Site-directed Spin Labeling EPR Research Articles

Structural Flexibility of Tau in Its Interaction with Microtubules as Viewed by Site-Directed Spin Labeling EPR Spectroscopy.

Tau is a microtubule-associated protein that belongs to the Intrinsically Disordered Proteins (IDPs) family. IDPs or Intrinsically Disordered Regions (IDRs) play key roles in protein interaction networks and their dysfunctions are often related to severe diseases. Defined by their lack of stable secondary and tertiary structures in physiological conditions while being functional, these proteins use their inherent structural flexibility to adapt to and interact with various binding partners. Knowledges on the structural dynamics of IDPs and their different conformers are crucial to finely decipher fundamental biological processes controlled by mechanisms such as conformational adaptations or switches, induced fit, or conformational selection events. Different mechanisms of binding have been proposed: among them, the so-called folding-upon-binding in which the IDP adopts a certain conformation upon interacting with a partner protein, or the formation of a "fuzzy" complex in which the IDP partly keeps its dynamical character at the surface of its partner. The dynamical nature and physicochemical properties of unbound as well as bound IDPs make this class of proteins particularly difficult to characterize by classical bio-structural techniques and require specific approaches for the fine description of their inherent dynamics.Among other techniques, Site-Directed Spin Labeling combined with Electron Paramagnetic Resonance (SDSL-EPR) spectroscopy has gained much interest in this last decade for the study of IDPs. SDSL-EPR consists in grafting a paramagnetic label (mainly a nitroxide radical) at selected site(s) of the macromolecule under interest followed by its observation using and/or combining different EPR strategies. These nitroxide spin labels detected by continuous wave (cw) EPR spectroscopy are used as perfect reporters or "spy spins" of their local environment, being able to reveal structural transitions, folding/unfolding events, etc. Another approach is based on the measurement of inter-label distance distributions in the 1.5-8.0nm range using pulsed dipolar EPR experiments, such as Double Electron-Electron Resonance (DEER) spectroscopy. The technique is then particularly well suited to study the behavior of Tau in its interaction with its physiological partner: microtubules (MTs). In this chapter we provide a detailed experimental protocol for the labeling of Tau protein and its EPR study while interacting with preformed (Paclitaxel-stabilized) MTs, or using Tau as MT inducer. We show how the choice of nitroxide label can be crucial to obtain functional information on Tau/tubulin complexes.

Site-directed spin label EPR studies of the structure and membrane interactions of the bacterial phospholipase ExoU.

Site-directed spin labeling (SDSL) has been invaluable in the analysis of protein structure and dynamics, and has been particularly useful in the study of membrane proteins. ExoU, an important virulence factor in Pseudomonas aeruginosa infections, is a bacterial phospholipase A2 that functions at the membrane - aqueous interface. Using SDSL methodology developed in the Hubbell lab, we find that the region surrounding the catalytic site of ExoU is buried within the tertiary structure of the protein in the soluble, apoenzyme state, but shows a significant increase in dynamics upon membrane binding and activation by ubiquitin. Continuous wave (CW) power saturation EPR studies show that the conserved serine hydrolase motif of ExoU localizes to the membrane surface in the active, holoenzyme state. SDSL studies on the C-terminal four-helix bundle (4HB) domain of ExoU similarly show a co-operative effect of ubiquitin binding and membrane association. CW power saturation studies of the 4HB domain indicate that two interhelical loops intercalate into the lipid bilayer upon formation of the holoenzyme state, anchoring ExoU at the membrane surface. Together these studies establish the orientation and localization of ExoU and the membrane surface, and illustrate the power of SDSL as applied to peripheral membrane proteins.

Electron paramagnetic resonance spectroscopic characterization of the human KCNE3 protein in lipodisq nanoparticles for structural dynamics of membrane proteins

One of the major challenges in solubilization of membrane proteins is to find the optimal physiological environment for their biophysical studies. EPR spectroscopy is a powerful biophysical technique for studying the structural and dynamic properties of macromolecules. However, the challenges in the membrane protein sample preparation and flexible motion of the spin label limit the utilization of EPR spectroscopy to a majority of membrane protein systems in a physiological membrane-bound state. Recently, lipodisq nanoparticles or styrene-maleic acid copolymer-lipid nanoparticles (SMALPs) have emerged as a membrane mimetic system for investigating the structural studies of membrane proteins. However, its detail characterization for membrane protein studies is still poorly understood. Recently, we characterized the potassium channel membrane protein KCNQ1 voltage sensing domain (KCNQ1-VSD) and KCNE1 reconstituted into lipodisq nanoparticles using EPR spectroscopy. In this study, the potassium channel accessory protein KCNE3 containing flexible N- and C-termini was encapsulated into proteoliposomes and lipodisq nanoparticles and characterized for studying its structural and dynamic properties using nitroxide based site-directed spin labeling EPR spectroscopy. CW-EPR lineshape analysis data indicated an increase in spectral line broadenings with the addition of the styrene-maleic acid (SMA) polymer which approaches close to the rigid limit providing a homogeneous stabilization of the protein-lipid complex. Similarly, EPR DEER measurements indicated an enhanced quality of distance measurements with an increase in the phase memory time (Tm) values upon incorporation of the sample into lipodisq nanoparticles, when compared to proteoliposomes. These results agree with the solution NMR structural structure of the KCNE3 and EPR studies of other membrane proteins in lipodisq nanoparticles. This study along with our earlier studies will provide the reference characterization data that will provide benefit to the membrane protein researchers for studying structural dynamics of challenging membrane proteins.

Hydrogen-Bonded Complexes of Neutral Nitroxide Radicals with 2-Propanol Studied by Multifrequency EPR/ENDOR

The hydrogen bond plays a key role in weak directional intermolecular interactions. It is operative in determining molecular conformation and aggregation, and controls the function of many chemical systems, ranging from inorganic, organic to biological molecules. Although an enormous amount of spectroscopic information has been collected about hydrogen-bond formation between molecules with closed-shell electronic configuration, the details of such interactions between open-shell radicals and closed-shell molecules are still rare. Here we report on an investigation of hydrogen-bonded complexes between pyrroline-type as well as piperidine-type neutral nitroxide radicals and an alcohol, i.e., 2-propanol. These nitroxide radicals are commonly used as EPR spin labels and probes. To obtain information on the geometry of the complexes and their electronic structure, multi-resonance EPR techniques at various microwave frequencies (X-, Q-, W-band, 244 GHz) have been employed in conjunction with DFT calculations. The planar five-membered ring system of the pyrroline-type nitroxide radical was found to form exclusively well-defined in-plane σ-type hydrogen-bonded complexes with one 2-propanol molecule in the first solvation shell in frozen solution. The measured hyperfine parameters of the hydrogen-bridge proton and the internal magnetic parameters describing the electron Zeeman and the electron-nuclear hyperfine and nuclear quadrupole interactions are in good agreement with values predicted by state-of-the-art DFT calculations. In contrast, multi-resonance EPR on the non-planar six-membered ring system of the piperidine-type nitroxide radical (TEMPOL) reveals a more complex situation, i.e., a mixture of a σ-type with, presumably, an out-of-plane π-type complex, both present in comparable fraction in frozen solution. For TEMPOL, the DFT calculations failed to predict magnetic interaction parameters that are in good agreement with experiment, apparently due to the considerable flexibility of the nitroxide and hydrogen-bonded complex. The detailed information about nitroxide/solvent complexes is of particular importance for Dynamic Nuclear Polarization (DNP) and site-directed spin-labeling EPR studies that employ nitroxides as polarizing agents or spin labels, respectively.

Structural dynamics of the chromo-shadow domain and chromodomain of HP1 bound to histone H3K9 methylated peptide, as measured by site-directed spin-labeling EPR spectroscopy

The structural dynamics of the chromo-shadow domain (CSD) and chromodomain (CD) of human HP1 proteins essential for heterochromatin formation were investigated at the nanosecond and nanometer scales by site-directed spin labeling electron paramagnetic resonance and pulsed double resonance spectroscopy. Distance measurements showed that the spin-labeled CSD of human HP1α and HP1γ tightly dimerizes. Unlike CD-CD interaction observed in fission yeast HP1 in an inactivated state (Canzio et al., 2013), the two CDs of HP1α and HP1γ were spatially separated from each other, dynamically mobile, and ready for a Brownian search for H3K9-tri-methyl(me3) on histones. Complex formation of the CD with H3K9me3 slowed dynamics of the domain due to a decreased diffusion constant. CSD mobility was significantly (∼1.3-fold) lower in full-length HP1α than in HP1γ, suggesting that the immobilized conformation of human HP1α shows an auto-inactivated state. Differential properties of HP1α and HP1γ to form the inactive conformation could be relevant to its physiological role in the heterochromatin formation in a cell.

Investigation of Structural Dynamics of the KCNE3 in a Lipid Bilayer Membrane using Site-Directed Spin Labeling EPR Spectroscopy

KCNE3, a single pass membrane protein, is responsible for controlling the function of multiple potassium channels, one of which is the KCNQ1 channel. The binding of KCNE3 with KCNQ1 is vital in transepithelial potassium ion recycling which has been associated with disorders such as cystic fibrosis. Although KCNE3 has great biological significance, its structural and dynamic information in a native like environment is poorly understood. In this study, we have utilized site-directed spin labeling electron paramagnetic resonance (SDSL-EPR) spectroscopic technique to investigate structural dynamics of KCNE3 in lipid bilayers. KCNE3 was overexpressed and purified in LMPG detergent micelles. MTSL spin label was introduced followed by the incorporation of the protein into POPC/POPG lipid bilayer. Continuous wave (CW)-EPR spectral lineshape analysis was performed on several spin labeling sites including transmembrane domain and extracellular domain of KCNE3 in LMPG detergent micelles and POPC/POPG lipid bilayes to obtain the spin label sidechain mobility and the rotational correlation times. Our EPR data indicated that the nitroxide spin label sidechains in liposomes have slower motion for the sites in the transmembrane domain of KCNE3 when compared to that in the extracellular region of KCNE3. Our results are consistent with the previously published solution NMR structure of KCNE3.

Effects of Fe2+/Fe3+ Binding to Human Frataxin and Its D122Y Variant, as Revealed by Site-Directed Spin Labeling (SDSL) EPR Complemented by Fluorescence and Circular Dichroism Spectroscopies.

Frataxin is a highly conserved protein whose deficiency results in the neurodegenerative disease Friederich’s ataxia. Frataxin’s actual physiological function has been debated for a long time without reaching a general agreement; however, it is commonly accepted that the protein is involved in the biosynthetic iron-sulphur cluster (ISC) machinery, and several authors have pointed out that it also participates in iron homeostasis. In this work, we use site-directed spin labeling coupled to electron paramagnetic resonance (SDSL EPR) to add new information on the effects of ferric and ferrous iron binding on the properties of human frataxin in vitro. Using SDSL EPR and relating the results to fluorescence experiments commonly performed to study iron binding to FXN, we produced evidence that ferric iron causes reversible aggregation without preferred interfaces in a concentration-dependent fashion, starting at relatively low concentrations (micromolar range), whereas ferrous iron binds without inducing aggregation. Moreover, our experiments show that the ferrous binding does not lead to changes of protein conformation. The data reported in this study reveal that the currently reported binding stoichiometries should be taken with caution. The use of a spin label resistant to reduction, as well as the comparison of the binding effect of Fe2+ in wild type and in the pathological D122Y variant of frataxin, allowed us to characterize the Fe2+ binding properties of different protein sites and highlight the effect of the D122Y substitution on the surrounding residues. We suggest that both Fe2+ and Fe3+ might play a relevant role in the context of the proposed FXN physiological functions.

Characterization of the ExoU activation mechanism using EPR and integrative modeling

ExoU, a type III secreted phospholipase effector of Pseudomonas aeruginosa, serves as a prototype to model large, dynamic, membrane-associated proteins. ExoU is synergistically activated by interactions with membrane lipids and ubiquitin. To dissect the activation mechanism, structural homology was used to identify an unstructured loop of approximately 20 residues in the ExoU amino acid sequence. Mutational analyses indicate the importance of specific loop amino acid residues in mediating catalytic activity. Engineered disulfide cross-links show that loop movement is required for activation. Site directed spin labeling EPR and DEER (double electron–electron resonance) studies of apo and holo states demonstrate local conformational changes at specific sites within the loop and a conformational shift of the loop during activation. These data are consistent with the formation of a substrate-binding pocket providing access to the catalytic site. DEER distance distributions were used as constraints in RosettaDEER to construct ensemble models of the loop in both apo and holo states, significantly extending the range for modeling a conformationally dynamic loop.

A non–GPCR-binding partner interacts with a novel surface on β-arrestin1 to mediate GPCR signaling

The multifaceted adaptor protein β-arr1 (β-arrestin1) promotes activation of focal adhesion kinase (FAK) by the chemokine receptor CXCR4, facilitating chemotaxis. This function of β-arr1 requires the assistance of the adaptor protein STAM1 (signal-transducing adaptor molecule 1) because disruption of the interaction between STAM1 and β-arr1 reduces CXCR4-mediated activation of FAK and chemotaxis. To begin to understand the mechanism by which β-arr1 together with STAM1 activates FAK, we used site-directed spin-labeling EPR spectroscopy-based studies coupled with bioluminescence resonance energy transfer-based cellular studies to show that STAM1 is recruited to activated β-arr1 by binding to a novel surface on β-arr1 at the base of the finger loop, at a site that is distinct from the receptor-binding site. Expression of a STAM1-deficient binding β-arr1 mutant that is still able to bind to CXCR4 significantly reduced CXCL12-induced activation of FAK but had no impact on ERK-1/2 activation. We provide evidence of a novel surface at the base of the finger loop that dictates non-GPCR interactions specifying β-arrestin-dependent signaling by a GPCR. This surface might represent a previously unidentified switch region that engages with effector molecules to drive β-arrestin signaling.

Probing the M1-M2 Interaction in Influenza a Virus using Site-Directed Spin Labeling EPR in Lipid Bilayer Nanodiscs

Influenza remains a serious global health threat and further characterization of key viral proteins is necessary in order to develop novel drugs and vaccines. Matrix Protein 1 (M1) and Matrix Protein 2 (M2) play key roles in the viral life cycle of Influenza A Virus. M2 is a 97-amino acid membrane bound protein which we have previously studied using site-directed spin labeling EPR and has been shown to have multiple conformational states as well as a critical role in the budding of new viral particles from infected cells. M1 is a 252-amino acid protein with multiple roles, including packaging viral RNA and recruiting it to the budozone through interactions with M2 and other viral membrane proteins. M1 oligomerizes through interactions with other M1 monomers inside the emerging virion. We are using a membrane scaffold protein nanodisc model system with incorporate M2 in order to study how interaction with full-length M1 alters M2 mobility and accessibility to relaxation agents. Nanodiscs allow access to intracellular domains of membrane-bound proteins and are compatible with a number of biophysical assays. Site-directed spin labeling EPR will provide residue-specific information about the conformational changes in M2 due to interactions with M1 and how this interaction contributes to viral budding in Influenza A.

Conformational Dynamics of Sensory Rhodopsin II in Nanolipoprotein and Styrene-Maleic Acid Lipid Particles.

Styrene-maleic acid lipid particles (SMALPs) provide stable water-soluble nanocontainers for lipid-encased membrane proteins. Possible effects of the SMA-stabilized lipid environment on the interaction dynamics between functionally coupled membrane proteins remain to be elucidated. The photoreceptor sensory rhodopsin II, NpSRII and its cognate transducer, NpHtrII, of Natronomonas pharaonis form a transmembrane complex, NpSRII2 /NpHtrII2 that plays a key role in negative phototaxis and provides a unique model system to study the light-induced transfer of a conformational signal between two integral membrane proteins. Photon absorption induces transient structural changes in NpSRII comprising an outward movement of helix F that cause further conformational alterations in NpHtrII. We applied site-directed spin labeling and time-resolved optical and EPR spectroscopy to compare the conformational dynamics of NpSRII2 /NpHtrII2 reconstituted in SMALPs with that of nanolipoprotein particle and liposome preparations. NpSRII and NpSRII2 /NpHtrII2 show similar photocycles in liposomes and nanolipoprotein particles. An accelerated decay of the M photointermediate found for SMALPs can be explained by a high local proton concentration provided by the carboxylic groups of the SMA polymer. Light-induced large-scale conformational changes of NpSRII2 /NpHtrII2 observed in liposomes and nanolipoprotein particles are affected in SMALPs, indicating restrictions of the protein's conformational freedom.

EPR Spectroscopic Studies of the Voltage Sensor Domain (Q1-VSD) of Human KCNQ1 Potassium Ion Channel in Lipid Bilayers

KCNQ1 is a voltage gated potassium ion channel expressed in several tissues of the body and modulated by another membrane protein KCNE1 (E1). Q1 is involved in the repolarization phase of cardiac action potential. Q1/E1 interaction slows down the activation kinetics required for proper channel and heart function. Mutations in Q1 can cause Long-QT syndrome resulting in atrial fibrillation, sudden infant death syndrome, cardiac arrhythmias and congenital deafness. Q1-VSD is important as the channel is activated by a change in voltage, which is sensed by the VSD. Around 40% of the >200 reported disease-related mutations arise from the amino acid substitutions in the VSD, making structural and functional studies of Q1-VSD important. The structure of a membrane protein varies from micelles to lipid bilayer environment. Site-directed spin labeling (SDSL) EPR is a powerful structural biology technique to study the structural dynamics and topology of membrane proteins in lipid bilayers. In this study, the structural dynamics and topology of several residues of Q1-VSD in the POPC/POPG vesicles were studied using CW-EPR line shape analysis and CW power saturation techniques. Spin labeled mutants F130C and F232C were found to be located inside the lipid bilayer, while Q147C and F222C were found to be solvent accessible. The study provides novel insights to the structural details of Q1-VSD reconstituted in POPC/POPG vesicles for more advanced structural and functional studies.

Site-Directed Spin Labeling EPR Studies on the Catalytic Aspartate Loop of Exou Upon Interaction with Ubiquitin and Membranes

Site-Directed Spin Labeling EPR Studies on the Catalytic Aspartate Loop of Exou Upon Interaction with Ubiquitin and Membranes

Proteins' Dynamics, Hydration and Conformational Changes Studied by EPR

Site-directed spin labeling EPR enables detection of conformational changes in proteins with almost no restriction in the environmental conditions. Key information for structural analysis is provided by changes in the dynamics of spin-labeled sites and by interspin distances between selected pairs of labels. Dynamics are detected by continuous wave EPR, interspin distances by pulse dipolar techniques (DEER or PELDOR being the most common). The long-range EPR distance constraints, combined with existing structural data at atomic level for one state of the protein, enable the creation of coarse-grained models of complex protein rearrangements in their physiological milieu. Each protein conformational transition due to oligomerization, ligand binding, transfer from a water to a membrane environment, etc. is also tightly coupled to rearrangements in the hydration water surrounding the different protein interfaces. Changes in local hydration dynamics and water accessibility can be monitored directly and with high precision at physiological temperature using Overhauser dynamic nuclear polarization (ODNP), which has several advantages with respect to other EPR techniques which provide water accessibility data. We will show examples of structural studies in which the changes in the protein and its surrounding water environment are observed. Examples will be given on ABC transporters, Bcl-2 proteins, and light receptors.

Characterization of KCNE1 inside Lipodisq Nanoparticles for EPR Spectroscopic Studies of Membrane Proteins.

EPR spectroscopic studies of membrane proteins in a physiologically relevant native membrane-bound state are extremely challenging due to the complexity observed in inhomogeneity sample preparation and dynamic motion of the spin-label. Traditionally, detergent micelles are the most widely used membrane mimetics for membrane proteins due to their smaller size and homogeneity, providing high-resolution structure analysis by solution NMR spectroscopy. However, it is often difficult to examine whether the protein structure in a micelle environment is the same as that of the respective membrane-bound state. Recently, lipodisq nanoparticles have been introduced as a potentially good membrane mimetic system for structural studies of membrane proteins. However, a detailed characterization of a spin-labeled membrane protein incorporated into lipodisq nanoparticles is still lacking. In this work, lipodisq nanoparticles were used as a membrane mimic system for probing the structural and dynamic properties of the integral membrane protein KCNE1 using site-directed spin labeling EPR spectroscopy. The characterization of spin-labeled KCNE1 incorporated into lipodisq nanoparticles was carried out using CW-EPR titration experiments for the EPR spectral line shape analysis and pulsed EPR titration experiment for the phase memory time (Tm) measurements. The CW-EPR titration experiment indicated an increase in spectral line broadening with the addition of the SMA polymer which approaches close to the rigid limit at a lipid to polymer weight ratio of 1:1, providing a clear solubilization of the protein-lipid complex. Similarly, the Tm titration experiment indicated an increase in Tm values with the addition of SMA polymer and approaches ∼2 μs at a lipid to polymer weight ratio of 1:2. Additionally, CW-EPR spectral line shape analysis was performed on six inside and six outside the membrane spin-label probes of KCNE1 in lipodisq nanoparticles. The results indicated significant differences in EPR spectral line broadening and a corresponding inverse central line width between spin-labeled KCNE1 residues located inside and outside of the membrane for lipodisq nanoparticle samples when compared to lipid vesicle samples. These results are consistent with the solution NMR structure of KCNE1. This study will be beneficial for researchers working on studying the structural and dynamic properties of membrane proteins.

Site-Directed Spin Labeling EPR Spectroscopy of the Cytoplasmic Tail of Influenza a M2

The M2 protein is a 97 residue homotetrameric, multifunctional ion channel that plays critical roles during the influenza infection cycle. While a variety of high-resolution biophysical techniques have been used to characterize the transmembrane domain (residues 22-46) and the juxtamembrane C-terminal region (46-62), less is known about the conformation and dynamics of the remaining residues of the C-terminal cytoplasmic tail. Here, we use site-directed spin labeling electron paramagnetic spectroscopy (SDSL-EPR) experiments to probe the secondary structure and membrane topology of cytoplasmic tail residues 60-80 when the protein is reconstituted into lipid bilayers. Cholesterol is essential for the role the C-terminal domain of the M2 protein plays in viral budding. SDSL-EPR data is collected in both in the presence and absence of cholesterol.

Site-Directed Spin-Label EPR Spectroscopy of M2 Protein in Lipid Bilayers of Different Curvature Generation Propensity

M2 is a homotetrameric membrane protein critical to generation of membrane curvature in the budding stage of the influenza A virus life cycle. We have designed and interpreted site-directed spin-label electron paramagnetic resonance spectroscopy (SDSL-EPR) experiments on the conformation and dynamics of M2 in lipid bilayers of different propensities for membrane curvature generation. We have obtained data for full length M2 protein spin labelled at multiple sites in the C-terminal juxtamembrane region, which forms a membrane associated amphipathic helix. Continuous wave (CW) and pulsed EPR spectra provide evidence that M2 adopts two conformational states in bilayers, and that the propensity of lipids to form membranes of differing curvature dictates the relative population of these states. Multicomponent simulation of CW-EPR spectra further show lipid bilayers of the highly curved cubic phases stabilize an immobilized conformation of M2, hypothesized to be relevant for curvature generation. We have also characterized the size distribution of large unilamellar vesicles of lipids of different curvature generation propensities alone and in the presence of full-length M2 using dynamic light scattering (DLS).

In Vitro Demonstration of Light-Driven Na+/H+ Pumping by a Microbial Rhodopsin

A new subfamily of rhodopsins was discovered in bacteria and proposed to function as dual function light-driven H+/Na+ pumps, ejecting Na+ from cells in the presence of Na+ and H+ in its absence (Inoue et al 2013. Nat. Commun. 4:1678). This proposal was based primarily on light-induced proton flux measurements in suspensions of E. coli cells expressing the pigments. However, because E. coli cells contain numerous proteins that mediate proton fluxes, indirect effects on proton movements involving endogenous bioenergetics components could not be excluded. Therefore, an in vitro system consisting of the purified pigment in the absence of other proteins was needed to assign the Na+ and H+ transport definitively. We expressed IAR from Indibacter alkaliphilus in E. coli cells and observed similar ion fluxes as reported for KR2 from Dokdonia eikasta reported earlier. We purified and reconstituted IAR into large unilamellar vesicles (LUVs), and demonstrated the proton flux criteria of light-dependent electrogenic Na+ pumping activity in vitro, namely Na+-dependent light-induced passive proton flux enhanced by protonophore. The proton flux was out of the LUV lumen, increasing lumenal pH. In contrast, illumination of the LUVs in a Na+-free suspension medium caused a decrease of lumenal pH, eliminated by protonophore, showing H+ pumping activity. The direction of proton fluxes indicated that IAR was inserted inside-out into the sealed LUV system, which we confirmed by site-directed spin-label EPR spectroscopy. The in vitro LUV system proves that the dual light-driven H+/Na+ pumping function of IAR is intrinsic to the single rhodopsin protein and enables study of the transport activities without perturbation by bioenergetics ion fluxes encountered in vivo. We are currently investigating ion fluxes through natural anion channelrhodopsins with this system.

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.

Influenza A M2 protein conformation depends on choice of model membrane.

While crystal and NMR structures exist of the influenza A M2 protein, there is disagreement between models. Depending on the requirements of the technique employed, M2 has been studied in a range of membrane mimetics including detergent micelles and membrane bilayers differing in lipid composition. The use of different model membranes complicates the integration of results from published studies necessary for an overall understanding of the M2 protein. Here we show using site-directed spin-label EPR spectroscopy (SDSL-EPR) that the conformations of M2 peptides in membrane bilayers are clearly influenced by the lipid composition of the bilayers. Altering the bilayer thickness or the lateral pressure profile within the bilayer membrane changes the M2 conformation observed. The multiple M2 peptide conformations observed here, and in other published studies, optimistically may be considered conformations that are sampled by the protein at various stages during influenza infectivity. However, care should be taken that the heterogeneity observed in published structures is not simply an artifact of the choice of the model membrane.