Transmembrane signaling is a fundamental process mediated by membrane proteins, which is not yet understood. The bacterial chemoreceptor family is an ideal system for investigating the molecular mechanism of transmembrane signaling. Although extensive studies of these proteins have yielded crystal structures of soluble fragments and evidence for subtle ligand-induced conformational changes, important questions remain regarding the mechanism of signal transmission across the membrane. Our work has employed a variety of methods to probe structure and function in bacterial chemoreceptors.

Transmembrane signaling by chemotaxis receptors: conformational changes

Solid-state NMR promises to be an important tool for characterizing structure and function in membrane proteins. We have established site-directed solid-state NMR as an approach capable of measuring local structure at sites throughout large membrane proteins. Application to the serine chemotaxis receptor (Tsr) provides the first distance measurements in the intact, membrane-bound receptor with sufficient resolution to measure the subtle changes thought to transmit the signal in this receptor. Results of interhelical distance measurements in the periplasmic domain of the intact receptor are consistent with the proposed ligand-induced piston model for the signaling mechanism (1). Distance measurements in the transmembrane domain are providing constraints to refine the structural model for this region and testing the proposed propagation of the piston via the transmembrane helices (2). Ligand-to-protein distance measurements have established the similarity of the ligand sites of the Ser and Asp receptors (3). Additional distance measurements in progress at sites throughout the receptor aim to:

• Map and follow the ligand-induced conformational change.

• Determine whether the conformational change is propagated by the transmembrane helices (an alternative is propagation via lateral interactions within receptor clusters).

• Determine whether ligand binding causes symmetric or asymmetric structural changes in the receptor dimer.

• Test for proposed contacts within receptor clusters.

• Determine what structural changes occur in the cytoplasmic domain upon receptor methylation.

These studies will help to reveal the structural basis of the mechanism of transmembrane signaling.

Transmembrane signaling by chemotaxis receptors: dynamics

A variety of biophysical studies of the cytoplasmic fragment of the Asp receptor have revealed some unusual properties and suggested a role for dynamics in the signaling mechanism. In a series of collaborative studies with Weis and coworkers we have demonstrated that

• Most of the cytoplasmic fragment has a dynamic tertiary structure (4), with a small core of stable structure (characterized by slowly exchanging amide protons).

• The very slow dissociation of dimers of the cytoplasmic fragment may involve unfolding of a large fraction of the protein (5).

• The size of the core of stable structure is changed dramatically by changes in only 1-3 residues. We propose the small stable core is the tip of the receptor (region 1 in figure at right), which has low B factors in the crystal structure. The core size is increased 2-fold by a single mutation (circled yellow site) suggesting stabilization of region 3. A pH change likely to protonate the 3 His residues (green) increases the core another 2-fold, suggesting electrostatic stabilization of region 2. Both of these changes are thought to stabilize the kinase-inactivating signaling state, suggesting that this flexible domain is poised for stabilization as part of the signaling mechanism (6).

Our efforts to apply solid-state NMR to such a complex system are also yielding technique developments which will expand the applicability of this tool to address important questions in biological systems. By combining the NMR with molecular biology we have devised the site-directed approach needed to move the measurements beyond synthetic peptides or active sites of large proteins (1-2). Our rotational resonance experiments have incorporated new approaches to make more accurate long distance measurements (7) and to correct for natural abundance contributions, making it possible to study sites throughout large proteins (2). In collaboration with Klaus Schmidt-Rohr we developed a novel spin diffusion method for measuring depths within membranes to characterize structure in membrane peptides and proteins (8). We have demonstrated the utility of spin diffusion for probing structures of membrane-bound peptides (9). Our review of recent magic-angle spinning studies of proteins illustrates how a number of laboratories are using solid-state NMR methods to address questions of fundamental interest and of medical importance in a large variety of systems (10).