[show abstract][hide abstract] ABSTRACT: HAMP domains are signal relay modules in >26,000 receptors of bacteria, eukaryotes, and archaea that mediate processes involved in chemotaxis, pathogenesis, and biofilm formation. We identify two HAMP conformations distinguished by a four- to two-helix packing transition at the C-termini that send opposing signals in bacterial chemoreceptors. Crystal structures of signal-locked mutants establish the observed structure-to-function relationships. Pulsed dipolar electron spin resonance spectroscopy of spin-labeled soluble receptors active in cells verify that the crystallographically defined HAMP conformers are maintained in the receptors and influence the structure and activity of downstream domains accordingly. Mutation of HR2, a key residue for setting the HAMP conformation and generating an inhibitory signal, shifts HAMP structure and receptor output to an activating state. Another HR2 variant displays an inverted response with respect to ligand and demonstrates the fine energetic balance between "on" and "off" conformers. A DExG motif found in membrane proximal HAMP domains is shown to be critical for responses to extracellular ligand. Our findings directly correlate in vivo signaling with HAMP structure, stability, and dynamics to establish a comprehensive model for HAMP-mediated signal relay that consolidates existing views on how conformational signals propagate in receptors. Moreover, we have developed a rational means to manipulate HAMP structure and function that may prove useful in the engineering of bacterial taxis responses.
[show abstract][hide abstract] ABSTRACT: Sodium and aspartate symporter from Pyrococcus horikoshii, Glt(Ph), is a homolog of the mammalian glutamate transporters, homotrimeric integral membrane proteins that control neurotransmitter levels in brain synapses. These transporters function by alternating between outward-facing and inward-facing states, in which the substrate binding site is oriented toward the extracellular space and the cytoplasm, respectively. Here we used double electron-electron resonance (DEER) spectroscopy to probe the structure and the state distribution of the subunits in the trimer in distinct hydrophobic environments of detergent micelles and lipid bilayers. Our experiments reveal a conformational ensemble of protomers that sample the outward-facing and inward-facing states with nearly equal probabilities, indicative of comparable energies, and independently of each other. On average, the distributions varied only modestly in detergent and in bilayers, but in several mutants unique conformations were stabilized by the latter.
[show abstract][hide abstract] ABSTRACT: We describe significantly improved long-distance measurements in biomolecules by use of the new multipulse double electron-electron spin resonance (DEER) illustrated with the example of a five-pulse DEER sequence. In this sequence, an extra pulse at the pump frequency is used compared with standard four-pulse DEER. The position of the extra pulse is fixed relative to the three pulses of the detection sequence. This significantly reduces the effect of nuclear spin-diffusion on the electron-spin phase relaxation, thereby enabling longer dipolar evolution times that are required to measure longer distances. Using spin-labeled T4 lysozyme at a concentration less than 50 μM, as an example, we show that the evolution time increases by a factor of 1.8 in protonated solution and 1.4 in deuterated solution to 8 and 12 μs, respectively, with the potential to increase them further. This enables a significant increase in the measurable distances, improved distance resolution, or both.
[show abstract][hide abstract] ABSTRACT: Bacterial receptors typically contain modular architectures with distinct functional domains that combine to send signals in response to stimuli. Although the properties of individual components have been investigated in many contexts, there is little information about how diverse sets of modules work together in full-length receptors. Here we investigate the architecture of Aer2, a soluble gas-sensing receptor that has emerged as a model for PAS and poly-HAMP domain signaling. The crystal structure of the heme-binding PAS domain in the ferric, ligand-free form, in comparison to the previously determined cyanide-bound state,identifies conformational changes induced by ligand bindingthat are likely essential forthe signaling mechanism.Heme-pocket alternations share some similarities with the heme-based PAS sensors FixL and EcDOS, but propagate to the Iβ-strand in a manner predicted to alter PAS-PAS associations and the downstream HAMP junction within full-length Aer2. SAXSof PAS and poly-HAMP domain fragments of increasing complexity allow unambiguous domain assignments and reveal a linear quaternary structure. The Aer2 PAS dimeric crystal structure fits well within ab initioSAXS molecular envelopes and pulsed-dipolar ESR measurements of inter-PAS distances confirm the crystallographic PAS arrangement within Aer2. Spectroscopic and pull-down assays fail to detect direct interactions between the PAS and HAMP domains. Overall, the Aer2 signaling mechanism differs from the E. coliAer paradigm, where side-on PAS-HAMP contacts are key. We propose an in-line model for Aer2 signaling, where ligand binding induces alterations in PAS domain structure and subunit association that is relayed through the poly-HAMP junction to downstream domains.
Journal of Molecular Biology 12/2012; · 3.91 Impact Factor
[show abstract][hide abstract] ABSTRACT: Lipoxygenase enzymes initiate diverse signaling pathways by specifically directing oxygen to different carbons of arachidonate and other polyunsaturated acyl chains, but structural origins of this specificity have remained unclear. We therefore determined the nature of the lipoxygenase interaction with the polar-end of a paramagnetic lipid by electron paramagnetic resonance spectroscopy. Distances between selected grid points on soybean seed lipoxygenase-1 (SBL1) and a lysolecithin spin-labeled on choline were measured by pulsed (electron) dipolar spectroscopy. The protein grid was designed by structure-based modeling so that five natural side chains were replaced with spin labels. Pairwise distances in 10 doubly spin-labeled mutants were examined by pulsed dipolar spectroscopy, and a fit to the model was optimized. Finally, experimental distances between the lysolecithin spin and each single spin site on SBL1 were also obtained. With these 15 distances, distance geometry localized the polar-end and the spin of the lysolecithin to the region between the two domains in the SBL1 structure, nearest to E236, K260, Q264, and Q544. Mutation of a nearby residue, E256A, relieved the high pH requirement for enzyme activity of SBL1 and allowed lipid binding at pH 7.2. This general approach could be used to locate other flexible molecules in macromolecular complexes.
[show abstract][hide abstract] ABSTRACT: 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.
Journal of the American Chemical Society 06/2012; 134(24):9950-2. · 10.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: Biologically important protein complexes often involve molecular interactions that are low affinity or transient. We apply pulsed dipolar electron spin resonance spectroscopy and site-directed spin labeling in what to our knowledge is a new approach to study aggregation and to identify regions on protein surfaces that participate in weak, but specific molecular interactions. As a test case, we have probed the self-association of the chemotaxis kinase CheA, which forms signaling clusters with chemoreceptors and the coupling protein CheW at the poles of bacterial cells. By measuring the intermolecular dipolar interactions sensed by spin-labels distributed over the protein surface, we show that the soluble CheA kinase aggregates to a small extent through interactions mediated by its regulatory (P5) domain. Direct dipolar distance measurements confirm that a hydrophobic surface at the periphery of P5 subdomain 2 associates CheA dimers in solution. This result is further supported by differential disulfide cross-linking from engineered cysteine reporter sites. We suggest that the periphery of P5 is an interaction site on CheA for other similar hydrophobic surfaces and plays an important role in structuring the signaling particle.
[show abstract][hide abstract] ABSTRACT: Channel and nonchannel forms of gramicidin A (GA) were studied by ESR in various lipid environments using new mono- and double-spin-labeled compounds. For GA channels, we demonstrate here how pulse dipolar ESR can be used to determine the orientation of the membrane-traversing molecule relative to the membrane normal and to study subtle effects of lipid environment on the interspin distance in the spin-labeled gramicidin channel. To study nonchannel forms of gramicidin, pulse dipolar ESR was used first to determine interspin distances corresponding to monomers and double-helical dimers of spin-labeled GA molecules in the organic solvents trifluoroethanol and octanol. The same distances were then observed in membranes. Since detection of nonchannel forms in the membrane is complicated by aggregation, we suppressed any dipolar spectra from intermolecular interspin distances arising from the aggregates by using double-labeled GA in a mixture with excess unlabeled GA. In hydrophobic mismatching lipids (L(β) phase of DPPC), gramicidin channels dissociate into free monomers. The backbone structure of the monomeric form is similar to a monomeric unit of the channel dimer. In addition to channels and monomers, the double-helical conformation of gramicidin is present in some membrane environments. In the gel phase of saturated phosphatidylcholines, the fraction of double helices increases in the following order: DLPC < DMPC < DSPC < DPPC. The equilibrium DHD/monomer ratio in DPPC was determined. In membranes, the double-helical form is present only in aggregates. In addition, we studied the effect of N-terminal substitution in the GA molecule upon channel formation. This work demonstrates how pulsed dipolar ESR may be utilized to study complex equilibria of peptides in membranes.
The Journal of Physical Chemistry B 01/2011; 115(1):176-85. · 3.61 Impact Factor
[show abstract][hide abstract] ABSTRACT: Alpha-synuclein (alphaS) is linked to Parkinson disease through its deposition in an amyloid fibril form within Lewy Body deposits, and by the existence of three alphaS point mutations that lead to early onset autosomal dominant Parkinsonism. The normal function of alphaS is thought to be linked to the ability of the protein to bind to the surface of synaptic vesicles. Upon binding to vesicles, alphaS undergoes a structural reorganization from a dynamic and disordered ensemble to a conformation consisting of a long extended helix. In the presence of small spheroidal detergent micelles, however, this extended helix conformation can convert into a broken helix state, in which a region near the middle of the helix unwinds to form a linker between the two resulting separated helices. Membrane-bound conformations of alphaS likely mediate the function of the protein, but may also play a role in the aggregation and toxicity of the protein. Here we have undertaken a study of the effects of the three known PD-linked mutations on the detergent- and membrane-bound conformations of alphaS, as well as factors that govern the transition of the protein between the extended helix and broken helix states. Using pulsed dipolar ESR measurements of distances up to 8.7 nm, we show that all three PD-linked alphaS mutants retain the ability to transition from the broken helix to the extended helix conformation. In addition, we find that the ratio of protein to detergent, rather than just the absolute detergent concentration, determines whether the protein adopts the broken or extended helix conformation.
Journal of Biological Chemistry 09/2010; 285(36):28261-74. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: α-Synuclein (αS) is linked to Parkinson disease through its deposition in an amyloid fibril form within Lewy Body deposits,
and by the existence of three αS point mutations that lead to early onset autosomal dominant Parkinsonism. The normal function
of αS is thought to be linked to the ability of the protein to bind to the surface of synaptic vesicles. Upon binding to vesicles,
αS undergoes a structural reorganization from a dynamic and disordered ensemble to a conformation consisting of a long extended
helix. In the presence of small spheroidal detergent micelles, however, this extended helix conformation can convert into
a broken helix state, in which a region near the middle of the helix unwinds to form a linker between the two resulting separated
helices. Membrane-bound conformations of αS likely mediate the function of the protein, but may also play a role in the aggregation
and toxicity of the protein. Here we have undertaken a study of the effects of the three known PD-linked mutations on the
detergent- and membrane-bound conformations of αS, as well as factors that govern the transition of the protein between the
extended helix and broken helix states. Using pulsed dipolar ESR measurements of distances up to 8.7 nm, we show that all
three PD-linked αS mutants retain the ability to transition from the broken helix to the extended helix conformation. In addition,
we find that the ratio of protein to detergent, rather than just the absolute detergent concentration, determines whether
the protein adopts the broken or extended helix conformation.
Journal of Biological Chemistry 09/2010; 285(36):28261-28274. · 4.65 Impact Factor
[show abstract][hide abstract] ABSTRACT: The signaling apparatus that controls bacterial chemotaxis is composed of a core complex containing chemoreceptors, the histidine autokinase CheA, and the coupling protein CheW. Site-specific spin labeling and pulsed dipolar ESR spectroscopy (PDS) have been applied to investigate the structure of a soluble ternary complex formed by Thermotoga maritima CheA (TmCheA), CheW, and receptor signaling domains. Thirty-five symmetric spin-label sites (SLSs) were engineered into the five domains of the CheA dimer and CheW to provide distance restraints within the CheA:CheW complex in the absence and presence of a soluble receptor that inhibits kinase activity (Tm14). Additional PDS restraints among spin-labeled CheA, CheW, and an engineered single-chain receptor labeled at six different sites allow docking of the receptor structure relative to the CheA:CheW complex. Disulfide cross-linking between selectively incorporated Cys residues finds two pairs of positions that provide further constraints within the ternary complex: one involving Tm14 and CheW and another involving Tm14 and CheA. The derived structure of the ternary complex indicates a primary site of interaction between CheW and Tm14 that agrees well with previous biochemical and genetic data for transmembrane chemoreceptors. The PDS distance distributions are most consistent with only one CheW directly engaging one dimeric Tm14. The CheA dimerization domain (P3) aligns roughly antiparallel to the receptor-conserved signaling tip but does not interact strongly with it. The angle of the receptor axis with respect to P3 and the CheW-binding P5 domains is bound by two limits differing by approximately 20 degrees . In one limit, Tm14 aligns roughly along P3 and may interact to some extent with the hinge region near the P3 hairpin loop. In the other limit, Tm14 tilts to interact with the P5 domain of the opposite subunit in an interface that mimics that observed with the P5 homologue CheW. The time domain ESR data can be simulated from the model only if orientational variability is introduced for the P5 and, especially, P3 domains. The Tm14 tip also binds beside one of the CheA kinase domains (P4); however, in both bound and unbound states, P4 samples a broad range of distributions that are only minimally affected by Tm14 binding. The CheA P1 domains that contain the substrate histidine are also broadly distributed in space under all conditions. In the context of the hexagonal lattice formed by trimeric transmembrane chemoreceptors, the PDS structure is best accommodated with the P3 domain in the center of a honeycomb edge.
[show abstract][hide abstract] ABSTRACT: Magnetic resonance force microscopy is a promising route to 3-dimensional nanoscale imaging of organic materials due to its high sensitivity and isotopic specificity. Labeling of proteins, DNA and biomolecular assemblies with free radical labels for inductive detection are well established techniques, although many of these radical's relaxation times are too short to support previously demonstrated techniques for single electron detection by magnetic resonance force microscopy. We report on our efforts toward sub-single electron sensitivity on organic radicals using batch fabricated 100 nm nickel nanorod tipped ultrasensitive cantilevers.
[show abstract][hide abstract] ABSTRACT: The double quantum coherence (DQC) echo signal for two coupled nitroxides separated by distances ≳10 Å, is calculated rigorously for the six-pulse sequence. Successive application of six pulses on the initial density matrix, with appropriate inter-pulse time evolution and coherence pathway selection leaves only the coherent pathways of interest. The amplitude of the echo signal following the last π pulse can be used to obtain a one-dimensional dipolar spectrum (Pake doublet), and the echo envelope can be used to construct the two-dimensional DQC spectrum. The calculations are carried out using the product space spanned by the two electron-spin magnetic quantum numbers m(1), m(2) and the two nuclear-spin magnetic quantum numbers M(1), M(2), describing e.g. two coupled nitroxides in bilabeled proteins. The density matrix is subjected to a cascade of unitary transformations taking into account dipolar and electron exchange interactions during each pulse and during the evolution in the absence of a pulse. The unitary transformations use the eigensystem of the effective spin-Hamiltonians obtained by numerical matrix diagonalization. Simulations are carried out for a range of dipolar interactions, D, and microwave magnetic field strength B for both fixed and random orientations of the two (14)N (and (15)N) nitroxides. Relaxation effects were not included. Several examples of one- and two-dimensional Fourier transforms of the time domain signals vs. dipolar evolution and spin-echo envelope time variables are shown for illustration. Comparisons are made between 1D rigorous simulations and analytical approximations. The rigorous simulations presented here provide insights into DQC ESR spectroscopy, they serve as a standard to evaluate the results of approximate theories, and they can be employed to plan future DQC experiments.
Applied Magnetic Resonance 12/2009; 36(2-4):237-258. · 0.83 Impact Factor
[show abstract][hide abstract] ABSTRACT: We report an approach that extends the applicability of ultrasensitive force-gradient detection of magnetic resonance to samples with spin-lattice relaxation times (T (1)) as short as a single cantilever period. To demonstrate the generality of the approach, which relies on detecting either cantilever frequency or phase, we used it to detect electron spin resonance from a T (1) = 1 ms nitroxide spin probe in a thin film at 4.2 K and 0.6 T. By using a custom-fabricated cantilever with a 4 microm-diameter nickel tip, we achieve a magnetic resonance sensitivity of 400 Bohr magnetons in a 1 Hz bandwidth. A theory is presented that quantitatively predicts both the lineshape and the magnitude of the observed cantilever frequency shift as a function of field and cantilever-sample separation. Good agreement was found between nitroxide T (1) 's measured mechanically and inductively, indicating that the cantilever magnet is not an appreciable source of spin-lattice relaxation here. We suggest that the new approach has a number of advantages that make it well suited to push magnetic resonance detection and imaging of nitroxide spin labels in an individual macromolecule to single-spin sensitivity.
Proceedings of the National Academy of Sciences 12/2009; 106(52):22251-6. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: A new method for measuring forces between small protein domains based on double electron-electron resonance (DEER) spectroscopy is demonstrated using a model peptide derived from the alpha-helical coiled-coil leucine zipper of yeast transcriptional activator GCN4. The equilibrium distribution of distances between two nitroxide spin labels rigidly attached to the helices of the dimer was determined by DEER and yielded a closing force of 100 +/- 10 pN between monomers, in excellent agreement with theoretical predictions.
Journal of the American Chemical Society 04/2009; 131(15):5374-5. · 10.68 Impact Factor
[show abstract][hide abstract] ABSTRACT: Neurotransmitter release at the synapse requires membrane fusion. The SNARE complex, composed of the plasma membrane t-SNAREs syntaxin 1A and SNAP-25 and the vesicle v-SNARE synaptobrevin, mediates the fusion of 2 membranes. Synaptic vesicles contain unusually high cholesterol, but the exact role of cholesterol in fusion is not known. In this study, cholesterol was found to stimulate SNARE-mediated lipid mixing of proteoliposomes by a factor of 5 at a physiological concentration. Surprisingly, however, the stimulatory effect was more pronounced when cholesterol was on the v-SNARE side than when it was on the t-SNARE side. Site-directed spin labeling and both continuous wave (CW) and pulsed EPR revealed that cholesterol induces a conformational change of the v-SNARE transmembrane domain (TMD) from an open scissors-like dimer to a parallel dimer. When the TMD was forced to form a parallel dimer by the disulfide bond, the rate was stimulated 2.3-fold even without cholesterol, supporting the relevance of the open-to-closed conformational change to the fusion activity. The open scissors-like conformation may be unfavorable for fusion and cholesterol may relieve this inhibitory factor.
Proceedings of the National Academy of Sciences 03/2009; 106(13):5141-6. · 9.74 Impact Factor
[show abstract][hide abstract] ABSTRACT: We apply pulsed dipolar ESR spectroscopy (Ku-band DEER) to elucidate the global conformation of the Parkinson's disease-associated protein, alpha-synuclein (alphaS) bound to small unilamellar phospholipid vesicles, rodlike SDS micelles, or lipid bicelles. By measuring distances as long as approximately 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 alphaS 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 interhelix linker. While the possibility of local helix discontinuities in the structure of membrane-bound alphaS 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.
Journal of the American Chemical Society 10/2008; 130(39):12856-7. · 10.68 Impact Factor