Challenges and approaches for assay development of membrane and membrane-associated proteins in drug discovery.

In addition to its role as a barrier between the cytoplasm and the extracellular milieu, the cell membrane is a scaffold for a diverse collection of receptors and enzymes. The organization afforded by this scaffold serves to ensure an efficient interaction between the components of the membrane. The desire to maintain this organization in solution is a challenge for the appropriate interrogation of these biochemical components.

Mobile loop mutations in an archaeal inositol monophosphatase: modulating three-metal ion assisted catalysis and lithium inhibition.

The inositol monophosphatase (IMPase) enzyme from the hyperthermophilic archaeon Methanocaldococcus jannaschii requires Mg(2+) for activity and binds three to four ions tightly in the absence of ligands: K(D) = 0.8 muM for one ion with a K(D) of 38 muM for the other Mg(2+) ions. However, the enzyme requires 5-10 mM Mg(2+) for optimum catalysis, suggesting substrate alters the metal ion affinity.

Template-directed self-assembly enhances RTK catalytic domain function.

Receptor tyrosine kinases have become important therapeutic targets because of their involvement in diseases, including cancer. Kinase domains, which are soluble and easily purified, have found widespread use in enzyme inhibitor assays, but these domains do not exhibit full function because they are isolated from the membrane. To address this shortcoming, the authors developed a simple method to restore biologically relevant function by assembling kinase domains on a nanometer-scale template, which imitates the membrane surface.

Receptor density balances signal stimulation and attenuation in membrane-assembled complexes of bacterial chemotaxis signaling proteins.

All cells possess transmembrane signaling systems that function in the environment of the lipid bilayer. In the Escherichia coli chemotaxis pathway, the binding of attractants to a two-dimensional array of receptors and signaling proteins simultaneously inhibits an associated kinase and stimulates receptor methylation--a slower process that restores kinase activity. These two opposing effects lead to robust adaptation toward stimuli through a physical mechanism that is not understood.

Template-directed assembly of signaling proteins: a novel drug screening and research tool.

A multitude of proteins reside at or near the cell membrane, which provides a unique environment for organizing and promoting assemblies of proteins that are involved in a variety of cellular signaling functions. Many of these proteins and pathways are implicated in disease. For example, strong links have been established between receptor tyrosine kinases and disease, most notably, cancer.

Formation and activity of template-assembled receptor signaling complexes.

Problems in membrane biology require methods to recreate the interactions between receptors and cytoplasmic signaling proteins at the membrane surface. Here, unilamellar vesicles composed of 1,2-dioleoyl-sn-glycero-3-phosphocholine and a nickel-chelating lipid were used as templates to direct the assembly of proteins from the Escherichia coli chemotaxis signaling pathway. The bacterial chemoreceptors are known to form clusters, which promote the binding of the adaptor protein (CheW) and the kinase (CheA).

Influence of resonance on the acidity of sulfides, sulfoxides, sulfones, and their group 16 congeners.

The influence of resonance on the acidities of dimethyl sulfide (DMS), dimethyl sulfoxide (DMSO), and dimethyl sulfone (DMSO2) and their group 16 congeners (DMXO(n) for X = Se, Te, Po and n = 0-2) is examined using ab initio methods and the natural bond orbital (NBO) and natural resonance theory (NRT) analyses. Gas-phase acidities are evaluated using B3LYP-optimized geometries with coupled cluster energies and complete basis set extrapolation. The acidity of the DMSO(n) molecules increases with increasing coordination of the central S atom.

Distributed subunit interactions in CheA contribute to dimer stability: a sedimentation equilibrium study.

The structural domains of the Escherichia coli CheA protein resemble 'beads on a string', since the N-terminal phosphate-accepting (P) domain is joined to the CheY/CheB-binding (B) domain through a flexible linker, and the B domain is in turn joined to the C-terminal dimerization/catalytic/regulatory domains by a second intervening linker. Dimerization occurs primarily via interactions between two dimerization domains, which is required for CheA trans-autophosphorylation. In this study, sedimentation equilibrium was used to demonstrate significant subunit interactions at secondary sites in the two naturally occurring (full-length and short) forms of CheA (CheA(1-654) or CheA(L), and CheA(98-654) or CheA(S)) by contrasting the dimerization of CheA(L) and CheA(S) to CheA(T), an engineered form that lacked the P domain entirely.

Template-directed assembly of receptor signaling complexes.

Transmembrane receptors in the signaling pathways of bacterial chemotaxis systems influence cell motility by forming noncovalent complexes with the cytoplasmic signaling proteins to regulate their activity. The requirements for receptor-mediated activation of CheA, the principal kinase of the Escherichia coli chemotaxis signaling pathway, were investigated using self-assembled clusters of a receptor fragment (CF) derived from the cytoplasmic domain of the aspartate receptor, Tar. Histidine-tagged Tar CF was assembled on the surface of sonicated unilamellar vesicles via a lipid containing the nickel-nitrilotriacetic acid moiety as a headgroup.