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In bacterial chemotaxis, an assembly of transmembrane receptors, the CheA histidine kinase and the adaptor protein CheW processes environmental stimuli to regulate motility. The structure of a Thermotoga maritima receptor cytoplasmic domain defines CheA interaction regions and metal ion-coordinating charge centers that undergo chemical modification to tune receptor response. Dimeric CheA-CheW, defined by crystallography and pulsed ESR, positions two CheWs to form a cleft that is lined with residues important for receptor interactions and sized to clamp one receptor dimer. CheW residues involved in kinase activation map to interfaces that orient the CheW clamps. CheA regulatory domains associate in crystals through conserved hydrophobic surfaces. Such CheA self-contacts align the CheW receptor clamps for binding receptor tips. Linking layers of ternary complexes with close-packed receptors generates a lattice with reasonable component ratios, cooperative interactions among receptors and accessible sites for modification enzymes.

We provide a review of current electron spin resonance (ESR) techniques for studying basic molecular mechanisms in membranes and proteins by using nitroxide spin labels. In particular, nitroxide spin label studies with high-field/high-frequency ESR and two-dimensional Fourier transform ESR enable one to accurately determine distances in biomolecules, unravel the details of the complex dynamics in proteins, characterize the dynamic structure of membrane domains, and discriminate between bulk lipids and boundary lipids that coat transmembrane peptides or proteins; these studies can also provide time resolution to studies of functional dynamics of proteins. We illustrate these capabilities with recent examples.

We apply pulsed dipolar ESR spectroscopy (Ku-band DEER) to elucidate the global conformation of the Parkinson's disease-associated protein, alpha-synuclein (αS) bound to small unilamellar phospholipid vesicles, rod-like SDS micelles, or lipid bicelles. By measuring distances as long as ~7 nm between introduced pairs of nitroxide spin labels, we show that distances are close to the expectations for a single continuous helix in all cases studied. In particular, we find distances of 7.5 nm between sites 24 and 72; 5.5 nm between sites 24 and 61; and 2 nm between sites 35 and 50. We conclude that αS does not retain a 'hairpin' structure with two antiparallel helices, as is known to occur with spheroidal micelles, in agreement with our earlier finding that the protein's geometry is determined by the surface topology rather than being constrained by the inter-helix linker. While the possibility of local helix discontinuities in the structure of membrane-bound αS remains, our data are more consistent with one intact helix. Importantly, we demonstrate that bicelles produce very similar results to liposomes, while offering a major improvement in experimentally accessible distance range and resolution, and thus are an excellent lipid membrane mimetic for the purpose of pulse dipolar ESR spectroscopy.Alpha-synuclein (αS) was originally discovered as a protein highly enriched in synaptosome preparations from the electric ray T. californica 1 and was later linked to both familial and sporadic Parkinson's disease (PD) through the discovery that αS point mutations or gene duplication/triplication cause familial PD and through the identification of αS as the major component of amyloid fibril aggregates present in the Lewy body deposits that are a diagnostic hallmark of PD. Both the normal function of αS and the precise relation between its aggregation and deposition in Lewy bodies and PD remain unclear. When isolated in solution, the protein is intrinsically disordered, but in the presence of lipid surfaces αS adopts a highly helical structure 2 that is believed to mediate its normal function(s). NMR-based characterization of this helical structure using detergent micelles as a membrane mimetic has shown that the protein E-mail: dae2005@med.cornell.edu; jhf@cornell.edu. adopts two extended surface-bound helices separated by a non-helical linker, that the helices are oriented in an antiparallel fashion, and that no inter-helical contacts are formed. 3-7 The slow tumbling rate of intact phospholipid vesicles precluded direct studies of the vesicle-bound conformation of αS using solution NMR methods, but it was proposed 3,8 that in the vesiclebound state, the two helices may become collinear and fuse into a single long surface-bound helix. Support for this possibility was provided by pulsed dipolar ESR (PDS) distance measurements of αS bound to different sized micelles, which showed that the helices splay further apart on the surface of larger micelles. 9 NIH Public AccessHere we use PDS, 10-13 namely 17.3 GHz DEER (cf. Su...

We report the use of a novel pulsed ESR technique for distance measurement, based on the detection of double quantum coherence (DQC), which yields high quality dipolar spectra, to significantly extend the range of measurable distances in proteins using nitroxide spin-labels. Eight T4 lysozyme (T4L) mutants, doubly labeled with methanethiosulfonate spin-label (MTSSL), have been studied using DQC-ESR at 9 and 17 GHz. The distances span the range from 20 A for the 65/76 mutant to 47 A for the 61/135 mutant. The high quality of the dipolar spectra also allows the determination of the distance distributions, the width of which can be used to set upper and lower bounds in future computational strategy. It is also demonstrated that the shape of these distributions can reveal the presence of multiple conformations of the spin-label, an issue of critical relevance to the structural interpretation of the distances. The distances and distributions found in this study are readily rationalized in terms of the known crystal structure, the characteristic conformers of the nitroxide side chains, and molecular modeling. This study sets the stage for the use of DQC-ESR for determining the tertiary structure of large proteins with just a small number of long-distance constraints.

Pulsed electron spin resonance (ESR) dipolar spectroscopy (PDS) in combination with site-directed spin labeling is unique in providing nanometer- range distances and distributions in biological systems. To date, most of the pulsed ESR techniques require frozen solutions at cryogenic temperatures to reduce the rapid electron spin relaxation rate and to prevent averaging of electron-electron dipolar interaction due to the rapid molecular tumbling. To enable measurements in liquid solution, we are exploring a triarylmethyl (TAM)-based spin label with a relatively long relaxation time where the protein is immobilized by attachment to a solid support. In this preliminary study, TAM radicals were attached via disulfide linkages to substituted cysteine residues at positions 65 and 80 or 65 and 76 in T4 lysozyme immobilized on Sepharose. Interspin distances determined using double quantum coherence (DQC) in solution are close to those expected from models, and the narrow distance distribution in each case indicates that the TAM-based spin label is relatively localized.

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