involved in a critical packing interaction. The buried face is further analyzed by disulfide scanning, which reveals three "lock-on" disulfides that covalently trap the receptor in its kinaseactivating state. Each of the lock-on disulfides crosslinks the buried faces of the two symmetric first methylation helices of the dimer, placing these helices in direct contact at the subunit interface. Comparative sequence analysis of 56 related receptors suggests that the identified helix is a conserved feature of this large receptor family, wherein it is likely to play a general role in adaptation and kinase regulation. Interestingly, the rapid rates and promiscuous nature of disulfide formation reactions within the scanned region reveal that the cytoplasmic domain of the full-length, membranebound receptor has a highly dynamic structure. Overall, the results demonstrate that cysteine and disulfide scanning can identify secondary structure elements and functionally important packing interfaces, even in proteins that are inaccessible to other structural methods.The aspartate receptor of Escherichia coli and Salmonella typhimurium is representative of a large family of cell-surface receptors that regulate two-component signaling pathways, which are widespread in prokaryotic and eukaryotic organisms (1-9). These receptors contain two putative transmembrane helices per subunit and, in all cases tested, form stable homodimers that signal via a transmembrane conformational change. Chimeric receptors containing the sensory domain of the aspartate receptor and the regulatory domain of another family member are functional, suggesting that members of this receptor family use a conserved mechanism of transmembrane signaling to regulate cytoplasmic histidine kinase activity (10 -12). More generally, conformational transmembrane signals ma...