Double electron-electron resonance (DEER) is now widely utilized to measure distance distributions in the 20-70Å range. DEER is frequently applied to biological systems that have multiple conformational states leading to complex distance distributions. These complex distributions raise issues regarding the best approach to analyze DEER data. A widely used method utilizes a priori background correction followed by Tikhonov regularization. Unfortunately, the underlying assumptions of this approach can impact the analysis. In this chapter, a method of analyzing DEER data is presented that is ideally suited to obtain these complex distance distributions. The approach allows the fitting of raw experimental data without a priori background correction as well as the rigorous determination of uncertainties for all fitting parameters. This same methodological approach can be used for the simultaneous or global analysis of multiple DEER data sets using variable ratios of a common set of components, thus allowing direct correlation of distance components with functionally relevant conformational and biochemical states. Examples are given throughout to highlight this robust fitting approach.

We report here specialized functions incorporated recently in the rigid-body docking software toolkit TagDock to utilize electron paramagnetic resonance derived (EPR-derived) interresidue distance measurements and spin-label accessibility data. The TagDock package extensions include a custom methanethiosulfonate spin label rotamer library to enable explicit, all-atom spin-label side-chain modeling and scripts to evaluate spin-label surface accessibility. These software enhancements enable us to better utilize the biophysical data routinely available from various spin-labeling experiments. To illustrate the power and utility of these tools, we report the refinement of an ankyrin:CDB3 complex model that exhibits much improved agreement with the EPR distance measurements, compared to model structures published previously.

Background: There are conflicting models for how intracellular Ca2+ allosterically regulates Na+/Ca2+ exchange. Results: The model of reorientation of the two Ca2+ binding domains upon Ca2+ binding in the NCX1.4 isoform is not supported by data for NCX1.1. Conclusion: There is motivation to further evaluate the mechanism(s) of Ca2+ activation of NCX1.1. Significance: Control of Ca2+ efflux in cardiomyocytes is vital for cardiac physiology. The cardiac Na+/Ca2+ exchanger (NCX1.1) serves as the primary means of Ca2+ extrusion across the plasma membrane of cardiomyocytes after the rise in intracellular Ca2+ during contraction. The exchanger is regulated by binding of Ca2+ to its intracellular domain, which contains two structurally homologous Ca2+ binding domains denoted as CBD1 and CBD2. NMR and x-ray crystallographic studies have provided structures for the isolated CBD1 and CBD2 domains and have shown how Ca2+ binding affects their structures and motional dynamics. However, structural information on the entire Ca2+ binding domain, denoted CBD12, and how binding of Ca2+ alters its structure and dynamics is more limited. Site-directed spin labeling has been employed in this work to address these questions. Electron paramagnetic resonance measurements on singly labeled constructs of CBD12 have identified the regions that undergo changes in dynamics as a result of Ca2+ binding. Double electron-electron resonance (DEER) measurements on doubly labeled constructs of CBD12 have shown that the β-sandwich regions of the CBD1 and CBD2 domains are largely insensitive to Ca2+ binding and that these two domains are widely separated at their N and C termini. Interdomain distances measured by DEER have been employed to construct structural models for CBD12 in the presence and absence of Ca2+. These models show that there is not a major change in the relative orientation of the two Ca2+ binding domains as a result of Ca2+ binding in the NCX1.1 isoform. Additional measurements have shown that there are significant changes in the dynamics of the F-G loop region of CBD2 that merit further characterization with regard to their possible involvement in regulation of NCX1.1 activity.

It has long been appreciated that the rotational diffusion coefficient (Dr) and hence the rotational correlation time (τr) of a transmembrane protein about its membrane normal axis is predicted to be highly sensitive to its cylindrical radius (e.g., Saffman and Delbrück (1Saffman P.G. Delbrück M. Brownian motion in biological membranes.Proc. Natl. Acad. Sci. USA. 1975; 72: 3111-3113Crossref PubMed Scopus (1366) Google Scholar)). Due to the highly viscous nature of a cellular membrane or a membrane bilayer, the correlation times for most transmembrane proteins are predicted to be in the microsecond or longer correlation time range. This time range is not readily accessible to classical spectroscopic techniques like time-resolved or frequency domain fluorescence anisotropy when using conventional fluorescence probes or by continuous-wave electron paramagnetic resonance (EPR) when using nitroxide spin labels. However, as shown by the early work of Hyde and Dalton (2Hyde J.S. Dalton L.R. Very slowly tumbling spin labels: adiabatic rapid passage.Chem. Phys. Lett. 1972; 16: 568-572Crossref Scopus (112) Google Scholar) and in the seminal study by Thomas et al. (3Thomas D.D. Dalton L.R. Hyde J.S. Rotational diffusion studied by passage saturation transfer electron paramagnetic resonance.J. Chem. Phys. 1976; 65: 3006-3024Crossref Scopus (315) Google Scholar), the range of motional sensitivity of EPR could be extended into the microsecond-to-millisecond time range by saturation transfer EPR (ST-EPR) spectroscopy using conventional nitroxide spin labels. Since its introduction, ST-EPR has been utilized to study the very slow rotational motions of a wide range of membrane proteins and other large protein assemblies. The work by James et al. (4James Z.M. McCaffrey J.E. Thomas D.D. et al.Protein-protein interactions in calcium transport regulation probed by saturation transfer electron paramagnetic resonance.Biophys. J. 2012; 103: 1370-1378Abstract Full Text Full Text PDF PubMed Scopus (26) Google Scholar) in this issue utilizes ST-EPR spectroscopy to characterize the full range of oligomeric interactions between the sarcoplasmic reticulum Ca2+-ATPase (SERCA) and its regulatory protein phospholamban (PLB). A particularly noteworthy aspect of the work is the incorporation of the rigidly coupled TOAC spin label into PLB so that changes in rotational diffusion upon interaction with SERCA could be readily observed independent of any local mobility of the probe. Using this basic approach, the authors were able to show that PLB regulation of SERCA does not involve changes in oligomeric state but instead are likely due to a structural rearrangement within the heterodimeric regulatory complex. This work highlights the power of utilizing methods like ST-EPR to characterize the rotational dynamics of interacting protein systems. The capabilities of modern commercial EPR spectrometers enable investigators to carry out ST-EPR measurements of very slow rotational motions with relative ease.

The signaling mechanisms that regulate CLC anion channels are poorly understood. Caenorhabditis elegans CLH-3b is a member of the CLC-1/2/Ka/Kb channel subfamily. CLH-3b is activated by meiotic cell-cycle progression and cell swelling. Inhibition is brought about by GCK-3 kinase-mediated phosphorylation of S742 and S747 located on a ∼176 amino acid disordered domain linking CBS1 and CBS2. Much of the inter-CBS linker is dispensable for channel regulation. However, deletion of a 14 amino acid activation domain encompassing S742 and S747 inhibits channel activity to the same extent as GCK-3. The crystal structure of CmCLC demonstrated that CBS2 interfaces extensively with an intracellular loop connecting membrane helices H and I, the C-terminus of helix D, and a short linker connecting helix R to CBS1. Point mutagenesis of this interface identified two highly conserved aromatic amino acid residues located in the H-I loop and the first α-helix (α1) of CBS2. Mutation of either residue to alanine rendered CLH-3b insensitive to GCK-3 inhibition. We suggest that the dephosphorylated activation domain normally interacts with CBS1 and/or CBS2, and that conformational information associated with this interaction is transduced through a conserved signal transduction module comprising the H-I loop and CBS2 α1.

The cardiac Na+/Ca2+ exchanger (NCX1.1) serves as the primary means of Ca2+ extrusion from cardiomyocytes following the rise in intracellular Ca2+ during contraction. The exchanger is regulated by binding of Ca2+ to the intracellular domain. This domain is composed of an α-catenin-like domain (CLD) that connects two structurally homologous Ca2+ binding domains (CBD1 and CBD2) to the transmembrane domain of the exchanger. NMR and X-ray crystallographic studies have provided structures for the isolated CBD1 and CBD2 domains and have suggested how Ca2+ binding alters their structures and motional dynamics.

The adaptor protein ankyrin-R interacts via its membrane binding domain with the cytoplasmic domain of the anion exchange protein (AE1) and via its spectrin binding domain with the spectrin-based membrane skeleton in human erythrocytes. This set of interactions provides a bridge between the lipid bilayer and the membrane skeleton, thereby stabilizing the membrane. Crystal structures for the dimeric cytoplasmic domain of AE1 (cdb3) and for a 12-ankyrin repeat segment (repeats 13-24) from the membrane binding domain of ankyrin-R (AnkD34) have been reported. However, structural data on how these proteins assemble to form a stable complex have not been reported. In the current studies, site-directed spin labeling, in combination with electron paramagnetic resonance (EPR) and double electron-electron resonance, has been utilized to map the binding interfaces of the two proteins in the complex and to obtain inter-protein distance constraints. These data have been utilized to construct a family of structural models that are consistent with the full range of experimental data. These models indicate that an extensive area on the peripheral domain of cdb3 binds to ankyrin repeats 18-20 on the top loop surface of AnkD34 primarily through hydrophobic interactions. This is a previously uncharacterized surface for binding of cdb3 to AnkD34. Because a second dimer of cdb3 is known to bind to ankyrin repeats 7-12 of the membrane binding domain of ankyrin-R, the current models have significant implications regarding the structural nature of a tetrameric form of AE1 that is hypothesized to be involved in binding to full-length ankyrin-R in the erythrocyte membrane.

The association between the cytoplasmic domain of band 3 (CDB3) and ankyrinR forms a critical link between the lipid bilayer of the erythrocyte membrane and its underlying spectrin cytoskeleton. This interaction is responsible for the remarkable mechanical stability of the erythrocyte membrane that is essential for the durability of the erythrocyte. While the structures of CDB3 [1] and ankD34 (repeats 13-24 from full length ankyrinR) [2] have been determined by X-ray crystallography, the structure of the CDB3-ankD34 complex has not been established. Using distance constraints from site-directed spin labeling (SDSL) and DEER spectroscopy, we propose a new structural model of CDB3-ankD34 complex modeled assuming rigid-body docking between the two proteins combined with rigorous modeling of the spin label. Unexpectedly, the new model generated by Rosetta docking calculations and filtered through multiple DEER distance constraints shows features which are quite different from the previously proposed docking model. The binding interface of CDB3 is widely scattered over its peripheral surface but the β6-β7 hairpin loop makes no direct contact with ankD34. Second, the binding interface of ankD34 resides on the opposite side of β-hairpin loops from the concave groove. The validity of our current model is also supported by a series of SDSL and cross-linking experiments where the binding interface of ankD34 was mapped by the model-guided scanning of a series of surface sites on ankD34. Supported by NIH P01 GM080513. [1] D. Zhang et al., Blood, 96, 2925 (2000) [2] P. Michaely et al., EMBO J., 21, 6387 (2002)

The cardiac Na+/Ca2+ exchanger (NCX1.1) serves as the primary means of Ca2+ extrusion from cardiomyocytes following the rise in intracellular Ca2+ during contraction. The exchanger is regulated by binding of Ca2+ to the intracellular domain. This domain is composed of an α-catenin-like domain (CLD) that connects two structurally homologous Ca2+ binding domains (CBD1 and CBD2) to the transmembrane domain of the exchanger. NMR and X-ray crystallographic studies have provided structures for the isolated CBD1 and CBD2 domains and have suggested how Ca2+ binding alters their structures and motional dynamics. It remains unknown how Ca2+ binding to the intact Ca2+ sensor signals the transmembrane domain to regulate exchanger activity. We have used site directed spin labeling to address this question. Conventional EPR experiments have shown that: 1) residues in, or near, the Ca2+ binding loops of CBD1 and CBD2 show decreased mobility upon Ca2+ binding; and 2) residues in the β-sandwich regions are insensitive to Ca2+ binding. Double Electron Electron Resonance (DEER) measurements on doubly labeled constructs revealed that: 1) the structure of the β-sandwich domains of CBD1 and CBD2 are not altered upon Ca2+ binding; 2) CBD1 and CBD2 do not lie lengthwise antiparallel in close proximity but rather residues in the distal ends that connect to the CLD are greater than 60 Å apart; and 3) residues nearer to the apex of the Ca2+ sensor are in close enough proximity to be measured by DEER and these distances are sensitive to Ca2+ binding. These studies support recent SAXS studies by Hilge et al. (PNAS 106:14333-8, 2009) and provide additional insight into a structural rearrangement of the intact Ca2+ sensor that may be involved in regulation of Na+/Ca2+ exchange.

A simulated continuous wave electron paramagnetic resonance spectrum of a nitroxide spin label can be obtained from the Fourier transform of a free induction decay. It has been previously shown that the free induction decay can be calculated by solving the time-dependent stochastic Liouville equation for a set of Brownian trajectories defining the rotational dynamics of the label. In this work, a quaternion-based Monte Carlo algorithm has been developed to generate Brownian trajectories describing the global rotational diffusion of a spin-labeled protein. Also, molecular dynamics simulations of two spin-labeled mutants of T4 lysozyme, T4L F153R1, and T4L K65R1 have been used to generate trajectories describing the internal dynamics of the protein and the local dynamics of the spin-label side chain. Trajectories from the molecular dynamics simulations combined with trajectories describing the global rotational diffusion of the protein are used to account for all of the dynamics of a spin-labeled protein. Spectra calculated from these combined trajectories correspond well to the experimental spectra for the buried site T4L F153R1 and the helix surface site T4L K65R1. This work provides a framework to further explore the modeling of the dynamics of the spin-label side chain in the wide variety of labeling environments encountered in site-directed spin labeling studies.

Previous studies have shown that a single P327R point mutation in the cytoplasmic domain of band 3 (cdb3) protein, known as band 3 Tuscaloosa, leads to a reduction in protein 4.2 content of the erythrocyte membrane and hemolytic anemia. Recent studies have shown that this point mutation does not dissociate the cdb3 dimer, nor does it lead to large-scale rearrangement of the protein structure (Bustos, S. P., and Reithmeier, R. A. F. (2006) Biochemistry 45, 1026-1034). To better define the structural changes in cdb3 that lead to the hemolytic anemia phenotype, site-directed spin labeling (SDSL), in combination with continuous wave electron paramagnetic resonance (EPR) and pulsed double electron-electron resonance (DEER) spectroscopies, has been employed in this study to compare the structure of the R327 variant with wild type P327 cdb3. It is confirmed that the P327R mutation does not dissociate the cdb3 dimer, nor does it change the spatial orientation of the two peripheral domains relative to the dimer interface. However, it does affect the packing of the C-terminal end of helix 10 of the dimerization arms in a subpopulation of cdb3 dimers, it leads to spectral changes at some residues in beta-strand 11 and in the N-terminal end of helix10, and it produces measurable spectral changes at other residues that are near the mutation site. The data indicate that the structural changes are subtle and are localized to one surface of the cdb3 dimer. The spectroscopic description of structural features of the P327R variant provides important clues about the location of one potential protein 4.2 binding surface on cdb3 as well as new insight into the structural basis of the membrane destabilization.

X-ray crystallography and nuclear magnetic resonance have been utilized to determine the atomic resolution structures of many proteins and protein domains. It has proven more challenging to apply these same techniques to determine the structures of large protein complexes. This is an important problem since the formation of protein complexes is often critical to regulating protein function. One way to make progress toward determining structural features of an assembly of proteins is to solve the structure of each member and then dock these individual structures together. Toward this goal, computational docking algorithms have been developed and employed with great success to predict important structural features in a wide range of protein-protein interactions. Recent work on the bacterial chemotaxis proteins CheA and CheW (reviewed in (1.Borbat, P. P., J. H. Freed, M. Simon, B. R. Crane, and A. B. Crane. 2007. Measuring distances by pulsed dipolar ESR spectroscopy: spin-labeled histidine kinases in two-component signaling systems. Methods Enzymol. In press.Google Scholar)) has demonstrated how long-range interprobe distance constraints between spin-labeled side chains can be obtained by modern pulsed electron paramagnetic resonance (EPR) methods and then utilized to guide the docking process. The work by Hilger et al. in the article on page 3675 utilizes a similar site-directed spin-labeling approach to determine the structure for the functional Na+/H+ antiporter dimer in a liposome starting from the known atomic resolution structure of the monomer. The authors describe a new scheme to account for the dimensions and flexibility of the spin-labeled side chains in the structure refinement process. As pointed out in several recent publications and as referenced in the article, this is an important step forward in using EPR-derived interprobe distances to build structural models since it is the distance between the unpaired electrons of the two interacting spin labels that is measured and not the distances between Cα carbons of the protein backbone. This work also points out that it is possible to utilize other methods including molecular dynamics to model the conformationally accessible space for the spin labels during the refinement process. Such work is being pursued in many laboratories around the world at this time. It is anticipated that continuing efforts along these lines will lead to the development of even more robust structure refinement tools in the future. However, the current work by Hilger et al., and references cited therein, demonstrate that site-directed spin labeling and modern EPR provides an important new capability for structural studies on large protein assemblies.

The cytoplasmic domain of the anion exchange protein (cdb3) serves as a critical organizing center for protein-protein interactions that stabilize the erythrocyte membrane. The structure of the central core of cdb3, determined by X-ray crystallography from crystals grown at pH 4.8, revealed a compact dimer for residues 55-356 and unresolved N- and C-termini on each monomer [Zhang et al. (2000) Blood 96, 2925-2933]. Given that previous studies had suggested a highly asymmetric structure for cdb3 and that pH dependent structural transitions of cdb3 have been reported, the structure of cdb3 in solution at neutral pH was investigated via site-directed spin labeling in combination with conventional electron paramagnetic resonance (EPR) and double electron electron resonance (DEER) spectroscopies. These studies show that the structure of the central compact dimer (residues 55-356) is indistinguishable from the crystal structure determined at pH 4.8. N-Terminal residues 1-54 and C-terminal residues 357-379 are dynamically disordered and show no indications of stable secondary structure. These results establish a structural model for cdb3 in solution at neutral pH which represents an important next step in characterizing structural details of the protein-protein interactions that stabilize the erythrocyte membrane.

Site directed spin labeling has emerged as a powerful technique for determining structural features of proteins. By incorporating two spin labeled side chains, distances between elements of secondary structure can be determined by quantitation of spin-spin interactions between the probes. Recent advances in methods for extracting both the distance between spin labeled probes and their relative orientations are providing the capability for testing and refining accurate structural models for a wide variety of proteins. These methods take advantage of multifrequency EPR and global non-linear data analysis tools. The capabilities of current analytical methods are described for three relevant models of spin-spin interactions and directions for further refinement of these methods for future applications are described. Reliable structural information can be obtained for interelectron distances ranging from 5 to 20 Å and up to 25 Å using perdeuterated probes.

We have examined the rotational mobility of SL-EGF, a bifunctional adduct of bis(sulfo-N-succinimidyl)-[(15)N,(2)H(16)]-doxyl-2-spiro-4'-pimelate and [Lys3,Tyr22]-murine epidermal growth factor, bound to the EGF receptor in A431 membrane vesicles. The linear EPR spectrum indicated that there was essentially no free SL-EGF in the bound complex preparation. To better define the rotational mobility of the SL-EGF bound to the EGF receptor, ST-EPR spectra were obtained at multiple Zeeman field modulation frequencies. Global analysis with a uniaxial rotational diffusion model of the ST-EPR data yielded two minima that have differences in rotational mobility and in orientation of the SL-EGF relative to the membrane normal axis. The rotational mobilities of the two rotational species are consistent with monomers and dimers or somewhat larger oligomers, such as trimers or tetramers, arguing against a role for higher order receptor clustering in receptor activation. Considering the two minima and previous observations that A431 membrane vesicles contain two distinguishable ligand-binding populations, the ST-EPR spectra were fit with a model having two uniaxial rotating species. This yielded two components that were similar to those obtained from the two original one-component fits, either fast or slow rotational mobility, with different orientations. The model-dependent results obtained suggest that there are potential conformational and rotational differences in the two populations and provide a plausible description for the origin of high- and low-affinity EGF-binding sites that can be tested in future experiments.

The rotational flexibility of the cytoplasmic domain of band 3, in the region that is proximal to the inner membrane surface, has been investigated using a combination of time-resolved optical anisotropy (TOA) and saturation-transfer electron paramagnetic resonance (ST-EPR) spectroscopies. TOA studies of rotational diffusion of the transmembrane domain of band 3 show a dramatic decrease in residual anisotropy following cleavage of the link with the cytoplasmic domain by trypsin (E. A. Nigg and R. J. Cherry, 1980, Proc. Natl. Acad. Sci. U.S.A. 77:4702–4706). This result is compatible with two independent hypotheses: 1) trypsin cleavage leads to dissociation of large clusters of band 3 that are immobile on the millisecond time scale, or 2) trypsin cleavage leads to release of a constraint to uniaxial rotational diffusion of the transmembrane domain. ST-EPR studies at X- and Q-band microwave frequencies detect rotational diffusion of the transmembrane domain of band 3 about the membrane normal axis of reasonably large amplitude that does not change upon cleavage with trypsin. These ST-EPR results are not consistent with dissociation of clusters of band 3 as a result of cleavage with trypsin. Global analyses of the ST-EPR data using a newly developed algorithm indicate that any constraint to rotational diffusion of the transmembrane domain of band 3 via interactions of the cytoplasmic domain with the membrane skeleton must be sufficiently weak to allow rotational excursions in excess of 32° full-width for a square-well potential. In support of this result, analyses of the TOA data in terms of restricted amplitude uniaxial rotational diffusion models suggest that the membrane-spanning domain of that population of band 3 that is linked to the membrane skeleton is constrained to diffuse in a square-well of ∼73° full-width. This degree of flexibility may be necessary for providing the unique mechanical properties of the erythrocyte membrane.

The orientation of the nitroxide moiety of an isotopically substituted spin-labeled derivative of dihydrostilbenedisulfonate ([15N,2H13]-SL-H2DADS-maleimide) covalently coupled at the extracellular stilbenedisulfonate binding site of the human erythrocyte anion exchange protein, band 3, has been determined relative to the membrane normal axis of intact cells. The X-band linear electron paramagnetic resonance (EPR) spectra of [15N,2H13]-SL-H2DADS-maleimide-labeled band 3 in intact erythrocytes oriented by flow through an EPR flat cell have been obtained for two orthogonal orientations of the sample in the DC magnetic field. Two different methods of analysis have provided very similar values for the angles alpha 1 and beta 1 which uniquely define the orientation of the nitroxide axis frame relative to the membrane normal axis. In the first approach, a variable fraction of the cells, f, were taken to be biconcave disks perfectly oriented relative to the flat cell surface with the remainder, 1-f, isotropically oriented. Simultaneous nonlinear least squares analysis of the spectra obtained at the two sample orientations yielded best fit values of f = 0.60, alpha 1 = 58 degrees, and beta 1 = 36 degrees. In the second approach, the EPR spectra of flow-oriented intact erythrocytes labeled with the fatty acid spin-label, [15N,2H12]-5-nitroxyl stearate, have been obtained at the two sample orientations. These two spectra have been used to determine a model-independent distribution of membrane normal orientations in the sample. Using this experimentally determined membrane normal orientation distribution, the EPR spectra of [15N,2H13]-SL-H2DADS-maleimide-labeled erythrocytes were then reanalyzed to obtain a second determination of the nitroxide orientation, alpha 1 = 61 degrees and beta 1 = 37 degrees. The orientation of the nitroxide with respect to the membrane normal axis determined in the present study is nearly identical to the orientation of the nitroxide with respect to the uniaxial rotational diffusion axis, alpha = 66 degrees and beta = 34 degrees, as determined from saturation transfer EPR (ST-EPR) studies [Hustedt, E. H., & Beth, A. H. (1995) Biophys. J. 69, 1409-1423]. This result supports the conclusion that the motion observed using ST-EPR spectroscopy is, in fact, the uniaxial rotational diffusion of band 3 about the membrane normal.

The separations between aromatic residues in the bait region and nitroxide spin labels attached to the thiol ester-forming residues (Cys949 and Gln952) in human alpha 2-macroglobulin (alpha 2M) have been determined from paramagnetic broadening effects of the spin labels on bait region 1H NMR signals. We found that both the Cys949 and Gln952 residues are within 11-17 A of the aromatic residues in the bait region, with closer approach of some residues to the Gln952 spin label than to the spin label attached to Cys949. A model of the location of bait regions and thiol esters within an alpha 2M half-molecule is proposed that places the bait regions in the central region of alpha 2M at the interface between the subunits.