A nonequilibrium allosteric model for receptor-kinase complexes: The role of energy dissipation in chemotaxis signaling.

The Escherichia coli chemotaxis signaling pathway has served as a model system for the adaptive sensing of environmental signals by large protein complexes. The chemoreceptors control the kinase activity of CheA in response to the extracellular ligand concentration and adapt across a wide concentration range by undergoing methylation and demethylation. Methylation shifts the kinase response curve by orders of magnitude in ligand concentration while incurring a much smaller change in the ligand binding curve. Here, we show that the disproportionate shift in binding and kinase response is inconsistent with equilibrium allosteric models. To resolve this inconsistency, we present a nonequilibrium allosteric model that explicitly includes the dissipative reaction cycles driven by adenosine triphosphate (ATP) hydrolysis. The model successfully explains all existing joint measurements of ligand binding, receptor conformation, and kinase activity for both aspartate and serine receptors. Our results suggest that the receptor complex acts as an enzyme: Receptor methylation modulates the ON-state kinetics of the kinase (e.g., phosphorylation rate), while ligand binding controls the equilibrium balance between kinase ON/OFF states. Furthermore, sufficient energy dissipation is responsible for maintaining and enhancing the sensitivity range and amplitude of the kinase response. We demonstrate that the nonequilibrium allosteric model is broadly applicable to other sensor-kinase systems by successfully fitting previously unexplained data from the DosP bacterial oxygen-sensing system. Overall, this work provides a nonequilibrium physics perspective on cooperative sensing by large protein complexes and opens up research directions for understanding their microscopic mechanisms through simultaneous measurements and modeling of ligand binding and downstream responses.

ATP Binding as a Key Target for Control of the Chemotaxis Kinase.

In bacterial chemotaxis, chemoreceptors in signaling complexes modulate the activity of two-component histidine kinase CheA in response to chemical stimuli. CheA catalyzes phosphoryl transfer from ATP to a histidinyl residue of its P1 domain. That phosphoryl group is transferred to two response regulators. Receptor control is almost exclusively at autophosphorylation, but the aspect of enzyme action on which that control acts is unclear. We investigated this by a kinetic analysis of activated kinase in signaling complexes. We found that phosphoryl transfer from ATP to P1 is an ordered sequential reaction in which the binding of ATP to CheA is the necessary first step; the second substrate, the CheA P1 domain, binds only to an ATP-occupied enzyme; and phosphorylated P1 is released prior to the second product, namely, ADP. We confirmed the crucial features of this kinetically deduced ordered mechanism by assaying P1 binding to the enzyme. In the absence of a bound nucleotide, there was no physiologically significant binding, but the enzyme occupied with a nonhydrolyzable ATP analog bound P1. Previous structural and computational analyses indicated that ATP binding creates the P1-binding site by ordering the "ATP lid." This process identifies the structural basis for the ordered kinetic mechanism. Recent mathematical modeling of kinetic data identified ATP binding as a focus of receptor-mediated kinase control. The ordered kinetic mechanism provides the biochemical logic of that control. We conclude that chemoreceptors modulate kinase by controlling ATP binding. Structural similarities among two-component kinases, particularly the ATP lid, suggest that ordered mechanisms and control of ATP binding are general features of two-component signaling.IMPORTANCE Our work provides important new insights into the action of the chemotaxis signaling kinase CheA by identifying the kinetic mechanism of its autophosphorylation as an ordered sequential reaction, in which the required first step is binding of ATP. These insights provide a framework for integrating previous kinetic, mathematical modeling, structural, simulation, and docking observations to conclude that chemoreceptors control the activity of the chemotaxis kinase by regulating binding of the autophosphorylation substrate ATP. Previously observed conformational changes in the ATP lid of the enzyme active site provide a structural basis for the ordered mechanism. Such lids are characteristic of two-component histidine kinases in general, suggesting that ordered sequential mechanisms and regulation by controlling ATP binding are common features of these kinases.

A dual regulation mechanism of histidine kinase CheA identified by combining network-dynamics modeling and system-level input-output data.

It is challenging to decipher molecular mechanisms in biological systems from system-level input-output data, especially for complex processes that involve interactions among multiple components. We addressed this general problem for the bacterial histidine kinase CheA, the activity of which is regulated in chemotaxis signaling complexes by bacterial chemoreceptors. We developed a general network model to describe the dynamics of the system, treating the receptor complex with coupling protein CheW and the P3P4P5 domains of kinase CheA as a regulated enzyme with two substrates, ATP and P1, the phosphoryl-accepting domain of CheA. Our simple network model allowed us to search hypothesis space systematically. For different and progressively more complex regulation schemes, we fit our models to a large set of input-output data with the aim of identifying the simplest possible regulation mechanisms consistent with the data. Our modeling and analysis revealed novel dual regulation mechanisms in which receptor activity regulated ATP binding plus one other process, either P1 binding or phosphoryl transfer between P1 and ATP. Strikingly, in our models receptor control affected the kinetic rate constants of substrate association and dissociation equally and thus did not alter the respective equilibrium constants. We suggest experiments that could distinguish between the two dual-regulation mechanisms. This systems-biology approach of combining modeling and a large input-output dataset should be applicable for studying other complex biological processes.

Spatial Restrictions in Chemotaxis Signaling Arrays: A Role for Chemoreceptor Flexible Hinges across Bacterial Diversity.

The chemotactic sensory system enables motile bacteria to move toward favorable environments. Throughout bacterial diversity, the chemoreceptors that mediate chemotaxis are clustered into densely packed arrays of signaling complexes. In these arrays, rod-shaped receptors are in close proximity, resulting in limited options for orientations. A recent geometric analysis of these limitations in Escherichia coli, using published dimensions and angles, revealed that in this species, straight chemoreceptors would not fit into the available space, but receptors bent at one or both of the recently-documented flexible hinges would fit, albeit over a narrow window of shallow bend angles. We have now expanded our geometric analysis to consider variations in receptor length, orientation and placement, and thus to species in which those parameters are known to be, or might be, different, as well as to the possibility of dynamic variation in those parameters. The results identified significant limitations on the allowed combinations of chemoreceptor dimensions, orientations and placement. For most combinations, these limitations excluded straight chemoreceptors, but allowed receptors bent at a flexible hinge. Thus, our analysis identifies across bacterial diversity a crucial role for chemoreceptor flexible hinges, in accommodating the limitations of molecular crowding in chemotaxis core signaling complexes and their arrays.

Flexible Hinges in Bacterial Chemoreceptors.

Transmembrane bacterial chemoreceptors are extended, rod-shaped homodimers with ligand-binding sites at one end and interaction sites for signaling complex formation and histidine kinase control at the other. There are atomic-resolution structures of chemoreceptor fragments but not of intact, membrane-inserted receptors. Electron tomography of in vivo signaling complex arrays lack distinct densities for chemoreceptor rods away from the well-ordered base plate region, implying structural heterogeneity. We used negative staining, transmission electron microscopy, and image analysis to characterize the molecular shapes of intact homodimers of the Escherichia coli aspartate receptor Tar rendered functional by insertion into nanodisc-provided E. coli lipid bilayers. Single-particle analysis plus tomography of particles in a three-dimensional matrix revealed two bend loci in the chemoreceptor cytoplasmic domain, (i) a short, two-strand gap between the membrane-proximal, four-helix-bundle HAMP (histidine kinases, adenylyl cyclases, methyl-accepting chemoreceptors, and phosphatases) domain and the membrane-distal, four-helix coiled coil and (ii) aligned glycines in the extended, four-helix coiled coil, the position of a bend noted in the previous X-ray structure of a receptor fragment. Our images showed HAMP bends from 0° to ∼13° and glycine bends from 0° to ∼20°, suggesting that the loci are flexible hinges. Variable hinge bending explains indistinct densities for receptor rods outside the base plate region in subvolume averages of chemotaxis arrays. Bending at flexible hinges was not correlated with the chemoreceptor signaling state. However, our analyses showed that chemoreceptor bending avoided what would otherwise be steric clashes between neighboring receptors that would block the formation of core signaling complexes and chemoreceptor arrays.IMPORTANCE This work provides new information about the shape of transmembrane bacterial chemoreceptors, crucial components in the molecular machinery of bacterial chemotaxis. We found that intact, lipid-bilayer-inserted, and thus functional homodimers of the Escherichia coli chemoreceptor Tar exhibited bends at two flexible hinges along their ∼200-Å, rod-like, cytoplasmic domains. One hinge was at the short, two-strand gap between the membrane-proximal, four-helix-bundle HAMP (histidine kinases, adenylyl cyclases, methyl-accepting chemoreceptors, and phosphatases) domain and the membrane-distal, four-helix coiled coil. The other hinge was at aligned glycines in the extended, four-helix coiled coil, where a bend had been identified in the X-ray structure of a chemoreceptor fragment. Our analyses showed that flexible hinge bending avoided structural clashes in chemotaxis core complexes and their arrays.

Bacterial chemotaxis: the early years of molecular studies.

This review focuses on the early years of molecular studies of bacterial chemotaxis and motility, beginning in the 1960s with Julius Adler's pioneering work. It describes key observations that established the field and made bacterial chemotaxis a paradigm for the molecular understanding of biological signaling. Consideration of those early years includes aspects of science seldom described in journals: the accidental findings, personal interactions, and scientific culture that often drive scientific progress.

Selective allosteric coupling in core chemotaxis signaling complexes.

Bacterial chemotaxis is mediated by signaling complexes that sense chemical gradients and direct bacteria to favorable environments by controlling a histidine kinase as a function of chemoreceptor ligand occupancy. Core signaling complexes contain two trimers of transmembrane chemoreceptor dimers, each trimer binding a coupling protein CheW and a protomer of the kinase dimer. Core complexes assemble into hexagons, and these form hexagonal arrays. The notable cooperativity and amplification in bacterial chemotaxis is thought to reflect allosteric interactions in cores, hexagons, and arrays, but little is known about this presumed allostery. We investigated allostery in core complexes assembled with two chemoreceptor species, each recognizing a different ligand. Chemoreceptors were inserted in Nanodiscs, which rendered them water soluble and allowed isolation of individual complexes. Neighboring dimers in receptor trimers influenced one another's operational ligand affinity, indicating allosteric coupling. However, this coupling did not include the key function of kinase inhibition. Our data indicated that only one receptor dimer could inhibit kinase as a function of ligand occupancy. This selective allosteric coupling corresponded with previously identified structural asymmetry: only one dimer in a trimer contacts kinase and only one CheW. We suggest one of these dimers couples ligand occupancy to kinase inhibition. Additionally, we found that kinase protomers are allosterically coupled, conveying inhibition across the dimer interface. Because kinase dimers connect core complex hexagons, allosteric communication across dimer interfaces provides a pathway for receptor-generated kinase inhibition in one hexagon to spread to another, providing a crucial step for the extensive amplification characteristic of chemotactic signaling.

Core unit of chemotaxis signaling complexes.

Bacterial chemoreceptors, histidine kinase CheA, and coupling protein CheW form clusters of chemotaxis signaling complexes. In signaling complexes kinase activity is enhanced several hundredfold and placed under receptor control. Activation is necessary to poise enzyme activity such that receptor control has physiologically relevant effects. Thus kinase activation can be considered the underlying core activity of signaling complexes. We defined the minimal physical unit that generates this activity using chemoreceptor Tar from Escherichia coli rendered water soluble by insertion into nanodiscs to (i) measure saturable binding of CheA and CheW to the smallest kinase-activating groups of receptor dimers and (ii) purify and characterize core units of signaling complexes. Purified complexes activated kinase almost as well as signaling complexes formed on arrays of receptors in isolated native membrane. Purified complexes contained two receptor trimers of dimers and two CheW for each CheA dimer, consistent with the approximately 1:1 CheACheW ratio determined by binding measurements. The 2:2:1 stoichiometry implied that CheA dimers, the enzymatically active form, connect two chemoreceptor trimers of dimers by interaction of one CheA protomer and a CheW with each trimer, an organization for which specific molecular interactions have previously been identified. The core unit associates six receptor dimers with a CheA dimer, providing sufficient capacity to account for much of the cooperativity and interdimer influence observed experimentally. We conclude that the 221 organization is the core structural and functional unit of chemotaxis signaling complexes and postulate that hexagonal arrays characteristic of signaling complexes are built from this unit.

Influence of membrane lipid composition on a transmembrane bacterial chemoreceptor.

Most bacterial chemoreceptors are transmembrane proteins. Although less than 10% of a transmembrane chemoreceptor is embedded in lipid, separation from the natural membrane environment by detergent solubilization eliminates most receptor activities, presumably because receptor structure is perturbed. Reincorporation into a lipid bilayer can restore these activities and thus functionally native structure. However, the extent to which specific lipid features are important for effective restoration is unknown. Thus we investigated effects of membrane lipid composition on chemoreceptor Tar from Escherichia coli using Nanodiscs, small (∼10-nm) plugs of lipid bilayer rendered water-soluble by an annulus of "membrane scaffold protein." Disc-enclosed bilayers can be made with different lipids or lipid combinations. Nanodiscs carrying an inserted receptor dimer have high protein-to-lipid ratios approximating native membranes and in this way mimic the natural chemoreceptor environment. To identify features important for functionally native receptor structure, we made Nanodiscs using natural and synthetic lipids, assaying extents and rates of adaptational modification. The proportion of functionally native Tar was highest in bilayers closest in composition to E. coli cytoplasmic membrane. Some other lipid compositions resulted in a significant proportion of functionally native receptor, but simply surrounding the chemoreceptor transmembrane segment with a lipid bilayer was not sufficient. Membranes effective in supporting functionally native Tar contained as the majority lipid phosphatidylethanolamine or a related zwitterionic lipid plus a rather specific proportion of anionic lipids, as well as unsaturated fatty acids. Thus the chemoreceptor is strongly influenced by its lipid environment and is tuned to its natural one.

Bacterial chemoreceptors: high-performance signaling in networked arrays.

Chemoreceptors are crucial components in the bacterial sensory systems that mediate chemotaxis. Chemotactic responses exhibit exquisite sensitivity, extensive dynamic range and precise adaptation. The mechanisms that mediate these high-performance functions involve not only actions of individual proteins but also interactions among clusters of components, localized in extensive patches of thousands of molecules. Recently, these patches have been imaged in native cells, important features of chemoreceptor structure and on-off switching have been identified, and new insights have been gained into the structural basis and functional consequences of higher order interactions among sensory components. These new data suggest multiple levels of molecular interactions, each of which contribute specific functional features and together create a sophisticated signaling device.

Resolving the binding-kinase discrepancy in bacterial chemotaxis: A nonequilibrium allosteric model and the role of energy dissipation.

The Escherichia coli chemotaxis signaling pathway has served as a model system for studying the adaptive sensing of environmental signals by large protein complexes. The chemoreceptors control the kinase activity of CheA in response to the extracellular ligand concentration and adapt across a wide concentration range by undergoing methylation and demethylation. Methylation shifts the kinase response curve by orders of magnitude in ligand concentration while incurring a much smaller change in the ligand binding curve. Here, we show that this asymmetric shift in binding and kinase response is inconsistent with equilibrium allosteric models regardless of parameter choices. To resolve this inconsistency, we present a nonequilibrium allosteric model that explicitly includes the dissipative reaction cycles driven by ATP hydrolysis. The model successfully explains all existing measurements for both aspartate and serine receptors. Our results suggest that while ligand binding controls the equilibrium balance between the ON and OFF states of the kinase, receptor methylation modulates the kinetic properties (e.g., the phosphorylation rate) of the ON state. Furthermore, sufficient energy dissipation is necessary for maintaining and enhancing the sensitivity range and amplitude of the kinase response. We demonstrate that the nonequilibrium allosteric model is broadly applicable to other sensor-kinase systems by successfully fitting previously unexplained data from the DosP bacterial oxygen-sensing system. Overall, this work provides a new perspective on cooperative sensing by large protein complexes and opens up new research directions for understanding their microscopic mechanisms through simultaneous measurements and modeling of ligand binding and downstream responses.

Chemotaxis kinase CheA is activated by three neighbouring chemoreceptor dimers as effectively as by receptor clusters.

Chemoreceptors are central to bacterial chemotaxis. These transmembrane homodimers form trimers of dimers. Trimers form clusters of a few to thousands of receptors. A crucial receptor function is 100-fold activation, in signalling complexes, of sensory histidine kinase CheA. Significant activation has been shown to require more than one receptor dimer but the number required for full activation was unknown. We investigated this issue using Nanodiscs, soluble, nanoscale (∼10 nm diameter) plugs of lipid bilayer, to limit the number of neighbouring receptors contributing to activation. Utilizing size-exclusion chromatography, we separated primary preparations of receptor-containing Nanodiscs, otherwise heterogeneous for number and orientation of inserted receptors, into fractions enriched for specific numbers of dimers per disc. Fractionated, clarified Nanodiscs carrying approximately five dimers per disc were as effective in activating kinase as native membrane vesicles containing many neighbouring dimers. At five independently inserted dimers per disc, every disc would have at least three dimers oriented in parallel and thus able act together as they would in native membrane. We conclude full kinase activation involves interaction of CheA with groups of three receptor dimers, presumably as a trimer of dimers, and that more extensive interactions among receptors are not necessary for full kinase activation.

Chemoreceptors in signalling complexes: shifted conformation and asymmetric coupling.

Bacterial chemotaxis is mediated by signalling complexes of chemoreceptors, histidine kinase CheA and coupling protein CheW. Interactions in complexes profoundly affect the kinase. We investigated effects of these interactions on chemoreceptors by comparing receptors alone and in complexes. Assays of initial rates of methylation indicated that signalling complexes shifted receptor conformation towards the methylation-on, higher-ligand-affinity, kinase-off state, tuning receptors for greater sensitivity. In contrast, transmembrane and conformational signalling within chemoreceptors was essentially unaltered, consistent with other evidence identifying receptor dimers as the fundamental units of such signalling. In signalling complexes, coupling of ligand binding to kinase activity is cooperative and the dynamic range of kinase control expanded > 100-fold by receptor adaptational modification. We observed no cooperativity in influence of ligand on receptor conformation, only on kinase activity. However, receptor modification generated increased dynamic range in a stepwise fashion, partly in coupling ligand to receptor conformation and partly in coupling receptor conformation to kinase activity. Thus, receptors and kinase were not equivalently affected by interactions in signalling complexes or by ligand binding and adaptational modification, indicating asymmetrical coupling between them. This has implications for mechanisms of precise adaptation. Coupling might vary, providing a previously unappreciated locus for sensory control.

A Selective Tether Recruits Activated Response Regulator CheB to Its Chemoreceptor Substrate.

Methylesterase/deamidase CheB is a key component of bacterial chemotaxis systems. It is also a prominent example of a two-component response regulator in which the effector domain is an enzyme. Like other response regulators, CheB is activated by phosphorylation of an aspartyl residue in its regulatory domain, creating an open conformation between its two domains. Studies of CheB in Escherichia coli and related organisms have shown that its enzymatic action is also enhanced by a pentapeptide-binding site for the enzyme at the chemoreceptor carboxyl terminus. Related carboxyl-terminal pentapeptides are found on >25,000 chemoreceptor sequences distributed across 11 bacterial phyla and many bacterial species, in which they presumably play similar roles. Yet, little is known about the interrelationship of CheB phosphorylation, pentapeptide binding, and interactions with its substrate methylesters and amides on the body of the chemoreceptor. We investigated by characterizing the binding kinetics of CheB to Nanodisc-inserted chemoreceptor dimers. The resulting kinetic and thermodynamic constants revealed a synergy between CheB phosphorylation and pentapeptide binding in which a phosphorylation mimic enhanced pentapeptide binding, and the pentapeptide served not only as a high-affinity tether for CheB but also selected the activated conformation of the enzyme. The basis of this selection was revealed by molecular modeling that predicted a pentapeptide-binding site on CheB which existed only in the open, activated enzyme. Recruitment of activated enzyme by selective tethering represents a previously unappreciated strategy for regulating response regulator action, one that may well occur in other two-component systems. IMPORTANCE Two-component signal transduction systems are a primary means by which bacteria sense and respond to their environment. Response regulators are key components of these systems. Phosphorylation of response regulators by cognate histidine kinases generate active conformations which act on specific targets, DNA sequences or proteins. The targets have been considered passive in this process. Our characterization of interaction between response regulator CheB and its target chemoreceptor revealed active participation of the target in response regulator action. We found that a pentapeptide sequence at the carboxyl terminus of Escherichia coli chemoreceptors is a selective tether that binds only phosphorylated CheB, thus selecting the form of this two-component enzyme active for covalent modification of the selecting chemoreceptor. Analogous pentapeptides are found on chemoreceptors in many bacterial species and are presumably also selective tethers. There may well be other, uncharacterized examples of active participation of target molecules in response to regulator action.

Concerted Differential Changes of Helical Dynamics and Packing upon Ligand Occupancy in a Bacterial Chemoreceptor.

Transmembrane receptors are central components of the chemosensory systems by which motile bacteria detect and respond to chemical gradients. An attractant bound to the receptor periplasmic domain generates conformational signals that regulate a histidine kinase interacting with its cytoplasmic domain. Ligand-induced signaling through the periplasmic and transmembrane domains of the receptor involves a piston-like helical displacement, but the nature of this signaling through the >200 Å four-helix coiled coil of the cytoplasmic domain had not yet been identified. We performed single-molecule Förster resonance energy transfer measurements on Escherichia coli aspartate receptor homodimers inserted into native phospholipid bilayers enclosed in nanodiscs. The receptors were labeled with fluorophores at diagnostic positions near the middle of the cytoplasmic coiled coil. At these positions, we found that the two N-helices of the homodimer were more distant, that is, less tightly packed and more dynamic than the companion C-helix pair, consistent with previous deductions that the C-helices form a stable scaffold and the N-helices are dynamic. Upon ligand binding, the scaffold pair compacted further, while separation and dynamics of the dynamic pair increased. Thus, ligand binding had asymmetric effects on the two helical pairs, shifting mean distances in opposite directions and increasing the dynamics of one pair. We suggest that this reflects a conformational change in which differential alterations to the packing and dynamics of the two helical pairs are coupled. These coupled changes could represent a previously unappreciated mode of conformational signaling that may well occur in other coiled-coil signaling proteins.

The chemoreceptor dimer is the unit of conformational coupling and transmembrane signaling.

Transmembrane chemoreceptors are central components in bacterial chemotaxis. Receptors couple ligand binding and adaptational modification to receptor conformation in processes that create transmembrane signaling. Homodimers, the fundamental receptor structural units, associate in trimers and localize in patches of thousands. To what degree do conformational coupling and transmembrane signaling require higher-order interactions among dimers? To what degree are they altered by such interactions? To what degree are they inherent features of homodimers? We addressed these questions using nanodiscs to create membrane environments in which receptor dimers had few or no potential interaction partners. Receptors with many, few, or no interaction partners were tested for conformational changes and transmembrane signaling in response to ligand occupancy and adaptational modification. Conformation was assayed by measuring initial rates of receptor methylation, a parameter independent of receptor-receptor interactions. Coupling of ligand occupancy and adaptational modification to receptor conformation and thus to transmembrane signaling occurred with essentially the same sensitivity and magnitude in isolated dimers as for dimers with many neighbors. Thus, we conclude that the chemoreceptor dimer is the fundamental unit of conformational coupling and transmembrane signaling. This implies that in signaling complexes, coupling and transmembrane signaling occur through individual dimers and that changes between dimers in a receptor trimer or among trimer-based signaling complexes are subsequent steps in signaling.

Methyltransferase CheR binds to its chemoreceptor substrates independent of their signaling conformation yet modifies them differentially.

Methylation of specific chemoreceptor glutamyl residues by methyltransferase CheR mediates sensory adaptation and gradient sensing in bacterial chemotaxis. Enzyme action is a function of chemoreceptor signaling conformation: kinase-off receptors are more readily methylated than kinase-on, a feature central to adaptational and gradient-sensing mechanisms. Differential enzyme action could reflect differential binding, catalysis or both. We investigated by measuring CheR binding to kinase-off and kinase-on forms of Escherichia coli aspartate receptor Tar deleted of its CheR-tethering, carboxyl terminus pentapeptide. This allowed characterization of the low-affinity binding of enzyme to the substrate receptor body, otherwise masked by high-affinity interaction with pentapeptide. We quantified the low-affinity protein-protein interactions by determining kinetic rate constants of association and dissociation using bio-layer interferometry and from those values calculating equilibrium constants. Whether Tar signaling conformations were shifted by ligand occupancy or adaptational modification, there was little or no difference between the two signaling conformations in kinetic or equilibrium parameters of enzyme-receptor binding. Thus, differential methyltransferase action does not reflect differential binding. Instead, the predominant determinants of binding must be common to different signaling conformations. Characterization of the dependence of association rate constants on Deybe length, a measure of the influence of electrostatics, implicated electrostatic interactions as a common binding determinant. Taken together, our observations indicate that differential action of methyltransferase on kinase-off and kinase-on chemoreceptors is not the result of differential binding and suggest it reflects differential catalytic propensity. Differential catalysis rather than binding could well be central to other enzymes distinguishing alternative conformations of protein substrates.

Analyzing transmembrane chemoreceptors using in vivo disulfide formation between introduced cysteines.

The sulfhydryl chemistry possible at the thiol group of cysteine provides a very useful tool for probing protein structure and function. The power of site-specific mutagenesis makes it possible to use this tool at essentially any position in a polypeptide sequence. The reactivity of introduced cysteines is often assessed in vitro, using purified proteins or cell extracts. However, it can be particularly informative to probe the protein of interest in vivo, in its native cellular environment. Our laboratory has used in vivo approaches extensively in studies of bacterial transmembrane chemoreceptors, particularly by utilizing disulfide formation between pairs of introduced cysteines to learn about structural organization and mechanisms of function. We have concentrated on experimental conditions in which the cellular system of interest remained functional and thus the protein we were characterizing maintained not only its native structure but also its natural interactions. For this reason, our studies of bacterial transmembrane chemoreceptors using disulfide formation in vivo have focused in large part on cysteines separated from the reducing environment of the cell interior, in transmembrane or periplasmic domains. In this chapter, we discuss the applications and limitation of these approaches as well as the details of experimental manipulations and data analysis.

Using Nanodiscs to create water-soluble transmembrane chemoreceptors inserted in lipid bilayers.

In this chapter we describe application of the emerging technology of Nanodiscs to chemoreceptors, a class of transmembrane proteins that presents many challenges to the investigator. Nanodiscs are soluble, nanoscale ( approximately 10nm diameter) particles of lipid bilayer surrounded by an annulus of amphipathic protein, the membrane scaffold protein. A transmembrane protein inserted in a Nanodisc is surrounded by a lipid bilayer much as it is prior to detergent solublization. Thus, the Nanodisc-inserted protein is in an environment that approximates its native state. Yet, that membrane protein is also water-soluble and segregated from other membrane proteins because the bilayer into which it is inserted is of very limited size and, with appropriate preparation, contains only a single protein. In a Nanodisc, the water-soluble, bilayer-inserted membrane protein can be purified by conventional techniques and analyzed for activities and interactions as a pure entity. Thus, Nanodisc technology has great promise for improving isolation, purification, and characterization of the many membrane proteins that are difficult to handle, become unstable, or lose native activity when surrounded by detergent instead of lipid bilayer. The technology has proven useful for the investigation of chemoreceptor activity as a function of oligomeric state.