The potential of spin labeling to reveal the dynamic dimension of macromolecules has been recognized since the dawn of the methodology in the 1960s. However, it was the development of pulsed electron paramagnetic resonance spectroscopy to detect dipolar coupling between spin labels and the availability of turnkey instrumentation in the 21st century that realized the full promise of spin labeling. Double electron-electron resonance (DEER) spectroscopy has seen widespread applications to channels, transporters, and receptors. In these studies, distance distributions between pairs of spin labels obtained under different biochemical conditions report the conformational states of macromolecules, illuminating the key movements underlying biological function. These experimental studies have spurred the development of methods for the rigorous analysis of DEER spectroscopic data along with methods for integrating these distributions into structural models. In this tutorial, we describe a model-based approach to obtaining a minimum set of components of the distance distribution that correspond to functionally relevant protein conformations with a set of fractional amplitudes that define the equilibrium between these conformations. Importantly, we review and elaborate on the error analysis reflecting the uncertainty in the various parameters, a critical step in rigorous structural interpretation of the spectroscopic data.

Distance distribution information obtained by pulsed dipolar EPR spectroscopy provides an important contribution to many studies in structural biology. Increasingly, such information is used in integrative structural modeling, where it delivers unique restraints on the width of conformational ensembles. In order to ensure reliability of the structural models and of biological conclusions, we herein define quality standards for sample preparation and characterization, for measurements of distributed dipole-dipole couplings between paramagnetic labels, for conversion of the primary time-domain data into distance distributions, for interpreting these distributions, and for reporting results. These guidelines are substantiated by a multi-laboratory benchmark study and by analysis of data sets with known distance distribution ground truth. The study and the guidelines focus on proteins labeled with nitroxides and on double electron-electron resonance (DEER aka PELDOR) measurements and provide suggestions on how to proceed analogously in other cases.

The continuous wave (CW) and pulse electron paramagnetic resonance (EPR) methods enable the measurement of distances between spin-labeled residues in biopolymers including proteins, providing structural information. Here we describe the CW EPR deconvolution/convolution method and the four-pulse double electron-electron resonance (DEER) approach for distance determination, which were applied to elucidate the organization of the BAK apoptotic pores formed in the lipid bilayers.

Given its ability to measure multicomponent distance distributions between electron-spin probes, double electron-electron resonance (DEER) spectroscopy has become a leading technique to assess the structural dynamics of biomolecules. However, methodologies to evaluate the statistical error of these distributions are not standard, often hampering a rigorous interpretation of the experimental results. Distance distributions are often determined from the experimental DEER data through a mathematical method known as Tikhonov regularization, but this approach makes rigorous error estimates difficult. Here, we build upon an alternative, model-based approach in which the distance probability distribution is represented as a sum of Gaussian components, and use propagation of errors to calculate an associated confidence band. Our approach considers all sources of uncertainty, including the experimental noise, the uncertainty in the fitted background signal, and the limited time span of the data collection. The resulting confidence band reveals the most and least reliable features of the probability distribution, thereby informing the structural interpretation of DEER experiments. To facilitate this interpretation, we also generalize the molecular simulation method known as ensemble-biased metadynamics (EBMetaD). This method, originally designed to generate maximal-entropy structural ensembles consistent with one or more probability distributions, now also accounts for the uncertainty in those target distributions exactly as dictated by their confidence bands. After careful benchmarks, we demonstrate the proposed techniques using DEER results from spin-labeled T4 lysozyme.

In mitochondrial apoptosis, Bak is activated by death signals to form pores of unknown structure on the mitochondrial outer membrane via homooligomerization. Cytochrome c and other apoptotic factors are released from the intermembrane space through these pores, initiating downstream apoptosis events. Using chemical crosslinking and double electron electron resonance (DEER)-derived distance measurements between specific structural elements in Bak, here we clarify how the Bak pore is assembled. We propose that previously described BH3-in-groove homodimers (BGH) are juxtaposed via the ‘α3/α5’ interface, in which the C-termini of helices α3 and α5 are in close proximity between two neighboring Bak homodimers. This interface is observed concomitantly with the well-known ‘α6:α6’ interface. We also mapped the contacts between Bak homodimers and the lipid bilayer based on EPR spectroscopy topology studies. Our results suggest a model for the lipidic Bak pore, whereby the mitochondrial targeting C-terminal helix does not change topology to accommodate the lining of the pore lumen by BGH.

Current distance measurements between spin-labels on multimeric protonated proteins using double electron-electron resonance (DEER) EPR spectroscopy are generally limited to the 15-60 Å range. Here we show how DEER experiments can be extended to dipolar evolution times of ca. 80 μs, permitting distances up to 170 Å to be accessed in multimeric proteins. The method relies on sparse spin-labeling, supplemented by deuteration of protein and solvent, to minimize the deleterious impact of multispin effects and substantially increase the apparent spin-label phase memory relaxation time, complemented by high sensitivity afforded by measurements at Q-band. We demonstrate the approach using the tetradecameric molecular machine GroEL as an example. Two engineered surface-exposed mutants, R268C and E315C, are used to measure pairwise distance distributions with mean values ranging from 20 to 100 Å and from 30 to 160 Å, respectively, both within and between the two heptameric rings of GroEL. The measured distance distributions are consistent with the known crystal structure of apo GroEL. The methodology presented here should significantly expand the use of DEER for the structural characterization of conformational changes in higher order oligomers.

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.

, 1168 (2012); 336 Science et al. Paul J. Barrett Domain and Binds Cholesterol The Amyloid Precursor Protein Has a Flexible Transmembrane This copy is for your personal, non-commercial use only. clicking here. colleagues, clients, or customers by , you can order high-quality copies for your If you wish to distribute this article to others here. following the guidelines can be obtained by Permission to republish or repurpose articles or portions of articles ): May 12, 2014 www.sciencemag.org (this information is current as of The following resources related to this article are available online at http://www.sciencemag.org/content/336/6085/1168.full.html version of this article at: including high-resolution figures, can be found in the online Updated information and services, http://www.sciencemag.org/content/suppl/2012/05/30/336.6085.1168.DC1.html can be found at: Supporting Online Material http://www.sciencemag.org/content/336/6085/1168.full.html#related found at: can be related to this article A list of selected additional articles on the Science Web sites http://www.sciencemag.org/content/336/6085/1168.full.html#ref-list-1 , 16 of which can be accessed free: cites 43 articles This article http://www.sciencemag.org/content/336/6085/1168.full.html#related-urls 18 articles hosted by HighWire Press; see: cited by This article has been http://www.sciencemag.org/cgi/collection/biochem Biochemistry subject collections: This article appears in the following

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.

An EPR membrane alignment technique was applied to measure distance and relative orientations between two spin labels on a protein oriented along the surface of the membrane. Previously we demonstrated an EPR membrane alignment technique for measuring distances and relative orientations between two spin labels using a dual TOAC-labeled integral transmembrane peptide (M2δ segment of Acetylcholine receptor) as a test system. In this study we further utilized this technique and successfully measured the distance and relative orientations between two spin labels on a membrane peripheral peptide (antimicrobial peptide magainin-2). The TOAC-labeled magainin-2 peptides were mechanically aligned using DMPC lipids on a planar quartz support, and CW-EPR spectra were recorded at specific orientations. Global analysis in combination with rigorous spectral simulation was used to simultaneously analyze data from two different sample orientations for both single- and double-labeled peptides. We measured an internitroxide distance of 15.3Å from a dual TOAC-labeled magainin-2 peptide at positions 8 and 14 that closely matches with the 13.3Å distance obtained from a model of the labeled magainin peptide. In addition, the angles determining the relative orientations of the two nitroxides have been determined, and the results compare favorably with molecular modeling. This study demonstrates the utility of the technique for proteins oriented along the surface of the membrane in addition to the previous results for proteins situated within the membrane bilayer.

Although there are competing hypotheses regarding the etiology of AD, mounting evidence shows that the genesis of β-amyloid (Aβ) peptides is usually a critical early event. Aβ is generated by proteolytic processing of the amyloid precursor protein (APP). The cleavage of APP by β- and γ-secretases leads to the production of the Aβ polypeptides. The toxicity of Aβ triggers the development of AD. APP has been widely reported to form homodimers that may play a role in regulating amyloidogenesis. However, it has been controversial as to whether homodimerization of APP is driven, in part, by GXXXG motifs present in its transmembrane (TM) domain. Previously, we reported that cholesterol binds to C99 (also known as βCTF) to form a 1:1 complex (Barrett et al., SCIENCE 2012). C99 is the C-terminal fragment the APP that is released by β-secretase and that serves of the substrate for cleavage by γ-secretase to release Aβ. Here, we examine the possible relationship of cholesterol binding to C99 and homodimerization of the protein with NMR, EPR and other methods. We find that C99 is more prone to forming dimers in POPC/POPG lipid bilayers than in detergent micelles. Mutations of the glycine in the GXXXG motifs reveal that these glycines are critical for dimerization. Addition of cholesterol to the vesicles stabilizes monomeric C99 and disfavors dimerization.

The multidomain pro-apoptotic Bcl-2 family proteins BAK and BAX are believed to form large oligomeric pores in the mitochondrial outer membrane during apoptosis. Formation of these pores results in the release of apoptotic factors including cytochrome c from the intermembrane space into the cytoplasm, where they initiate the cascade of events that lead to cell death. Using the site-directed spin labeling method of electron paramagnetic resonance (EPR) spectroscopy, we have determined the conformational changes that occur in BAK when the protein targets to the membrane and forms pores. The data showed that helices α1 and α6 disengage from the rest of the domain, leaving helices α2-α5 as a folded unit. Helices α2-α5 were shown to form a dimeric structure, which is structurally homologous to the recently reported BAX "BH3-in-groove homodimer." Furthermore, the EPR data and a chemical cross-linking study demonstrated the existence of a hitherto unknown interface between BAK BH3-in-groove homodimers in the oligomeric BAK. This novel interface involves the C termini of α3 and α5 helices. The results provide further insights into the organization of the BAK oligomeric pores by the BAK homodimers during mitochondrial apoptosis, enabling the proposal of a BAK-induced lipidic pore with the topography of a "worm hole."

The 99-residue transmembrane C-terminal domain (C99, also known as β-CTF) of the amyloid precursor protein (APP) is the product of the β-secretase cleavage of the full-length APP and is the substrate for γ-secretase cleavage. The latter cleavage releases the amyloid-β polypeptides that are closely associated with Alzheimer's disease. C99 is thought to form homodimers; however, the free energy in favor of dimerization has not previously been quantitated. It was also recently documented that cholesterol forms a 1:1 complex with monomeric C99 in bicelles. Here, the affinities for both homodimerization and cholesterol binding to C99 were measured in bilayered lipid vesicles using both electron paramagnetic resonance (EPR) and Förster resonance energy transfer (FRET) methods. Homodimerization and cholesterol binding were seen to be competitive processes that center on the transmembrane G₇₀₀XXXG₇₀₄XXXG₇₀₈ glycine-zipper motif and adjacent Gly709. On one hand, the observed Kd for cholesterol binding (Kd = 2.7 ± 0.3 mol %) is on the low end of the physiological cholesterol concentration range in mammalian cell membranes. On the other hand, the observed K(d) for homodimerization (K(d) = 0.47 ± 0.15 mol %) likely exceeds the physiological concentration range for C99. These results suggest that the 1:1 cholesterol/C99 complex will be more highly populated than C99 homodimers under most physiological conditions. These observations are of relevance for understanding the γ-secretase cleavage of C99.

A membrane alignment technique has been used to measure the distance between two TOAC nitroxide spin labels on the membrane-spanning M2δ, peptide of the nicotinic acetylcholine receptor (AChR), via CW-EPR spectroscopy. The TOAC-labeled M2δ peptides were mechanically aligned using DMPC lipids on a planar quartz support, and CW-EPR spectra were recorded at specific orientations. Global analysis in combination with rigorous spectral simulation was used to simultaneously analyze data from two different sample orientations for both single- and double-labeled peptides. We measured an internitroxide distance of 14.6 Å from a dual TOAC-labeled AChR M2δ peptide at positions 7 and 13 that closely matches with the 14.5 Å distance obtained from a model of the labeled AChR M2δ peptide. In addition, the angles determining the relative orientation of the two nitroxides have been determined, and the results compare favorably with molecular modeling. The global analysis of the data from the aligned samples gives much more precise estimates of the parameters defining the geometry of the two labels than can be obtained from a randomly dispersed sample.

C99 is the transmembrane carboxyl-terminal domain of the amyloid precursor protein that is cleaved by γ-secretase to release the amyloid-β polypeptides, which are associated with Alzheimer's disease. Nuclear magnetic resonance and electron paramagnetic resonance spectroscopy show that the extracellular amino terminus of C99 includes a surface-embedded "N-helix" followed by a short "N-loop" connecting to the transmembrane domain (TMD). The TMD is a flexibly curved α helix, making it well suited for processive cleavage by γ-secretase. Titration of C99 reveals a binding site for cholesterol, providing mechanistic insight into how cholesterol promotes amyloidogenesis. Membrane-buried GXXXG motifs (G, Gly; X, any amino acid), which have an established role in oligomerization, were also shown to play a key role in cholesterol binding. The structure and cholesterol binding properties of C99 may aid in the design of Alzheimer's therapeutics.

Site-directed spin-labeling electron paramagnetic resonance (SDSL EPR) provides insight into the local structure and motion of a spin probe strategically attached to a molecule. When a second spin is introduced to the system, macromolecular information can be obtained through measurement of inter-spin distances either by continuous wave (CW) or pulsed electron double resonance (ELDOR) techniques. If both methodologies are considered, inter-spin distances of 8-80 Å can be experimentally determined. However, there exists a region at the upper limit of the conventional X-band (9.5 GHz) CW technique and the lower limit of the four-pulse double electron-electron resonance (DEER) experiment where neither method is particularly reliable. The work presented here utilizes L-band (1.9 GHz) in combination with non-adiabatic rapid sweep (NARS) EPR to address this opportunity by increasing the upper limit of the CW technique. Because L-band linewidths are three to seven times narrower than those at X-band, dipolar broadenings that are small relative to the X-band inhomogeneous linewidth become observable, but the signal loss, due to the frequency dependence of the Boltzmann factor, has made L-band especially challenging. NARS has been shown to increase sensitivity by a factor of five, and overcomes much of this loss, making L-band distance determination more feasible. Two different systems are presented, and distances of 18-30 Å have been experimentally determined at physiologically relevant temperatures. Measurements are in excellent agreement with a helical model and values determined by DEER.

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.

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.