Double Electron-electron Resonance Research Articles

Energy transduction and alternating access of the mammalian ABC transporter P-glycoprotein.

ATP binding cassette (ABC) transporters of the exporter class harness the energy of ATP hydrolysis in the nucleotide-binding domains (NBDs) to power the energetically uphill efflux of substrates by a dedicated transmembrane domain (TMD). Although numerous investigations have described the mechanism of ATP hydrolysis and defined the architecture of ABC exporters, a detailed structural dynamic understanding of the transduction of ATP energy to the work of substrate translocation remains elusive. Here we used double electron-electron resonance and molecular dynamics simulations to describe the ATP- and substrate-coupled conformational cycle of the mouse ABC efflux transporter P-glycoprotein (Pgp; also known as ABCB1), which has a central role in the clearance of xenobiotics and in cancer resistance to chemotherapy. Pairs of spin labels were introduced at residues selected to track the putative inward-facing to outward-facing transition. Our findings illuminate how ATP energy is harnessed in the NBDs in a two-stroke cycle and elucidate the consequent conformational motion that reconfigures the TMD, two critical aspects of Pgp transport mechanism. Along with a fully atomistic model of the outward-facing conformation in membranes, the insight into Pgp conformational dynamics harmonizes mechanistic and structural data into a novel perspective on ATP-coupled transport and reveals mechanistic divergence within the efflux class of ABC transporters.

Intersubunit distances in full-length, dimeric, bacterial phytochrome Agp1, as measured by pulsed electron-electron double resonance (PELDOR) between different spin label positions, remain unchanged upon photoconversion

Bacterial phytochromes are dimeric light-regulated histidine kinases that convert red light into signaling events. Light absorption by the N-terminal photosensory core module (PCM) causes the proteins to switch between two spectrally distinct forms, Pr and Pfr, thus resulting in a conformational change that modulates the C-terminal histidine kinase region. To provide further insights into structural details of photoactivation, we investigated the full-length Agp1 bacteriophytochrome from the soil bacterium Agrobacterium fabrum using a combined spectroscopic and modeling approach. We generated seven mutants suitable for spin labeling to enable application of pulsed EPR techniques. The distances between attached spin labels were measured using pulsed electron-electron double resonance spectroscopy to probe the arrangement of the subunits within the dimer. We found very good agreement of experimental and calculated distances for the histidine-kinase region when both subunits are in a parallel orientation. However, experimental distance distributions surprisingly showed only limited agreement with either parallel- or antiparallel-arranged dimer structures when spin labels were placed into the PCM region. This observation indicates that the arrangements of the PCM subunits in the full-length protein dimer in solution differ significantly from that in the PCM crystals. The pulsed electron-electron double resonance data presented here revealed either no or only minor changes of distance distributions upon Pr-to-Pfr photoconversion.

Molecular Breakdown of Double Electron-Electron Resonance Data with Atomistic Simulations

Double Electron-Electron Resonance (DEER) spectroscopy has become a landmark technique to investigate the structure and dynamics of membrane proteins, in different functional states and physiological conditions. From DEER experiments it is possible to obtain any given number of probability distributions for distances between spin labels attached to biomolecules. In contrast to diffraction methods or NMR spectroscopy, DEER is neither limited by the need of crystallization nor by the protein size. This notwithstanding, it is often not straightforward to interpret DEER data, as it reflects a plethora of protein conformations and rotameric states of the spin labels. Several strategies to disentangle this variability and to derive a clear structural interpretation have been put forward recently. Some of them entail the calculation of distance distributions from approximate protein models and rotamer libraries for the spin-labels. Other more rigorous approaches, such as Ensemble-Biased MetaDynamics (EBMetaD), are based on atomistic simulation methods, specifically designed to reproduce the DEER distributions exactly and with a minimal bias. Both kinds of approaches, however, rely on the probability distributions that are inferred from the actual measured data, and do not take into account the experimental noise. Here, we present a powerful and simple approach to minimally bias an atomistic simulation to sample a conformational ensemble that directly reproduces the DEER time-signal within the experimental uncertainty. The method is based on the maximum-entropy principle and extends the EBMetaD approach. We assess the performance of the method using spin-labeled T4 lysozyme in explicit water. The results demonstrate the accuracy and efficiency of the method. In summary, we propose a novel, rigorous technique to directly interpret experimental data from DEER measurements in terms of physically realistic conformational ensembles.

Guiding Double Electron Electron Resonance Experiments using Information Theory and Molecular Dynamics Simulations

Flexible proteins present a unique set of challenges for refinement with either computational or experimental techniques: because these proteins occupy a large conformational space, standard molecular dynamics (MD) simulations can be prohibitively expensive, and high-resolution experimental techniques like x-ray crystallography intentionally capture only small set of low energy states. A new approach is therefore needed to capture the conformational ensembles of flexible proteins. Double electron-electron resonance (DEER) spectroscopy, an experimental technique that measures distance distributions between pairs of spin-labeled residues on a protein, provides quantitative information about less populated but still potentially important conformational states. However, because DEER experiments are costly and time consuming, only a small subset of pairs can be measured, often chosen arbitrarily. If instead of selecting an arbitrary or random subset, we select a subset of pairs such that they provide the maximum amount of information about the conformational ensemble, DEER could become an even more powerful tool for refinement. We have therefore developed 1) a new information-theoretic metric that ranks pairs of residues based on how well they refine a conformational ensemble and 2) a new methodology which identifies a set of highly informative pairs that perform well under this metric. Specifically, we incorporate DEER distance distributions into restrained-ensemble MD simulations, utilize the data from these simulations to identify a set of maximally-informative and minimally-redundant pairs, measure the pairs with DEER, and demonstrate the use of these pairs in refining the conformational ensemble of a flexible protein.

A Reactive, Rigid GdIII Labeling Tag for In‐Cell EPR Distance Measurements in Proteins

AbstractThe cellular environment of proteins differs considerably from in vitro conditions under which most studies of protein structures are carried out. Therefore, there is a growing interest in determining dynamics and structures of proteins in the cell. A key factor for in‐cell distance measurements by the double electron–electron resonance (DEER) method in proteins is the nature of the used spin label. Here we present a newly designed GdIII spin label, a thiol‐specific DOTA‐derivative (DO3MA‐3BrPy), which features chemical stability and kinetic inertness, high efficiency in protein labelling, a short rigid tether, as well as favorable spectroscopic properties, all are particularly suitable for in‐cell distance measurements by the DEER method carried out at W‐band frequencies. The high performance of DO3MA‐3BrPy‐GdIII is demonstrated on doubly labelled ubiquitin D39C/E64C, both in vitro and in HeLa cells. High‐quality DEER data could be obtained in HeLa cells up to 12 h after protein delivery at in‐cell protein concentrations as low as 5–10 μm.

A Reactive, Rigid GdIII Labeling Tag for In‐Cell EPR Distance Measurements in Proteins

The cellular environment of proteins differs considerably from in vitro conditions under which most studies of protein structures are carried out. Therefore, there is a growing interest in determining dynamics and structures of proteins in the cell. A key factor for in-cell distance measurements by the double electron-electron resonance (DEER) method in proteins is the nature of the used spin label. Here we present a newly designed GdIII spin label, a thiol-specific DOTA-derivative (DO3MA-3BrPy), which features chemical stability and kinetic inertness, high efficiency in protein labelling, a short rigid tether, as well as favorable spectroscopic properties, all are particularly suitable for in-cell distance measurements by the DEER method carried out at W-band frequencies. The high performance of DO3MA-3BrPy-GdIII is demonstrated on doubly labelled ubiquitin D39C/E64C, both in vitro and in HeLa cells. High-quality DEER data could be obtained in HeLa cells up to 12 h after protein delivery at in-cell protein concentrations as low as 5-10 μm.

Ph-Induced Oligomerization of the Voltage Dependent Anion Channel

The Voltage-Dependent Anion Channel (VDAC) is the most abundant protein in the outer mitochondrial membrane facilitating the passage of ions and metabolites between the cytoplasm and the intermembrane space. In addition to its critical role in bioenergetics, VDAC is capable of modulating the organelle's permeability, implicating VDAC in metabolic stress and mitochondria-mediated apoptotic cell death. VDAC functions as a monomer but exhibits various oligomeric states, as shown by electron microscopy and atomic force microscopy. Oligomerization of VDAC can be linked to apoptosis and therefore is crucial for understanding mitochondrial regulation. Double Electron-Electron Resonance (DEER) on 4 singly spin-labeled mutants of mVDAC1 revealed a specific pH-induced dimerization. These inter-molecular distances were used to generate a model of a dimer interface that contains Glutamate 73. Mutation of Glutamate 73, to either Alanine or Glutamine, prevented pH induced oligomerization confirming the vital role of this residue. This dimer, induced by acidification, differs from any oligomeric form of VDAC previously described and likely plays a significant role in mitochondrial regulation and apoptosis in the response to cytosolic acidification during cellular stress.

Structural Insights into Sodium-Dependent Sugar Transporters and their Inhibition Mechanism

Sodium-dependent glucose transporters (SGLTs) are members of the large solute carrier (SLC) family of proteins that exploit the sodium ion concentration gradient to transport a myriad of small molecules across the plasma membrane. In humans, there are six SGLT subtypes labeled 1-6 that are expressed widely in the small intestine, kidney, lung, muscle, and brain. Due to their role in sugar reabsorption, SGLTs are currently exploited as drug targets for the treatment of type 2 diabetes, especially hSGLT2, which is responsible for 98% of glucose reabsorption in the kidneys. Current inhibitors are chemical derivatives of the naturally occurring small molecule phlorizin, which is expressed in the bark of fruit trees, such as apple and pear. The structural basis of binding is not known, in part, because high-resolution structures of mammalian SGLTs do not exist. However, the inward-facing structure of the bacterial homologue from vibrio parahaemolyticus (vSGLT) has been solved both in apo and in complex with galactose, and our collaborators recently solved the outward-facing structure of a closely related homologue (unpublished). Here we combine homology modeling, virtual screening techniques and molecular dynamics simulations to achieve two goals: 1) model the outward-facing state of SGLTs, and 2) predict the binding mode of phlorizin and its derivatives hSGLT1 and 2. As a result, the spectroscopic data (double electron-electron resonance) probing outward facing state of SGLTs validate our homology model and mutagenesis studies testing binding to hSGLT1 and 2 are in agreement with our predicted binding modes.

Sucrose and the Lipid Environment Modulate Conformational Heterogeneity in the Glutamate Transporter Homologue Gltph

Gltph is a sodium dependent aspartate transporter that is structurally homologous to glutamate transporters; as a result, it provides a model for excitatory amino acid transporters (EAATs) that facilitate glutamate reuptake. Crystal structures suggest a model for transport but do not provide information on biologically relevant intermediate states or effects of the lipid bilayer on the transport cycle. In the present work, distance distributions were measured for Gltph in three different bilayer systems though site directed spin labelling (SDSL) and double electron-electron resonance (DEER), and membrane depths were measured through SDSL and power saturation to determine if the crystal structures are representative of biologically relevant conformations. Transport domain distances were measured across the subunits of the trimer, and the protective osmolyte sucrose was used to modulate conformational populations. Distance distributions for T375R1 revealed populations with average distances that were close to crystal structure predictions. However, narrowing of the distance distributions and a shift to shorter distances in the presence of substrate suggest that there are biologically relevant states that are not seen in the crystal structures. Sucrose mediated stabilization of the inward facing state suggests increased lipid contacts, and membrane depth measurements showed that T375R1 comes into contact with the lipid environment in the presence of substrate, which suggests a currently unknown role for lipids during the transport cycle. This work was supported by NIGMS, GM035215.

Spectroscopic Studies of a Bacterial Cyclic Nucleotide-Gated Ion Channel

Cyclic nucleotide-gated (CNG) channels are members of the Kv channel superfamily and play a crucial role in the phototransduction and olfactory transduction pathways, where they generate the initial electrical signal following sensory transduction. CNG channels are directly regulated by cyclic nucleotides, which bind to a C-terminal cyclic nucleotide-binding domain (CNBD) connected to the pore by an intervening C-linker domain. Mutagenesis and crosslinking experiments indicate that the C-terminal region (C-linker/CNBD) undergoes a conformation change to transduce cyclic nucleotide binding into channel activation, though the exact nature of this change remains unclear. To address this question, we first sought a stable and well-behaved bacterial CNG channel suitable for spectroscopic studies. We used fluorescence-detection size-exclusion chromatography to screen a library of bacterial orthologs, which identified several candidates that could be expressed and purified at high levels. Flux studies of liposome-reconstituted channels demonstrated that these orthologs are fully functional and are activated by cyclic nucleotides. We then mutated the endogenous Cys residues in these channels before introducing Cys residues throughout the C-terminal region for spin label and fluorophore attachment. Inter-subunit double electron-electron resonance (DEER) measurements will be used to detect large-scale structural changes in these channels induced by cyclic nucleotide binding, while transition metal FRET (tmFRET) will be used to characterize subtle, localized changes to channel structure following activation. Our ultimate goal is to use these two spectroscopic techniques to characterize small- and large-scale rearrangements induced by cyclic nucleotide binding, allowing us to generate a complete structural model for CNG channel activation.

Principles of Energy Transduction and Alternating Access of ABC Exporters

ATP binding cassette (ABC) transporters to drive the energetically uphill translocation of substrates including the export of amphiphilic molecules out of the cell by harnessing the energy of ATP hydrolysis. Dedicated motor domains, the nucleotide binding domains (NBDs), bind and turn ATP over to power conformational rearrangements in the transmembrane domain (TMD) which binds and transports substrates. Although numerous investigations have defined the architecture and described the ATP-coupled conformational rearrangements for a number of ABC transporters, a general model of the transduction of ATP energy to the work of substrate remains elusive. We utilized double electron-electron resonance (DEER) and molecular dynamics (MD) simulations to describe the ATP- and substrate-coupled conformational cycle of the mammalian ABC efflux transporter P-glycoprotein (Pgp), or ABCB1, which has been extensively studied owing to its role in the clearance of xenobiotics and clinical implications in cancer resistance to chemotherapy. Pairs of spin labels were introduced at residues selected to track the putative inward-facing (IF) to outward-facing (OF) transition. Our findings illuminate how ATP energy is harnessed in the NBDs in a two-stroke cycle and elucidate the consequent conformational motion that reconfigures the TMD, two critical aspects of Pgp transport mechanism. Along with a novel, fully atomistic model of the OF conformation in membrane, the insight into Pgp conformational dynamics harmonizes mechanistic and structural data into a novel perspective on ATP-coupled transport. When considered in the context of previous studies of ABC exporters, these findings reveal mechanistic divergence within the efflux class of ABC transporters.

The Alpha-Synuclein Fibril Fold - Comparing Models from Electron Paramagnetic Resonance and NMR

Amyloid fibrils and plaques are hallmarks of neurodegenerative diseases. In Parkinson's disease, plaques (Lewy bodies) consist predominantly of the α-synuclein (αS) protein. To understand aggregation and elucidate the role of mutants in the disease, the molecular architecture of αS fibrils needs to be known. By double electron-electron paramagnetic resonance (DEER), nm-distance constraints can be determined. This is done by DEER on fibrils of doubly spin-labeled αS-variants, diamagnetically diluted with wild-type αS to suppress intermolecular interactions. Intramolecular distances in three pairs (56/69, 56/90 and 69/90) are reported. An approach to derive a model for the fibril-fold from sparse distance data assuming parallel β-sheets is described. Using the DEER distances as input, a model was derived with three strands, comprising residues 56 to 90, in which the strands consist of eight to twelve residues each. Details are described in[1], where also the viability of such a simple model is discussed.Comparison to structural models in the literature given in[1] is augmented in the present contribution by the discussion of the recently published NMR structure of αS fibrils.[2] This structure enables us to compare our constraints directly with the NMR-derived structure, using the MMM model for spin-label attachment.[3][1] Hashemi Shabestari et al. Appl Magn Reson 46, 369 (2015) DOI 10.1007/s00723-014-0622-7[2] Tuttle et al. Nat Struct Mol Biol. 23, 409 (2016)[3] Polyhach et al. Physical Chemistry Chemical Physics 13 2356 (2011)

Artefact suppression in 5-pulse double electron electron resonance for distance distribution measurements.

A 5-pulse version of the Double Electron Electron Resonance (DEER) experiment with Carr-Purcell delays and an additional pump pulse has been shown to significantly extend the experimentally accessible distance range in cases where nuclear spin diffusion dominates electron spin phase memory loss [Borbat et al., J. Phys. Chem. Lett., 2013, 4, 170]. We show that the sequence also prolongs coherence decay for spin labels in or near lipid bilayers, where this decay is mono-exponential. Compared to 4-pulse DEER, 5-pulse DEER suffers from additional artefacts that stem from pulse imperfection and excitation band overlap. Only some of these artefacts can be suppressed by phase cycling and the remaining ones have hindered widespread utilization of the method. Here, we report previously unknown additional artefact contributions stemming from overlap between the excitation bands of the microwave pulses that introduce additional dipolar evolution pathways. Experimental conditions are analyzed in detail that suppress these as well as the already known artefacts. Such suppression results in data that contain at most the partial excitation artefact, which can be deliberately shifted in time by a change in pulse timing without affecting the wanted contribution.

Reliable nanometre-range distance distributions from 5-pulse double electron electron resonance.

Double electron electron resonance (DEER) enables determination of distance distributions in the nanometre range. A recently introduced 5-pulse version of this experiment prolongs the electron spin coherence lifetime and thus provides improved sensitivity or an extended distance range, but suffers from artefacts due to partial excitation and excitation band overlap. In particular, the partial excitation artefact is hard to eliminate experimentally at frequencies where DEER is most sensitive or on spectrometers that provide only monochromatic pulses. Here, a data post-processing method is introduced that removes the partial excitation artefact without relying on previous knowledge of its amplitude and without sensitivity loss. The method is based on acquisition of two traces with shifted positions of the artefact and computation of the artefact shape from the difference of the two traces. Artefact removal was successfully tested both on simulated and experimental data. It was found to be stable for a variety of distance distributions and down to low signal-to-noise ratios in the presence of moderate background decay. The artefact correction method also performs well in the regime of rather strong partial excitation artefacts that is usually encountered with rectangular monochromatic pump pulses on widely available commercial spectrometers.

On the use of the Cu2+-iminodiacetic acid complex for double histidine based distance measurements by pulsed ESR.

Cu2+ based distance measurements using the double-histidine (dHis) motif by pulsed ESR present an attractive strategy to obtain precise, narrow distance distributions that can be easily related to protein backbone structure (Cunningham et al., Angew. Chem., Int. Ed., 2015, 54, 633). The Cu2+-ion is introduced as a complex with the iminodiacetic acid (IDA) chelating agent, which enhances binding selectivity to the two histidine residues that are site-selectively placed on the protein through mutagenesis. However, initial results of this method produced weak dipolar modulations. To enhance applicability of the double histidine motif using IDA, we perform a systematic examination of the possible causes of these weak dipolar modulations. We examine the efficiency of the Cu2+-ion to form the Cu2+-IDA complex in solution. In addition, we analyze the selectivity of Cu2+-IDA binding to dHis sites at both α-helical and β-strand environments. Our results indicate that the dHis motif on the β-sheet sites have high affinity towards Cu2+-IDA while the dHis sites on α-helices show poor affinity for the metal-ion complex. We are able to use our new findings to optimize conditions to maximize dHis loading while minimizing both free Cu2+ and unbound Cu2+-IDA complex in solution, allowing us to double the sensitivity of the Double Electron-Electron Resonance (DEER) experiment. Finally, we illustrate how Cu2+-based CW-ESR and DEER can be combined to obtain information on populations of different Cu2+-complexes in solution.

Spin labelling for integrative structure modelling: a case study of the polypyrimidine-tract binding protein 1 domains in complexes with short RNAs.

A combined method, employing NMR and EPR spectroscopies, has demonstrated its strength in solving structures of protein/RNA and other types of biomolecular complexes. This method works particularly well when the large biomolecular complex consists of a limited number of rigid building blocks, such as RNA-binding protein domains (RBDs). A variety of spin labels is available for such studies, allowing for conventional as well as spectroscopically orthogonal double electron-electron resonance (DEER) measurements in EPR. In this work, we compare different types of nitroxide-based and Gd(iii)-based spin labels attached to isolated RBDs of the polypyrimidine-tract binding protein 1 (PTBP1) and to short RNA fragments. In particular, we demonstrate experiments on spectroscopically orthogonal labelled RBD/RNA complexes. For all experiments we analyse spin labelling, DEER method performance, resulting distance distributions, and their consistency with the predictions from the spin label rotamers analysis. This work provides a set of intra-domain calibration DEER data, which can serve as a basis to start structure determination of the full length PTBP1 complex with an RNA derived from encephalomycarditis virus (EMCV) internal ribosomal entry site (IRES). For a series of tested labelling sites, we discuss their particular advantages and drawbacks in such a structure determination approach.

Computing distance distributions from dipolar evolution data with overtones: RIDME spectroscopy with Gd(iii)-based spin labels.

Extraction of distance distributions between high-spin paramagnetic centers from relaxation induced dipolar modulation enhancement (RIDME) data is affected by the presence of overtones of dipolar frequencies. As previously proposed, we account for these overtones by using a modified kernel function in Tikhonov regularization analysis. This paper analyzes the performance of such an approach on a series of model compounds with the Gd(iii)-PyMTA complex serving as paramagnetic high-spin label. We describe the calibration of the overtone coefficients for the RIDME kernel, demonstrate the accuracy of distance distributions obtained with this approach, and show that for our series of Gd-rulers RIDME technique provides more accurate distance distributions than Gd(iii)-Gd(iii) double electron-electron resonance (DEER). The analysis of RIDME data including harmonic overtones can be performed using the MATLAB-based program OvertoneAnalysis, which is available as open-source software from the web page of ETH Zurich. This approach opens a perspective for the routine use of the RIDME technique with high-spin labels in structural biology and structural studies of other soft matter.

Conformation-based assay of tau protein aggregation.

Amyloid fiber-forming proteins are predominantly intrinsically disordered proteins (IDPs). The protein tau, present mostly in neurons, is no exception. There is a significant interest in the study of tau protein aggregation mechanisms, given the direct correlation between the deposit of β-sheet structured neurofibrillary tangles made of tau and pathology in several neurodegenerative diseases, including Alzheimer's disease. Among the core unresolved questions is the nature of the initial step triggering aggregation, with increasing attention placed on the question whether a conformational change of the IDPs plays a key role in the early stages of aggregation. Specifically, there is growing evidence that a shift in the conformation ensemble of tau is involved in its aggregation pathway, and might even dictate structural and pathological properties of mature fibers. Yet, because IDPs lack a well-defined 3D structure and continuously exchange between different conformers, it has been technically challenging to characterize their structural changes on-pathway to aggregation. Here, we make a case that double spin labeling of the β-sheet stacking region of tau combined with pulsed double electron-electron resonance spectroscopy is a powerful method to assay conformational changes occurring during the course of tau aggregation, by probing intramolecular distances around aggregation-prone domains. We specifically demonstrate the potential of this approach by presenting recent results on conformation rearrangement of the β-sheet stacking segment VQIINK (known as PHF6*) of tau. We highlight a canonical shift of the conformation ensemble, on-pathway and occurring at the earliest stage of aggregation, toward an opening of PHF6*. We expect this method to be applicable to other critical segments of tau and other IDPs.

Analytical solution of the PELDOR inverse problem using the integral Mellin transform.

We describe a new model-free approach to solve the inverse problem in pulsed double electron-electron resonance (PELDOR, also known as DEER) spectroscopy and obtain the distance distribution function between two radicals from time-domain PELDOR data. The approach is based on analytical solutions of the Fredholm integral equations of the first kind using integral Mellin transforms to provide the distance distribution function directly. The approach appears to confine the noise in the computed distance distribution to short distances and does not introduce systematic distortions. Thus, the proposed analysis method can be a useful supplement to current methods to determine complicated distance distributions.

ESR分光法による光化学系IIの酸素発生系Mnクラスターの構造解析

Electronic structure of Manganese cluster in photosystem II was investigated by Electron Spin Resonance (ESR) spectroscopy. Spin projections on Mn ions in S2 state manganese cluster were determined by Pulsed Electron-Electron Double Resonance (PELDOR). Protons surrounded manganese cluster were detected by Electron Nuclear Double Resonance (ENDOR). Based on obtained spin projections, ENDOR signals were assigned to the water molecules ligated to manganese cluster. PELDOR technique was applied to the determination of the high affinity Mn2+ site in apo-photosystem II, which is the key site of photo-activation process of manganese cluster.