Spin Labeling Electron Paramagnetic Resonance Research Articles

The Assembly of a Multisubunit Photosynthetic Membrane Protein Complex: A Site-Specific Spin Labeling EPR Spectroscopic Study of the PsaC Subunit in Photosystem I

The assembly of the PsaC subunit in the photosystem I (PS I) complex was studied using site-specific spin labeling electron paramagnetic resonance (EPR) spectroscopic techniques. The binding was monitored from the perspective of a reporter spin label attached to either the native C34(C) or the engineered C75(C) residue of wild-type PsaC (PsaC(WT)). Three distinct stages of PsaC assembly were analyzed: unbound PsaC, the P(700)-F(X)/PsaC complex, and the P(700)-F(X)/PsaC/PsaD complex. The changes in the EPR spectral line shape and the rotational correlation time of the spin label when PsaC(WT) binds to the PS I core are consistent with the conformational changes that are expected to occur during the assembly process. The addition of the PsaD subunit to the P(700)-F(X)/PsaC(WT-C34) complex induces further EPR spectral changes, which indicate that the presence of PsaD affects the orientation of the PsaC subunit on the PS I core. The binding of several PsaC variants, each lacking one or more key binding contacts with the PsaA/PsaB heterodimer, was monitored using a reporter spin label at C34(C). Our results indicate that the absence of the PsaC-PsaA/PsaB binding contacts causes PsaC to bind in an altered configuration on the PS I core. In particular, the removal of the entire C-terminus (PsaC(C-term)) causes PsaC to dock in a significantly different orientation when compared to the wild-type protein, as indicated by the EPR spectrum of the P(700)-F(X)/PsaC(C-term-C34) complex. Because the PsaC(C-term) variant retains only the symmetric network of PsaC-PsaA/PsaB ionic contacts, the altered EPR spectrum could, in principle, reflect a fraction of reaction centers that contain PsaC bound in the 180 degrees-rotated, C(2)-symmetry-related configuration. The results of this study are used to provide a comprehensive, stepwise mechanism for the binding of PsaC on the PS I core.

Molecular Details of Bax Activation, Oligomerization, and Membrane Insertion

Bax and Bid are pro-apoptotic members of the Bcl-2 protein family. Upon cleavage by caspase-8, Bid activates Bax. Activated Bax inserts into the mitochondrial outer membrane forming oligomers which lead to membrane poration, release of cytochrome c, and apoptosis. The detailed mechanism of Bax activation and the topology and composition of the oligomers are still under debate. Here molecular details of Bax activation and oligomerization were obtained by application of several biophysical techniques, including atomic force microscopy, cryoelectron microscopy, and particularly electron paramagnetic resonance (EPR) spectroscopy performed on spin-labeled Bax. Incubation with detergents, reconstitution, and Bid-triggered insertion into liposomes were found to be effective in inducing Bax oligomerization. Bid was shown to activate Bax independently of the stoichiometric ratio, suggesting that Bid has a catalytic function and that the interaction with Bax is transient. The formation of a stable dimerization interface involving two Bcl-2 homology 3 (BH3) domains was found to be the nucleation event for Bax homo-oligomerization. Based on intermolecular distance determined by EPR, a model of six adjacent Bax molecules in the oligomer is presented where the hydrophobic hairpins (helices alpha5 and alpha6) are equally spaced in the membrane and the two BH3 domains are in close vicinity in the dimer interface, separated by >5 nm from the next BH3 pairs.

Site-Directed Spin Labeling Electron Paramagnetic Resonance Studies of Flap Conformations and Flexibility in Multiple HIV-1 Protease Variants

Human immunodeficiency virus type 1 protease (HIV-1PR) is a 99 amino acid homodimeric aspartic protease that plays an essential role in the maturation and life cycle of the retrovirus HIV-1, as it functions in regulating post-translational processing of viral polyproteins gag and gag-pol. Accessibility of substrate to the active site is mediated by the conformational changes within two β-hairpins, or flaps. Site-directed spin labeling (SDSL) in conjunction with continuous wave and pulsed electron paramagnetic resonance spectroscopy was used to monitor the conformations of the flaps in HIV-1PR. Six inactive (D25N) HIV-1PR constructs were purified and spin labels were incorporated into the flaps at the aqueous exposed sites K55 and K55’. Constructs included Subtypes B, C, F, circulating recombinant form CRF01_A/E, and patient isolates V6 and MDR 769. For all constructs, two naturally occurring cysteine residues (C67 and C95) were substituted to alanine residues to allow for site-specific labeling as well as to avoid intramolecular disulfide cross-linking. B, F, C, and CRF01_A/E constructs contained three stabilizing mutations that provide protection from autocatalytic cleavage: Q7K, L33I, and L63I. Pulsed EPR results have shown that sequence variations within the subtypes of HIV-1 protease alter the average flap conformations within the apo-enzymes. With detailed data and error analysis, these altered distance profiles can be understood as shifts in the conformational sampling of four distinct HIV-1PR conformations, with some states having enhanced flexibility or structural instability, which may play an important role in viral fitness and drug-resistance. Additionally, DEER data was collected on each construct in the presence of nine different FDA-approved protease inhibitors and a non-hydrolysable substrate mimic called Ca-P2. Continuous wave EPR was used to monitor the autoproteolysis of active (D25) Subtype F and CRF01_A/E constructs.

Structure and Dynamics of the Mobile Domain of Troponin I by SDSL-EPR

The Troponin (Tn) molecular switch contains highly dynamic regions which allow for Ca2+ induced conformational changes to be propagated through to the thin filament. The Mobile domain (Md) of TnI, a secondary thin filament binding domain, is a key player in this process. The functional importance of the Md is also highlighted through the clustering of cardiomyopathy mutations. Structural elucidation of this region by traditional methods is often limited by the absence of key thin filament binding partners. Current Md models describe a highly dynamic region with either a nascent or a well-defined structure. We have utilized Site-Directed Spin Labeling Electron Paramagnetic Resonance (SDSL-EPR) to elucidate the structure of the Md upon interaction with the thin filament. EPR mobility measurements from cysteine scanning of the Md (res. 175-206) in the reconstituted thin filament show a highly dynamic domain in the +Ca2+ (ON) state. A decrease in the mobility occurs in the -Ca2+ (OFF) state, indicating interaction with the thin filament. Further, trends in the mobility of the EPR label reveal two helical structural components within the Md (res. 175-179 & 192-202). Conventional EPR methods were used to measure three interspin distances (176/178, 176/179 & 176/180) which further confirm this assignment. Double Electron-Electron Resonance (DEER) was used to measure the longer interspin distance (178/206) and found that the Md exists in an extended conformation (34±26A). An extended helical structural model for the interaction of the Md with the thin filament through electrostatic bonding is proposed. Residues involved in cardiomyopathy are found clustered at the interacting interface.

Biophysical Studies of Cecropin-Mellitin Antimicrobial Peptides with Improved Selectivity

Antimicrobial peptides (AMPs) have received much attention as models for the development of antibiotics capable of meeting the challenge of drug-resistant infections. Our research has focused on a linear, 15-residue cecropin-mellitin (CM) hybrid peptide designated CM15. In these studies we compare the biological activities and membrane interactions of CM15 and lysine substituted derivates designed to have optimized amphipathicity upon membrane binding. Previous studies have shown that these lysine enriched peptides maintain the high antimicrobial activity of the parent peptide but have substantially decreased hemolytic effects (Sato and Feix, Antimicrob. Agents Chemother. 52, 4463, 2008). Using model membranes to understand the differences that govern peptide-membrane interaction, we have performed a series of biophysical experiments including circular dichroism (CD), fluorescence, and site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy. These experiments establish the structures of the membrane-bound peptides, determine the extent of membrane lysis, and allow determination of partition coefficients and depth of penetration. Our results show that differences in red cell hemolysis can be reconstructed using model membranes, and provide insights into the mechanism of membrane disruption. This work was supported by award number R01GM068829 from the National Institute Of General Medical Sciences.

SDSL-EPR Study of a C-terminal Segment of Myelin Basic Protein in a Myelin Mimetic Environment

Myelin basic protein (MBP) is a peripheral membrane protein and a major component of human central nervous system myelin. It is a multifunctional, highly positively charged, intrinsically disordered protein that undergoes extensive post-translational modifications. Its multifunctionality includes among others, binding of cytoskeletal proteins to a membrane surface and polymerization and bundling of cytoskeletal proteins. These interactions are modulated by interaction of MBP with Ca2+-calmodulin. We report here the use of site-directed spin labeling and continuous wave power saturation electron paramagnetic resonance spectroscopy (SDSL-EPR) to examine the conformation of segment A141-L154 of a recombinant 18.5 kDa murine isoform of MBP in a reconstituted membrane environment. This segment overlaps the primary calmodulin binding region (T147-D158) of MBP and is predicted, on the basis of helical wheel constructs, to form an amphipathic alpha helix. Solution NMR investigations also showed this region to exist as a transient alpha helix in 30% TFE. Our measurements using SDSL-EPR reveal that this region forms an extended helix with a period of ∼3.8 residues per turn and with a tilt angle of ∼4.3 degrees with respect to the plane of the lipid bilayer. The N-terminal end of the helix appears to be buried more deeply in the membrane bilayer than the C-terminal end. The C-terminal region of the segment bears two Lys residues whose side chains interact with the lipid head groups causing this end to be more exposed. The accessibility of the C-terminal region of A141-L154, which forms a part of the primary calmodulin binding segment, could facilitate interaction of this region with cytosolic proteins viz., calmodulin and modulate cytoskeletal binding to the oligodendrocyte plasma membrane.(Supported by CIHR, NSERC and MS Society of Canada)

EPR Spectroscopic Studies on the Structural and Dynamic Properties of Human KCNE1 Membrane Protein in Lipid Bilayers

The KCNE1 membrane protein regulates KCNQ1, which forms the voltage-gated potassium channel in the human heart. Mutations in these genes are responsible for the human genetic disease, long QT syndrome. However, the structure of KCNE1 in a lipid bilayer and biophysical basis for KCNE1's modulation of KCNQ1 are not completely understood. Recent research from Sanders group using solution NMR indicates the transmembrane domain of KCNE1 is a curved alpha-helix and is flanked by intra- and extracellular domains comprised of alpha-helices joined by flexible linkers (Kang et al., Biochemistry 2008 47:7999-8006). Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy has emerged as a well-established method to study the structural properties of membrane proteins. Our objective is to use advanced EPR spectroscopic techniques including CW and pulsed EPR spectroscopy to obtain structural parameters of the KCNE1 protein in a bilayer. To date, we have successfully over-expressed KCNE1 in E. coli and reconstituted the protein into POPG/POPC lipid bilayers. Electrophysiological experiments confirm KCNE1 can co-assemble with the channel protein, KCNQ1 and are fully functional. Electrophysiological experiments further confirm that KCNE1 is inserted correctly into the lipid-bilayers. Additionally we have also demonstrated potential use of DEER (Double Electron-Electron Resonance) and CW-EPR power saturation experiments for distance and depth measurements of KCNE1 in lipid bilayers, respectively.

Opposite Movements of the External Gate in Glutamate Transporters upon Binding Different Cotransported Ligands Measured by EPR

Hairpin two (HP2) has been proposed as the extracellular gate of glutamate transporters. To test this hypothesis, we use double site-directed spin-labeling electron paramagnetic resonance spectroscopy on the bacterial transporter GltPh to examine conformational changes in HP2. Surprisingly, the two co-ligands Na+ and aspartate induce opposite movements of HP2. We find that Na+ binding to the apo state of the transporter opens the extracellular gate, while the subsequent binding of aspartate closes the gate. In addition, using voltage clamp fluorometry on the mammalian excitatory amino acid transporter EAAT3, we confirm that the opposite conformational changes of HP2 induced by Na+ and amino acid substrates also occur in mammalian amino acid transporters. Our findings are consistent with HP2 comprising the extracellular gate of glutamate transporters, and that Na+ binding opens and stabilizes the extracellular gate thereby allowing for amino acid substrate binding.

Rosettaepr: Developing Protein Structure Prediction Methods using Sparse SDSL-EPR Data

Site-Directed Spin-Labeling Electron Paramagnetic Resonance (SDSL-EPR), in combination with the Rosetta protein folding algorithm (Rohl et al 2004), could serve as an alternative method in structure elucidation of proteins that continue to evade traditional techniques, such as x-ray crystallography and NMR. A spin-label motion-on-a-cone model was used during de novo folding of T4-lysozyme and αA-crystallin, which resulted in full-atom models at 1.0A and 2.6A to the crystal structures, respectively (Alexander et al 2008). This spin-label model and already-existing EPR distance data have been used to generate EPR distance and accessibility knowledge-based potentials, which can be implemented as folding constraints into Rosetta. In addition, we have introduced a rotamer library of the methanethiosulfonate spin-label (MTSSL). Spin-label rotamers have been derived from conformations observed in crystal structures of spin-labeled T4-lysozyme. The method was benchmarked using a set of proteins where the spin-label was positioned at various levels of exposure. The results indicate that the method is able to recover important aspects of spin-label orientation with up to 0.4A RMSD. In particular, experimental distances and distance distributions observed for T4-lysozyme were reproduced with relatively high accuracy.

Reduced Cooperative Segmental Motion and Hindered Hydrogen Bonding of Poly(ethylene oxide) Intercalated in Layered Silicates: Deductions Revealed by Simulating ESR Spectra of Spin-Labeled PEO and by XRD, DSC, and TGA Measurements

The structure and dynamics of poly(ethylene oxide) (MW 400, PEO400) intercalated in the galleries of a layered fluoromica clay were investigated by spin-label electron spin resonance (ESR), XRD, DSC, and TGA. PEO400 was end-labeled by attachment of a nitroxide radical and intercalated in 0.78 nm clay galleries. The ESR spectra of intercalated PEO400 showed two motional components, fast and slow, which were attributed to polymer segments weakly and strongly interacting with the clay interface, respectively, while a single spectral component was measured for bulk PEO400. The rotational diffusion coefficient for the polymer chain segment, RS, was determined by simulating the temperature variation of the ESR spectra, based on the “macroscopic order with microscopic disorder” (MOMD) model. The effect of the molecular weight was assessed by comparison with the previous study of PEO4000 (PEO4K [Miwa, Y.; et al. Macromolecules 2008, 41, 4701−4708]. The values of log RS for bulk PEO400 and PEO4K at 330 K were 8.8 ...

Aggregation of Transmembrane Peptides Studied by Spin-Label EPR

The structure and function of membrane proteins is partly determined by the interaction of these proteins with the lipids of the membrane. Peptides forming single membrane-spanning alpha-helices, such as the WALP peptide (acetyl-GWWL(AL)(n)WWA-amide), are good models for such interactions. This interaction can be studied by investigating the aggregation of peptides. If the peptides remain isolated in the membrane, the peptide-lipid interaction dominates, if the peptides aggregate, the peptide-peptide interaction is stronger. The intrinsic dynamics and the disordered nature of the system require new approaches to determine eventual aggregation. We performed electron paramagnetic resonance (EPR) on spin-labeled WALP (SL-WALP) in the gel and the liquid-crystalline phases of two different phospholipids, the saturated DPPC (1,2-dipalmitoyl-sn-glycero-3-phosphocholine), and the unsaturated DOPC (1,2-dioleoyl-sn-glycero-3-phosphocholine). At low temperatures (120 K) where both lipids are in the gel phase, less extensive aggregation is observed for the peptide in DOPC as compared to DPPC. Together with previous data on aggregation of WALP peptides from atomic force microscopy and fluorescence spectroscopy at 294 K ( Sparr ; et al. Biochemistry 2005 , 44 , 2 -10 ), the results suggest that at 120 K WALP peptides form line aggregates in DOPC and cluster aggregates in DPPC. In the liquid-crystalline phase of both lipids, signatures of aggregation are absent, showing that in this phase the peptide can be accommodated by either lipid. It can be concluded that the lipid phase, in this case gel or liquid-crystalline, is a more important determinant for peptide aggregation than whether the lipid is saturated (DPPC) or unsaturated (DOPC). In view of the gel-phase-like behavior of some membrane phases in physiological systems the methodology should be relevant.

Electron spin resonance in membrane research: protein-lipid interactions from challenging beginnings to state of the art.

Conventional electron paramagnetic resonance (EPR) spectra of lipids that are spin-labelled close to the terminal methyl end of the acyl chains are able to resolve the lipids directly contacting the protein from those in the fluid bilayer regions of the membrane. This allows determination of both the stoichiometry of lipid–protein interaction (i.e., number of lipid sites at the protein perimeter) and the selectivity of the protein for different lipid species (i.e., association constants relative to the background lipid). Spin-label EPR data are summarised for 20 or more different transmembrane peptides and proteins, and 7 distinct species of lipids. Lineshape simulations of the two-component conventional spin-label EPR spectra allow estimation of the rate at which protein-associated lipids exchange with those in the bulk fluid regions of the membrane. For lipids that do not display a selectivity for the protein, the intrinsic off-rates for exchange are in the region of 10 MHz: less than 10× slower than the rates of diffusive exchange in fluid lipid membranes. Lipids with an affinity for the protein, relative to the background lipid, have off-rates for leaving the protein that are correspondingly slower. Non-linear EPR, which depends on saturation of the spectrum at high radiation intensities, is optimally sensitive to dynamics on the timescale of spin-lattice relaxation, i.e., the microsecond regime. Both progressive saturation and saturation transfer EPR experiments provide definitive evidence that lipids at the protein interface are exchanging on this timescale. The sensitivity of non-linear EPR to low frequencies of spin exchange also allows the location of spin-labelled membrane protein residues relative to those of spin-labelled lipids, in double-labelling experiments.

Site-Directed Spin-Labeling Study of the Light-Harvesting Complex CP29

The topology of the long N-terminal domain (∼100 amino-acid residues) of the photosynthetic Lhc CP29 was studied using electron spin resonance. Wild-type protein containing a single cysteine at position 108 and nine single-cysteine mutants were produced, allowing to label different parts of the domain with a nitroxide spin label. In all cases, the apoproteins were either solubilized in detergent or they were reconstituted with their native pigments (holoproteins) in vitro. The spin-label electron spin resonance spectra were analyzed in terms of a multicomponent spectral simulation approach, based on hybrid evolutionary optimization and solution condensation. These results permit to trace the structural organization of the long N-terminal domain of CP29. Amino-acid residues 97 and 108 are located in the transmembrane pigment-containing protein body of the protein. Positions 65, 81, and 90 are located in a flexible loop that is proposed to extend out of the protein from the stromal surface. This loop also contains a phosphorylation site at Thr81, suggesting that the flexibility of this loop might play a role in the regulatory mechanisms of the light-harvesting process. Positions 4, 33, 40, and 56 are found to be located in a relatively rigid environment, close to the transmembrane protein body. On the other hand, position 15 is located in a flexible region, relatively far away from the transmembrane domain.

Exploring the Gating Mechanism of the Glutamate Transporter GltPh

Glutamate acts as the primary neurotransmitter in the mammalian central nervous system. Clearance of this neurotransmitter from the synapse is accomplished by a family of glutamate transporters known as EAATs. These proteins move synaptic glutamate across the cell membrane into the cell against its concentration gradient. It has long been speculated that the mechanism for transport involves the movement of extracellular and intracellular gates, providing “alternating access” to a substrate binding site. Recently, the crystal structure of a related bacterial transporter, GltPh, was solved, revealing two helical hairpins (HP1 and HP2) which have been proposed to contribute to these gates. HP2 lies on the extracellular face of the transporter, and a number of studies have shown that this hairpin can adopt multiple conformations that either provide or restrict access to the substrate binding site. However, to date there is no structural information regarding the conformational changes that must occur within HP1 in order to provide the substrate a path to the intracellular solution. Here we use the technique of site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy to explore the structure of HP1 (residues 258-290) in purified GltPh reconstituted into proteoliposomes. This technique provides an opportunity to probe the dynamic structure of HP1 in a functional protein embedded in a physiological lipid environment.

Membrane Protein Frustration: Protein Incorporation into Hydrophobic Mismatched Binary Lipid Mixtures

Bacteriophage M13 major coat protein was reconstituted in different nonmatching binary lipid mixtures composed of 14:1PC and 22:1PC lipid bilayers. Challenged by this lose-lose situation of hydrophobic mismatch, the protein-lipid interactions are monitored by CD and site-directed spin-label electron spin resonance spectroscopy of spin-labeled site-specific single cysteine mutants located in the C-terminal protein domain embedded in the hydrophobic core of the membrane (I39C) and at the lipid-water interface (T46C). The CD spectra indicate an overall α-helical conformation irrespective of the composition of the binary lipid mixture. Spin-labeled protein mutant I39C senses the phase transition in 22:1PC, in contrast to spin-labeled protein mutant T46C, which is not affected by the transition. The results of both CD and electron spin resonance spectroscopy clearly indicate that the protein preferentially partitions into the shorter 14:1PC both above and below the gel-to-liquid crystalline phase transition temperature of 22:1PC. This preference is related to the protein tilt angle and energy penalty the protein has to pay in the thicker 22:1PC. Given the fact that in Escherichia coli, which is the host for M13 bacteriophage, it is easier to find shorter 14 carbon acyl chains than longer 22 carbon acyl chains, the choice the M13 coat protein makes seems to be evolutionary justified.

Orientation and Peptide−Lipid Interactions of Alamethicin Incorporated in Phospholipid Membranes: Polarized Infrared and Spin-Label EPR Spectroscopy

Alamethicin is a 20-residue peptaibiotic that induces voltage-dependent ion channels in lipid membranes. The mode by which alamethicin inserts into membranes was investigated using measurements of peptide-lipid interactions by spin-label electron paramagnetic resonance (EPR) and of peptide orientation by polarized infrared (IR) spectroscopy. In fluid membranes, spin-labeled stearic acid shows no evidence of a specific motionally restricted population of lipid chains, such as that found at the intramembranous surface of integral membrane proteins or oligomeric assemblies of transmembrane alpha-helices. In agreement with recent results from TOAC-substituted alamethicin analogues, native alamethicin is predominantly monomeric in fluid lipid membranes and presents an intramembrane surface that integrates well with the lipid chains but is insufficiently extensive to induce specific motional restriction. Channel formation takes place by transient association of transmembrane monomers. In aligned fluid membranes, alamethicin exhibits a large tilt in short chain-length lipids that decreases first rapidly with increasing chain-length and then more gradually for the lipids with longer chains. This macroscopically low order contrasts with the high local order, relative to the local membrane normal, that is found by EPR for alamethicins spin-labeled with TOAC. The macroscopic behavior is consistent with predictions for the chain-length dependence of elastic bending fluctuations of the membrane surface, which was invoked recently to explain the spontaneous insertion of beta-barrel proteins in short-chain lipid membranes.

Analysis of side chain rotational restrictions of membrane-embedded proteins by spin-label ESR spectroscopy

Site-directed spin-labeling electron spin resonance (SDSL-ESR) is a promising tool for membrane protein structure determination. Here we propose a novel way to translate the local structural constraints gained by SDSL-ESR data into a low-resolution structure of a protein by simulating the restrictions of the local conformational spaces of the spin label attached at different protein sites along the primary structure of the membrane-embedded protein. We test the sensitivity of this approach for membrane-embedded M13 major coat protein decorated with a limited number of strategically placed spin labels employing high-throughput site-directed mutagenesis. We find a reasonably good agreement of the simulated and the experimental data taking a protein conformation close to the one determined by fluorescence resonance energy transfer analysis [P.V. Nazarov, R.B.M. Koehorst, W.L. Vos, V.V. Apanasovich, M.A. Hemminga, FRET study of membrane proteins: determination of the tilt and orientation of the N-terminal domain of M13 major coat protein, Biophys. J. 92 (2007) 1296–1305].

Structural and Dynamic Study of the Tetramerization Region of Non-Erythroid α-Spectrin: A Frayed Helix Revealed by Site-Directed Spin Labeling Electron Paramagnetic Resonance

The N-terminal region of alpha-spectrin is responsible for its association with beta-spectrin in a heterodimer, forming functional tetramers. Non-erythroid alpha-spectrin (alphaII-spectrin) has a significantly higher association affinity for beta-spectrin than the homologous erythroid alpha-spectrin (alphaI-spectrin). We have previously determined the solution structure of the N-terminal region of alphaI-spectrin by NMR methods, but currently no structural information is available for alphaII-spectrin. We have used cysteine scanning, spin labeling electron paramagnetic resonance (EPR), and isothermal titration calorimetry (ITC) methods to study the tetramerization region of alphaII-spectrin. EPR data clearly show that, in alphaII-spectrin, the first nine N-terminal residues were unstructured, followed by an irregular helix (helix C'), frayed at the N-terminal end, but rigid at the C-terminal end, which merges into the putative triple-helical structural domain. The region corresponding to the important unstructured junction region linking helix C' to the first structural domain in alphaI-spectrin was clearly structured. On the basis of the published model for aligning helices A', B', and C', important interactions among residues in helix C' of alphaI- and alphaII-spectrin and helices A' and B' of betaI- and betaII-spectrin are identified, suggesting similar coiled coil helical bundling for spectrin I and II in forming tetramers. The differences in affinity are likely due to the differences in the conformation of the junction regions. Equilibrium dissociation constants of spin-labeled alphaII and betaI complexes from ITC measurements indicate that residues 15, 19, 37, and 40 are functionally important residues in alphaII-spectrin. Interestingly, all four corresponding homologous residues in alphaI-spectrin (residues 24, 28, 46, and 49) have been reported to be clinically significant residues involved in hematological diseases.

Functionally important ATP binding and hydrolysis sites in Escherichia coli MsbA.

ATP-binding cassette (ABC) transporters make up one of the largest classes of proteins found in nature, and their ability to move a variety of substrates across the membrane using energy from the binding or hydrolysis of ATP is essential to an array of human pathologies and to bacterial viability. MsbA is an essential ABC transporter that specifically transports lipid A across the inner membranes of Gram-negative organisms such as Escherichia coli. The exact mechanisms of function during the binding and hydrolysis of ATP at the molecular level remain unclear. The studies presented and summarized in this work directly address the role and local dynamics of specific residues within the conserved ABC motifs in E. coli MsbA using in vivo growth and biochemical activity assays coupled with site-directed spin labeling electron paramagnetic resonance (EPR) spectroscopy motional and accessibility analysis. This first comprehensive analysis of the specific residues in these motifs within MsbA indicates that closure of the dimer interface does not occur upon ATP binding in this transporter.

Salt-driven Equilibrium between Two Conformations in the HAMP Domain from Natronomonas pharaonis: THE LANGUAGE OF SIGNAL TRANSFER?

HAMP domains (conserved in histidine kinases, adenylyl cyclases, methyl-accepting chemotaxis proteins, and phosphatases) perform their putative function as signal transducing units in diversified environments in a variety of protein families. Here the conformational changes induced by environmental agents, namely salt and temperature, on the structure and function of a HAMP domain of the phototransducer from Natronomonas pharaonis (NpHtrII) in complex with sensory rhodopsin II (NpSRII) were investigated by site-directed spin labeling electron paramagnetic resonance. A series of spin labeled mutants were engineered in NpHtrII157, a truncated analog containing only the first HAMP domain following the transmembrane helix 2. This truncated transducer is shown to be a valid model system for a signal transduction domain anchored to the transmembrane light sensor NpSRII. The HAMP domain is found to be engaged in a "two-state" equilibrium between a highly dynamic (dHAMP) and a more compact (cHAMP) conformation. The structural properties of the cHAMP as proven by mobility, accessibility, and intra-transducer-dimer distance data are in agreement with the four helical bundle NMR model of the HAMP domain from Archaeoglobus fulgidus.