The bacterial chemoreceptor complex governs signal detection and the upstream elements of chemotactic behavior, but the detailed molecular mechanism is still unclear. We have assembled nativelike functional arrays of an aspartate receptor cytoplasmic fragment (CF) with its two cytoplasmic protein partners (CheA and CheW) for solid-state nuclear magnetic resonance (NMR) studies of structural changes involved in signaling. In this initial study of the uniformly 13C- and 15N-enriched CF in these >13.8 MDa size arrays, residue-type assignments are made for amino acids that together make up 90% of the protein. We demonstrate that homo- and heteronuclear two-dimensional spectra are consistent with structure-based chemical shift predictions: a number of major assignable correlations are consistent with the predominantly α-helical secondary structure, and minor correlations are consistent with the disordered C-terminal tail. Sub-parts per million line widths and spectral changes upon freezing of samples suggest these arrays are structurally homogeneous and sufficiently immobilized for efficient solid-state NMR.

The goal of understanding mechanisms of transmembrane signaling, one of many key life processes mediated by membrane proteins, has motivated numerous studies of bacterial chemotaxis receptors. Ligand binding to the receptor causes a piston motion of an α helix in the periplasmic and transmembrane domains, but it is unclear how the signal is then propagated through the cytoplasmic domain to control the activity of the associated kinase CheA. Recent proposals suggest that signaling in the cytoplasmic domain involves opposing changes in dynamics in different subdomains. However, it has been difficult to measure dynamics within the functional system, consisting of extended arrays of receptor complexes with two other proteins, CheA and CheW. We have combined hydrogen exchange mass spectrometry with vesicle template assembly of functional complexes of the receptor cytoplasmic domain to reveal that there are significant signaling-associated changes in exchange, and these changes localize to key regions of the receptor involved in the excitation and adaptation responses. The methylation subdomain exhibits complex changes that include slower hydrogen exchange in complexes in a kinase-activating state, which may be partially consistent with proposals that this subdomain is stabilized in this state. The signaling subdomain exhibits significant protection from hydrogen exchange in complexes in a kinase-activating state, suggesting a tighter and/or larger interaction interface with CheA and CheW in this state. These first measurements of the stability of protein subdomains within functional signaling complexes demonstrate the promise of this approach for measuring functionally important protein dynamics within the various physiologically relevant states of multiprotein complexes.

The transmembrane signaling mechanism of bacterial chemotaxis receptors is thought to involve changes in receptor conformation and dynamics. The receptors function in ternary complexes with two other proteins, CheA and CheW, that form extended membrane-bound arrays. Previous studies have shown that attractant binding induces a small (∼2 Å) piston displacement of one helix of the periplasmic and transmembrane domains toward the cytoplasm, but it is not clear how this signal propagates through the cytoplasmic domain to control the kinase activity of the CheA bound at the membrane-distal tip, nearly 200 Å away. The cytoplasmic domain has been shown to be highly dynamic, which raises the question of how a small piston motion could propagate through a dynamic domain to control CheA kinase activity. To address this, we have developed a method for measuring dynamics of the receptor cytoplasmic fragment (CF) in functional complexes with CheA and CheW. Hydrogen–deuterium exchange mass spectrometry (HDX-MS) measurements of global exchange of the CF demonstrate that the CF exhibits significantly slower exchange in functional complexes than in solution. Because the exchange rates in functional complexes are comparable to those of other proteins with similar structures, the CF appears to be a well-structured protein within these complexes, which is compatible with its role in propagating a signal that appears to be a tiny conformational change in the periplasmic and transmembrane domains of the receptor. We also demonstrate the feasibility of this protocol for local exchange measurements by incorporating a pepsin digest step to produce peptides with 87% sequence coverage and only 20% back exchange. This method extends HDX-MS to membrane-bound functional complexes without detergents that may perturb the stability or structure of the system.

Many cellular processes involve interactions between membrane-associated proteins, and those interactions are enhanced by membrane association. We have used cross-linking reactions to compare the extent and specificity of protein interactions in solution versus on a membrane surface. Cysteine mutants of a soluble cytoplasmic fragment (CF) of the aspartate receptor, a transmembrane receptor involved in bacterial chemotaxis, are used in disulfide bond formation with the thiol-specific oxidant diamide and chemical cross-linking reactions with the trifunctional maleimide TMEA. CF binding to membranes is mediated by its N-terminal His tag binding to vesicles containing a nickel-chelating lipid, so cross-linking reactions conducted in the presence and absence of vesicles differ only in whether CF is bound to the vesicles or is free in solution. For multiple Cys throughout the CF, membrane association is shown to increase the rate and extent of these reactions. Cross-linking specificity, which is measured as the preference for cross-linking between Cys near each other in the native structure, is also enhanced by membrane association. These results provide an experimental demonstration that membrane binding enhances protein–protein interactions, an important consideration for understanding processes involving membrane-associated proteins. The experiments further demonstrate the importance of cross-linking conditions for these reactions that are often used to probe protein structure and dynamics and the potential of membrane association to restore native interactions of membrane-associated proteins for cross-linking studies.

Binding of attractant to bacterial chemotaxis receptors initiates a transmembrane signal that inhibits the kinase CheA bound ∼300 Å distant at the other end of the receptor. Chemoreceptors form large clusters in many bacterial species, and the extent of clustering has been reported to vary with signaling state. To test whether ligand binding regulates kinase activity by modulating a clustering equilibrium, we measured the effects of two-dimensional receptor concentration on kinase activity in proteoliposomes containing the purified Escherichia coli serine receptor reconstituted into vesicles over a range of lipid:protein molar ratios. The IC50 of kinase inhibition was unchanged despite a 10-fold change in receptor concentration. Such a change in concentration would have produced a measurable shift in the IC50 if receptor clustering were involved in kinase regulation, based on a simple model in which the receptor oligomerization and ligand binding equilibria are coupled. These results indicate that the primary signal, ligand control of kinase activity, does not involve a change in receptor oligomerization state. In combination with previous work on cytoplasmic fragments assembled on vesicle surfaces [Besschetnova, T. Y., et al. (2008) Proc. Natl. Acad. Sci. U.S.A.105, 12289–12294], this suggests that binding of ligand to chemotaxis receptors inhibits the kinase by inducing a conformational change that expands the membrane area occupied by the receptor cytoplasmic domain, without changing the number of associated receptors in the signaling complex.

The receptor dimers that mediate bacterial chemotaxis form high-order signaling complexes with CheW and the kinase CheA. From the packing arrangement in two crystal structures of different receptor cytoplasmic fragments, two different models have been proposed for receptor signaling arrays: the trimers-of-dimers and hedgerow models. Here we identified an interdimer distance that differs substantially in the two models, labeled the atoms defining this distance through isotopic enrichment, and measured it with 19F−13C REDOR. This was done in two types of receptor samples: isolated bacterial membranes containing overexpressed, intact receptor and soluble receptor fragments reconstituted into kinase-active signaling complexes. In both cases, the distance found was not compatible with the receptor dimer−dimer contacts observed in the trimers-of-dimers or in the hedgerow models. Comparisons of simulated and observed REDOR dephasing were used to deduce a closest approach distance at this interface, which provides a constraint for the possible arrangements of receptor assemblies.

An inexpensive, easily synthesized calixarene:fluorotoluene host:guest inclusion complex has been designed for optimization and calibration of solid-state NMR measurements of carbon–fluorine distances using Rotational Echo DOuble Resonance (REDOR). Complexation of the fluorotoluene with the calixarene host separates the molecules such that simple two-spin behavior is observed for one site with a 4.08 Å carbon–fluorine distance. Fluorotoluene dynamics within the calixarene matrix cause motional averaging of the dipolar couplings, which makes it possible to easily optimize REDOR experiments and test their accuracy for relatively long distance measurements (>6.6 Å). This provides a new tool for accurate REDOR measurements of long carbon–fluorine distances, which have important applications in the characterization of fluorine-containing drugs, proteins, and polymers.