SDSL EPR Research Articles

Protein protein interactions of KCNQ1 and KCNE1 observed via SDSL EPR line shape analysis

KCNQ1, also known as Kv7.1, is a voltage gated potassium channel that associates with the KCNE protein family. Mutations in this protein has been found to cause a variety of diseases including long QT syndrome, a type of cardiac arrythmia where the QT interval observed on an electrocardiogram is longer than normal. This condition is often aggravated during strenuous exercise and can cause fainting spells or sudden death. KCNE1 is an ancillary protein that interacts with KCNQ1 in the membrane at varying molar ratios.

Recent Developments in Electron Paramagnetic Resonance for Spectroscopic Applications

This review investigated the developments of Electron paramagnetic resonance such as SDSL EPR, pulsed/CW ENDOR, high frequency/ high-field EPR, and ESEEM in biological systems such as photosynthesis, metalloproteins, radical enzymes, and phospholipid membranes. EPR spectroscopy act as a powerful tool to calculate the dynamics and structure of the biological systems from the paramagnetic centers. Applying the EPR measurements on the biomolecules like calmodulin, pyruvate kinase, nitrogenase, bromoperoxidase can also help understand their intermolecular behavior. The better insight into the detailed kinetics of the protein in the radical state can be evaluated quickly with the EPR spectroscopy than that of the XRD or NMR techniques. Hence, the specific potentials, principles, technicality, developments, and limitations, of EPR spectroscopy are highlighted here to understand its applications in the research and development of bio-medicine.

A metabolically engineered spin-labeling approach for studying glycans on cells.

Metabolic glycan engineering (MGE) coupled with nitroxide spin-labeling (SL) was utilized to investigate the heterogeneous environment of cell surface glycans in select cancer and normal cells. This approach exploited the incorporation of azides into cell surface glycans followed by a click reaction with a new nitroxide spin label. Both sialic acid and N-acetylglucosamine (GlcNAc) were targeted for spin labelling. Although each of these moieties experiences a diverse and heterogeneous glycan environment, their EPR spectra and hence mobility are both characterized as a linear combination of two distinct spectra where one component reflects a highly mobile or uncrowded micro-environment with the second component reflecting more restricted motion, reflective of increased crowding and packing within the glycocalyx. What differs among the spectra of the targeted glycans is the relative percentage of each component, with sialic acid moieties experiencing on average an ∼80% less crowded environment, where conversely GlcNAc/GalNAz labeled sites reported on average a ∼50% more crowded environment. These distinct environments are consistent with the organization of sugar moieties within cellular glycans where some residues occur close to the cell membrane/protein backbone (i.e. more restricted) and others are more terminal in the glycan (i.e. more mobile). Strikingly, different cell lines displayed varied relative populations of these two components, suggesting distinctive glycan packing, organization, and composition of different cells. This work demonstrates the capability of SDSL EPR to be a broadly useful tool for studying glycans on cells, and interpretation of the results provides insights for distinguishing the differences and changes in the local organization and heterogeneity of the cellular glycocalyx.

Probing Conformational Motions Underlying Anesthetic Drug Actions in a Ligand-Gated Ion Channel

General anesthetics and ethanol exert their functional effects on pentameric ligand-gated ion channels (pLGICs) by binding to distinct allosteric sites that are far from the agonist binding sites. While recent high-resolution structures of prokaryotic and eukaryotic pLGICs, solved in the presence of these drugs have illuminated their binding sites, the structural mechanisms underlying how these drugs enhance or inhibit pLGIC function are largely unknown. Recently, a single residue variant in the bacterial pLGIC, GLIC, was identified that is potentiated by ethanol (PNAS 108:12149-12154, 2011). Taking advantage of this GLIC ethanol-sensitive mutant, we are using site directed spin labeling electron paramagnetic resonance (SDSL EPR) spectroscopy to study motions underlying ethanol potentiation of GLIC currents. Previous studies have shown that agonist-mediated channel opening is associated with an inward tilting of the extracellular binding domain (ECD). Here, we examined if, and how, the ECD moves in response to ethanol binding in the transmembrane channel domain. Individual cysteine residues were introduced in the ECD of an ethanol-sensitive GLIC construct (F14’A) at the top of β-strands (β1, 2, 5, 6, 8), labeled with the spin probe MTSL and ethanol-induced motions were measured using double electron-electron resonance (DEER) spectroscopy and compared to agonist (proton)-induced gating motions. DEER distance distributions for spin labels attached to N19C (β1), S93C (β5) and Q101C (β6) revealed that the attached probes moved closer together at pH 3.0, indicating that the inner β-sheet of GLIC ECD tilts inwards upon agonist activation. Interestingly, DEER distance distributions for N19 (β1) and S93 (β5) showed no significant changes in the presence of ethanol, indicating that structural mechanisms underlying ethanol allosteric drug modulation of pLGIC function are different than agonist-mediated motions that lead to channel activation.

Production of recombinant human tektin 1, 2, and 4 and in vitro assembly of human tektin 1.

Proteins predicted to be composed of large stretches of coiled-coil structure have often proven difficult to crystallize for structural determination. We have successfully applied EPR spectroscopic techniques to the study of the structure and assembly of full-length human vimentin assembled into native 11 nm filaments, in physiologic solution, circumventing the limitations of crystallizing shorter peptide sequences. Tektins are a small family of highly alpha helical filamentous proteins found in the doublet microtubules of cilia and related structures. Tektins exhibit several similarities to intermediate filaments (IFs): moderate molecular weight, highly alpha helical, hypothesized to be coiled-coil, and homo- and heteromeric assembly into long smooth filaments. In this report, we show the application of IF research methodologies to the study of tektin structure and assembly. To begin in vitro studies, expression constructs for human tektins 1, 2, and 4 were synthesized. Recombinant tektins were produced in E. coli and purified by chromatography. Preparations of tektin 1 successfully formed filaments. The recombinant human tektin 1 was used to produce antibodies which recognized an antigen in mouse testes, most likely present in sperm flagella. Finally, we report the creation of seven mutants to analyze predictions of coiled-coil structure in the rod 1A domain of tektin 1. Although this region is predicted to be coiled-coil, our EPR analysis does not reflect the parallel, in register, coiled-coil structure as demonstrated in vimentin and kinesin. These results document that tektin can be successfully expressed and assembled in vitro, and that SDSL EPR techniques can be used for structural analysis.

Structural Dynamics of the MscL C-terminal Domain

The large conductance mechanosensitive channel (MscL), acts as an osmoprotective emergency valve in bacteria by opening a large, water-filled pore in response to changes in membrane tension. In its closed configuration, the last 36 residues at the C-terminus form a bundle of five α-helices co-linear with the five-fold axis of symmetry. Here, we examined the structural dynamics of the C-terminus of EcMscL using site-directed spin labelling electron paramagnetic resonance (SDSL EPR) spectroscopy. These experiments were complemented with computational modelling including molecular dynamics (MD) simulations and finite element (FE) modelling. Our results show that under physiological conditions, the C-terminus is indeed an α-helical bundle, located near the five-fold symmetry axis of the molecule. Both experiments and computational modelling demonstrate that only the top part of the C-terminal domain (from the residue A110 to E118) dissociates during the channel gating, while the rest of the C-terminus stays assembled. This result is consistent with the view that the C-terminus functions as a molecular sieve and stabilizer of the oligomeric MscL structure as previously suggested.

Structural Dynamics of the MSCL C-Terminal Domain

The large conductance mechanosensitive channel (MscL), acts as an osmoprotective emergency valve in bacteria by opening a large, water-filled pore in response to changes in membrane tension. In its closed configuration, the last 36 residues at the C-terminus form a bundle of five α-helices co-linear with the five-fold axis of symmetry. Here, we examined the structural dynamics of the C-terminus of EcMscL using site-directed spin labelling electron paramagnetic resonance (SDSL EPR) spectroscopy. These experiments were complemented with computational modelling including molecular dynamics (MD) simulations and finite element (FE) modelling. Our results show that under physiological conditions, the C-terminus is indeed an α-helical bundle, located near the five-fold symmetry axis of the molecule. Both experiments and computational modelling demonstrate that only the top part of the C-terminal domain (from the residue A110 to E118) dissociates during the channel gating, while the rest of the C-terminus stays assembled. This result is consistent with the view that the C-terminus functions as a molecular sieve and stabilizer of the oligomeric MscL structure as previously suggested.

Nanoscale lipid membrane mimetics in spin-labeling and electron paramagnetic resonance spectroscopy studies of protein structure and function

AbstractCellular membranes and associated proteins play critical physiological roles in organisms from all life kingdoms. In many cases, malfunction of biological membranes triggered by changes in the lipid bilayer properties or membrane protein functional abnormalities lead to severe diseases. To understand in detail the processes that govern the life of cells and to control diseases, one of the major tasks in biological sciences is to learn how the membrane proteins function. To do so, a variety of biochemical and biophysical approaches have been used in molecular studies of membrane protein structure and function on the nanoscale. This review focuses on electron paramagnetic resonance with site-directed nitroxide spin-labeling (SDSL EPR), which is a rapidly expanding and powerful technique reporting on the local protein/spin-label dynamics and on large functionally important structural rearrangements. On the other hand, adequate to nanoscale study membrane mimetics have been developed and used in conjunction with SDSL EPR. Primarily, these mimetics include various liposomes, bicelles, and nanodiscs. This review provides a basic description of the EPR methods, continuous-wave and pulse, applied to spin-labeled proteins, and highlights several representative applications of EPR to liposome-, bicelle-, or nanodisc-reconstituted membrane proteins.

Radial Tilting of the Extracellular Domain of GLIC Revealed by EPR Spectroscopy

Signaling in the brain depends on rapid opening and closing of pentameric ligand-gated ion channels (pLGICs). Defects in these proteins are linked to a variety of diseases including myasthenia, epilepsy and sleep-disorders. In addition, these proteins are therapeutic targets for various drugs including anti-convulsants, anxiolytics, anesthetics and sedatives. While recent crystal structures of prokaryotic and eukaryotic pLGICs solved in detergent micelles have shed light on the molecular architecture of these proteins, describing their conformational dynamics in physiological lipids is essential for understanding their function. Crystal structures of GLIC and the glutamate-activated chloride channel predict that agonist-mediated channel opening is associated with a quaternary twisting and a radial un-blooming (inward tilting) of the extracellular binding domain (ECD). Using site directed spin labeling electron paramagnetic resonance (SDSL EPR) spectroscopy and functional GLIC channels reconstituted in liposomes, we are testing these gating predictions. Residues located at the top of β-strands (β1, 2, 5, 6, 8) were mutated to cysteine, labeled with the spin probe MTSL and proton-induced gating motions were measured using continuous wave (CW) and double electron-electron resonance (DEER) spectroscopy. CW EPR spectroscopy data showed the spin label attached to I92C, S93C, V94C (β5), T99C and Q101C (β6) moved into a slightly more constrained/immobile environment at pH 3.0 as compared to pH 7.6. DEER distance distributions for K48 (β2), S93 (β5), T99 and Q101 (β6) revealed the probes moved closer together at pH 3.0, suggesting that the inner β-sheet of the extracellular domain of GLIC tilt inwards upon agonist activation.

Elucidating the design principles of photosynthetic electron-transfer proteins by site-directed spin labeling EPR spectroscopy

Site-directed spin labeling electron paramagnetic resonance (SDSL EPR) spectroscopy is a powerful tool to determine solvent accessibility, side-chain dynamics, and inter-spin distances at specific sites in biological macromolecules. This information provides important insights into the structure and dynamics of both natural and designed proteins and protein complexes. Here, we discuss the application of SDSL EPR spectroscopy in probing the charge-transfer cofactors in photosynthetic reaction centers (RC) such as photosystem I (PSI) and the bacterial reaction center (bRC). Photosynthetic RCs are large multi-subunit proteins (molecular weight≥300kDa) that perform light-driven charge transfer reactions in photosynthesis. These reactions are carried out by cofactors that are paramagnetic in one of their oxidation states. This renders the RCs unsuitable for conventional nuclear magnetic resonance spectroscopy investigations. However, the presence of native paramagnetic centers and the ability to covalently attach site-directed spin labels in RCs makes them ideally suited for the application of SDSL EPR spectroscopy. The paramagnetic centers serve as probes of conformational changes, dynamics of subunit assembly, and the relative motion of cofactors and peptide subunits. In this review, we describe novel applications of SDSL EPR spectroscopy for elucidating the effects of local structure and dynamics on the electron-transfer cofactors of photosynthetic RCs. Because SDSL EPR Spectroscopy is uniquely suited to provide dynamic information on protein motion, it is a particularly useful method in the engineering and analysis of designed electron transfer proteins and protein networks. This article is part of a Special Issue entitled Biodesign for Bioenergetics — the design and engineering of electronic transfer cofactors, proteins and protein networks, edited by Ronald L. Koder and J.L. Ross Anderson.

Selective Modification of Lipid Bilayer Dynamics and Fluidity Properties by Kl4 (Mimetic of Surfactant Protein B)

KL4, a 21 residue mimetic of surfactant protein B, is effective in the treatment of infant respiratory distress syndrome (RDS) and functions by lowering aveolar surface tension and promoting oxygen exchange. Here we compare the results from previous NMR investigations to those of fluorescence characterization of the effects of KL4 on the lipid dynamics and organization in liposomes. We utilized a pyrene phospholipid analog to investigate the effect of KL4 on lipid organization and acyl chain dynamics by monitoring changes in excimer-to-monomer (Ie/Im) ratio. This experiment probes the environment of the hydrophobic core of DPPC/POPG and POPC/POPG liposomes. An average decrease of ∼27-40% and ∼0-10% in Ie/Im was observed in the DPPC/POPG and POPC/POPG LUVs, respectively, with increasing peptide concentration (0.5 to 5 mol%). This decrease is directly proportional to a lowered probability of excimer formation, which is highly dependent on proximal interactions of an excited monomer with a pyrene moiety at ground state.The ability of the peptide to modify membrane fluidity properties was studied via anisotropy measurements of a rhodamine-labeled phospholipid. A steady increase in the order was observed in the DPPC/POPG liposomes with relatively constant fluorescence intensity, while collisional quenching was observed in the POPC/POPG liposomes. These observations agree with NMR observations and proposed mechanisms of peptide-mediated lipid trafficking. Further studies on the orientation and penetration of the peptide were interrogated using power saturation SDSL EPR.

Inter-spin distance determination using L-band (1–2 GHz) non-adiabatic rapid sweep electron paramagnetic resonance (NARS EPR)

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 8–80Å can be experimentally determined. However, there exists a region at the upper limit of the conventional X-band (9.5GHz) 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 (1.9GHz) 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 [1]. Two different systems are presented, and distances of 18–30Å have been experimentally determined at physiologically relevant temperatures. Measurements are in excellent agreement with a helical model and values determined by DEER.

Site-Directed Spin Labeling Reveals Pentameric Ligand-Gated Ion Channel Gating Motions

Pentameric ligand-gated ion channels (pLGICs) are neurotransmitter-activated receptors that mediate fast synaptic transmission. In pLGICs, the flexible loops in each subunit that connect the extracellular binding domain (loops 2, 7, 9) to the transmembrane channel domain (M2-M3 loop) are essential for coupling ligand binding to channel gating. Comparing the recent crystal structures of two bacterial pLGIC homologues, ELIC and GLIC, suggests channel opening is associated with rearrangements in these loops but the specifics of these motions remain unknown. Here, using site-directed spin labeling electron paramagnetic resonance (SDSL EPR) spectroscopy, we measured proton-induced movements of loop 2, loop 9, and M2-M3 loop in functional GLIC channels reconstituted into liposomes. The loops undergo significant proton-dependent motion, with loop 2 and loop 9 moving inward toward the channel lumen, and the M2-M3 loop moving at least 5 angstrom outward away from the channel lumen. The movements are consistent with the gating motions predicted by comparing the ELIC and GLIC structures, and suggest the ELIC structure represents the resting state of pLGICs.

Conformational Changes in the Novel Redox Sensor Protein HbpS Studied by Site-Directed Spin Labeling and Its Turnover in Dependence on the Catalase-Peroxidase CpeB

To establish conditions to study the oligomeric assembly of heme-binding protein (HbpS) in solution by applying the tools of site-directed spin labeling combined with pulse electron paramagnetic resonance (SDSL EPR) spectroscopy, as well as to analyze redox stress-based conformational changes in HbpS subunits within the oligomer in solution. In vivo elucidation of molecular mechanisms that control the downregulation of the novel redox-system HbpS-SenS-SenR. Using a set of specifically generated HbpS mutants, and SDSL EPR spectroscopy, we show the octomeric assembly of HbpS in solution, and demonstrate that iron-mediated stress induces conformational changes in HbpS subunits within the octamer. We further demonstrate that the catalase-peroxidase CpeB protects HbpS from hydrogen peroxide (H(2)O(2))-mediated oxidative attack in vivo. Moreover, chromosomal inactivation of cpeB results in an enhanced sensitivity of the mutant to redox-cycling compounds. SDSL EPR has been used in this work for the first time to monitor redox-mediated conformational changes in a redox-sensing protein in solution. This work substantially explains redox-dependent dynamics in HbpS at the atomic level, and presents novel molecular mechanisms supporting downregulation of a signaling cascade. Iron-mediated stress induces movements of subunits within the HbpS octomeric assembly. We suggest a motion of the C-terminal α-helix toward the preceding helical segment. These events upregulate the activity of the HbpS-SenS-SenR system, in which HbpS acts as an accessory element. The mycelia-associated CpeB, under the control of HbpS-SenS-SenR, protects the extracellular HbpS from oxidation in vivo. Thus, de novo synthesized HbpS proteins downregulate the HbpS-SenS-SenR signaling cascade.

Conformational Analysis of the Partially Disordered Measles Virus N TAIL-XD Complex by SDSL EPR Spectroscopy

To characterize the structure of dynamic protein systems, such as partly disordered protein complexes, we propose a novel approach that relies on a combination of site-directed spin-labeled electron paramagnetic resonance spectroscopy and modeling of local rotation conformational spaces. We applied this approach to the intrinsically disordered C-terminal domain of the measles virus nucleoprotein (N TAIL) both free and in complex with the X domain (XD, aa 459–507) of the viral phosphoprotein. By comparing measured and modeled temperature-dependent restrictions of the side-chain conformational spaces of 12 SL cysteine-substituted N TAIL variants, we showed that the 490–500 region of N TAIL is prestructured in the absence of the partner, and were able to quantitatively estimate, for the first time to our knowledge, the extent of the α-helical sampling of the free form. In addition, we showed that the 505–525 region of N TAIL conserves a significant degree of freedom even in the bound form. The latter two findings provide a mechanistic explanation for the reported rather high affinity of the N TAIL-XD binding reaction. Due to the nanosecond timescale of X-band EPR spectroscopy, we were also able to monitor the disordering in the 488–525 region of N TAIL, in particular the unfolding of the α-helical region when the temperature was increased from 281 K to 310 K.

To Determine the Structure of Vimentin Head Domain Using SDSL-EPR Approach

Cysteines were placed in vimentin head spanning 1-108 positions using basic genetic engineering approaches. The mutants were expressed in bacteria, purified by FPLC, and spin-labeled using O-87500 [MTSL-d15]. Labeled proteins were assembled into filaments by dialysis from 8M urea and filament assembly was checked by electron microscopy. EPR measurements were carried out in JEOL X-band spectrometer fitted with a loop-gap resonator. The SDSL EPR spectral data indicated that 48/50 positions tested displayed spectral broadening indicating the close proximity of these positions (<2.1 nm). Mixing protein spin labeled at a given residue, with protein labeled at sites slightly “up”- or downstream, decreased the level of dipolar interaction in all cases. This data provide strong evidence that the polypeptide backbones of head domains remain in close proximity, forming a symmetric structure which folds back on the rod domain, bringing residues 17 of the head and 137 of the rod into close proximity. By monitoring in vitro assembly process, we show that head-head interactions occur early in the assembly process. We show that spin labels in the region of residues 50 and 103 display high level of mobility at protofilament stage of assembly but becoming more compacted and motionally constrained upon assembly into intact filaments, suggesting that head domain structure is dynamic and changes during assembly. Finally, because the vimentin head domain is the major site of phosphorylation, we compared specific spin-spin interactions in the phosphorylated state, and have identified distinct structural changes resulting from phosphorylation of head domain. This report presents the first evidence-based structural model for the head domain of any IF protein. We provide data which shows that head domain structure is dynamic, changing with both assembly into filaments, but also with phosphorylation, a physiologic regulator of vimentin assembly/disassembly.

Conformational Flexibility of the GM2 Activator Protein Loop Regions Investigated By Site Directed Spin Labeling EPR Spectroscopy

The GM2 activator protein (GM2AP) is an essential component in the degradation pathway of neuronal gangliosides. GM2AP is a required accessory protein for the hydrolytic conversion of GM2 to GM3 by a water soluble hydrolase. The X-ray structure of GM2AP reveals a β-cup topology with multiple conformations of the protein within the unit cell. Because the crystal structures show different conformations of the putative membrane binding loops, we have utilized site-directed spin labeling to investigate conformational flexibility of these loops for protein in solution and bound with GM2 ligand. As such, a series of single and double CYS mutants (still with original 8 CYS in 4 disulfide bridges) have been generated and spin labeled with MTSL. EPR spectra of spin labeled GM2AP were collected with and without GM2 ligand, and no significant changes in the EPR lineshape were seen. EPR spectra were simulated for spin labels located in the loop regions and reveal multiple component fits, while those in the backside of the β-cup beta strands have single component fits. For certain sites in the mobile loops, spectra were acquired as a function of temperature. From these lineshape simulations, the activation energy for the conformational change has been determined. The SDSL EPR results indicate that the multiple conformations observed in the crystallographic unit cell are populated in solution and represent conformational flexibility of the protein; which is not necessarily altered by binding to lipid ligands. Additionally, spin labeled protein was analyzed by mass spectrometry to confirm proper formation of the four disulfide linkages and addition of only one spin label at the mutant cysteine.

3P-003 SDSL EPRによる距離測定におけるスピンラベル分子の運動性の影響(蛋白質-構造,第47回日本生物物理学会年会)

3P-003 SDSL EPRによる距離測定におけるスピンラベル分子の運動性の影響(蛋白質-構造,第47回日本生物物理学会年会)