Spin Labeling EPR Research Articles

Natural terpenes II. Concentration-dependent profile of effects on dynamic organization of biological and model membranes.

Monoterpenes (MTs), the major constituents of plant essential oils, cover a broad spectrum of biological activities through their interaction with biomembranes. MTs are highly hydrophobic substances with a net electrical dipole, but are not clearly amphipathic. As a result, they aggregate at increasing concentrations in aqueous media, and in membrane environments their behavior changes from dynamics modulators to disruptors. In the present work we have tried to find the boundaries between these conditions. Using spin-label electron paramagnetic resonance (EPR) spectroscopy, we studied the effect of a wide range of concentrations of camphor, cineole, thymol, menthol and geraniol on the dynamics of phospholipid model membranes, with or without cholesterol, organized in different initial lyotropic phases, as well as on synaptosomal membranes from bovine brain cortex. EPR spectra of two fatty acid spin probes incorporated into the membranes (5-SASL and 12-SASL) provided information on molecular order and mobility at different bilayer depths. Qualitative changes in the spectra indicated transitions between monomeric and aggregated MTs, as well as membranes with different degrees of order and disrupted membranes. Analyzed in conjunction with the kinetics of [3H]flunitrazepam membrane binding, either non-specifically to the membrane or specifically to the GABAA receptor, the present results allowed us to define 200μM as the concentration limit to safely evaluate MT effects in non-disrupted biological and model membranes. These findings may be helpful in the interpretation and design of pharmacological assays and provide concentration references that allow correlating a variety of biophysical and pharmacological data in the literature.

Probing the inner structure and dynamics of pH-sensitive block copolymer nanoparticles with nitroxide radicals using scattering and EPR techniques

This research emphasizes the crucial role of electron paramagnetic resonance (EPR) spectroscopy in nanoparticle analysis, showcasing its distinctive ability to probe molecular-level details. We demonstrate the synthesis and self-assembly of a new class of pH-responsive amphiphilic diblock copolymers, specifically poly[N-(2-hydroxypropyl)-methacrylamide]-block-poly[2-(diisopropylamino)ethyl methacrylate] (PHPMA-b-PDPA), incorporating 2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPO) radicals covalently bound to the hydrophilic PHPMA segment. The copolymer synthesis involved a three-step process, combining reversible addition − fragmentation chain transfer (RAFT) polymerization with carbodiimide (DCC) chemistry. TEMPO radical-containing nanoparticles (RNPs) were created using microfluidic (MF) nanoprecipitation, a technique essential for generating uniform nanoparticles with predictable biodistribution and cellular uptake. Adjusting MF protocol parameters allowed fine-tuning of RNP sizes. EPR spectroscopy confirmed the formation of core–shell RNPs, with features aligning closely with those obtained from scattering techniques like dynamic light scattering (DLS), static light scattering (SLS), small-angle X-ray scattering (SAXS), and cryo-transmission electron microscopy (cryo-TEM). EPR spectroscopy emerges as a potent tool for molecular-level analysis of colloidal polymer systems. This study highlights the EPR-spin label method’s efficacy in probing the internal structural features and dynamics of core–shell nanoparticles, especially their response to pH-triggered disassembly. The findings open new avenues for the use of pH-responsive TEMPO-labeled diblock copolymers in controlled drug delivery applications.

Probing conformational dynamics of DNA binding by CO-sensing transcription factor, CooA

The transcription factor CooA is a CRP/FNR (cAMP receptor protein/ fumarate and nitrate reductase) superfamily protein that uses heme to sense carbon monoxide (CO). Allosteric activation of CooA in response to CO binding is currently described as a series of discrete structural changes, without much consideration for the potential role of protein dynamics in the process of DNA binding. This work uses site-directed spin-label electron paramagnetic resonance spectroscopy (SDSL-EPR) to probe slow timescale (μs-ms) conformational dynamics of CooA with a redox-stable nitroxide spin label, and IR spectroscopy to probe the environment at the CO-bound heme. A series of cysteine substitution variants were created to selectively label CooA in key functional regions, the heme-binding domain, the 4/5-loop, the hinge region, and the DNA binding domain. The EPR spectra of labeled CooA variants are compared across three functional states: Fe(III) “locked off”, Fe(II)-CO “on”, and Fe(II)-CO bound to DNA. We observe changes in the multicomponent EPR spectra at each location; most notably in the hinge region and DNA binding domain, broadening the description of the CooA allosteric mechanism to include the role of protein dynamics in DNA binding. DNA-dependent changes in IR vibrational frequency and band broadening further suggest that there is conformational heterogeneity in the active WT protein and that DNA binding alters the environment of the heme-bound CO.

Designing surface exposed sites on Bacillus subtilis lipase A for spin-labeling and hydration studies

Spin-labeling with electron paramagnetic resonance spectroscopy (EPR) is a facile method for interrogating macromolecular flexibility, conformational changes, accessibility, and hydration. Within we present a computationally based approach for the rational selection of reporter sites in Bacillus subtilis lipase A (BSLA) for substitution to cysteine residues with subsequent modification with a spin-label that are expected to not significantly perturb the wild-type structure, dynamics, or enzymatic function. Experimental circular dichroism spectroscopy, Michaelis-Menten kinetic parameters and EPR spectroscopy data validate the success of this approach to computationally select reporter sites for future magnetic resonance investigations of hydration and hydration changes induced by polymer conjugation, tethering, immobilization, or amino acid substitution in BSLA. Analysis of molecular dynamic simulations of the impact of substitutions on the secondary structure agree well with experimental findings. We propose that this computationally guided approach for choosing spin-labeled EPR reporter sites, which evaluates relative surface accessibility coupled with hydrogen bonding occupancy of amino acids to the catalytic pocket via atomistic simulations, should be readily transferable to other macromolecular systems of interest including selecting sites for paramagnetic relaxation enhancement NMR studies, other spin-labeling EPR studies or any method requiring a tagging method where it is desirable to not alter enzyme stability or activity.

Water concentration and dielectric constant gradients along the transmembrane peptide-lipid interface revealed by spin-labeling EPR methods

Water concentration and dielectric constant gradients along the transmembrane peptide-lipid interface revealed by spin-labeling EPR methods

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.

A disulfide chaperone knockout facilitates spin labeling and pulse EPR spectroscopy of outer membrane transporters.

Pulse EPR measurements provide information on distances and distance distributions in proteins but require the incorporation of pairs of spin labels that are usually attached to engineered cysteine residues. In previous work, we demonstrated that efficient in vivo labeling of the Escherichia coli outer membrane vitamin B12 transporter, BtuB, could only be achieved using strains defective in the periplasmic disulfide bond formation (Dsb) system. Here, we extend these in vivo measurements to FecA, the E. coli ferric citrate transporter. As seen for BtuB, pairs of cysteines cannot be labeled when the protein is present in a standard expression strain. However, incorporating plasmids that permit an arabinose induced expression of FecA into a strain defective in the thiol disulfide oxidoreductase, DsbA, enables efficient spin-labeling and pulse EPR of FecA in cells. A comparison of the measurements made on FecA in cells with measurements made in reconstituted phospholipid bilayers suggests that the cellular environment alters the behavior of the extracellular loops of FecA. In addition to these in situ EPR measurements, the use of a DsbA minus strain for the expression of BtuB improves the EPR signals and pulse EPR data obtained in vitro from BtuB that is labeled, purified, and reconstituted into phospholipid bilayers. The in vitro data also indicate the presence of intermolecular BtuB-BtuB interactions, which had not previously been observed in a reconstituted bilayer system. This result suggests that in vitro EPR measurements on other outer membrane proteins would benefit from protein expression in a DsbA minus strain.

Spin-Lattice Relaxation Rates of Lipid Spin Labels as a Measure of Their Rotational Diffusion Rates in Lipid Bilayer Membranes.

The spin-lattice relaxation rate (T1−1) of lipid spin labels obtained from saturation recovery EPR measurements in deoxygenated membranes depends primarily on the rate of the rotational diffusion of the nitroxide moiety within the lipid bilayer. It has been shown that T1−1 also can be used as a qualitative convenient measure of membrane fluidity that reflects local membrane dynamics; however, the relation between T1−1 and rotational diffusion coefficients was not provided. In this study, using data previously presented for continuous wave and saturation recovery EPR measurements of phospholipid analog spin labels, one-palmitoyl-2-(n-doxylstearoyl)phosphatidylcholine in 1,2-dimyristoyl-sn-glycero-3-phosphorylcholine/cholesterol membranes, we show that measured T1−1 values are linear functions of rotational diffusion of spin labels. Thus, these linear relationships can be used to transfer T1−1 values into spin label rotational rates as a precise description of membrane fluidity. This linearity is independent through the wide range of conditions including lipid environment, depth in membrane, local hydrophobicity, and the anisotropy of rotational motion. Transferring the spin-lattice relaxation rates into the rotational diffusion coefficients makes the results obtained from saturation recovery EPR spin labeling easy to understand and readily comparable with other membrane fluidity data.

Comparative evaluation of spin-label modeling methods for protein structural studies

Site-directed spin-labeling electron paramagnetic resonance spectroscopy is a powerful technique for the investigation of protein structure and dynamics. Accurate spin-label modeling methods are essential to make full quantitative use of site-directed spin-labeling electron paramagnetic resonance data for protein modeling and model validation. Using a set of double electron-electron resonance data from seven different site pairs on maltodextrin/maltose-binding protein under two different conditions using five different spin labels, we compare the ability of two widely used spin-label modeling methods, based on accessible volume sampling and rotamer libraries, to predict experimental distance distributions. We present a spin-label modeling approach inspired by canonical side-chain modeling methods and compare modeling accuracy with the established methods.

Multilamellar Liposomes as a Model for Biological Membranes: Saturation Recovery EPR Spin-Labeling Studies.

EPR spin labeling has been used extensively to study lipids in model membranes to understand their structures and dynamics in biological membranes. The lipid multilamellar liposomes, which are the most commonly used biological membrane model, were prepared using film deposition methods and investigated with the continuous wave EPR technique (T2-sensitive spin-labeling methods). These investigations provided knowledge about the orientation of lipids, their rotational and lateral diffusion, and their rate of flip-flop between bilayer leaflets, as well as profiles of membrane hydrophobicity, and are reviewed in many papers and book chapters. In the early 1980s, the saturation recovery EPR technique was introduced to membrane studies. Numerous T1-sensitive spin-label methods were developed to obtain detailed information about the three-dimensional dynamic membrane structure. T1-sensitive methods are advantageous over T2-sensitive methods because the T1 of spin labels (1–10 μs) is 10 to 1000 times longer than the T2, which allows for studies of membrane dynamics in a longer time–space scale. These investigations used multilamellar liposomes also prepared using the rapid solvent exchange method. Here, we review works in which saturation recovery EPR spin-labeling methods were applied to investigate the properties of multilamellar lipid liposomes, and we discuss their relationships to the properties of lipids in biological membranes.

Protonation of model ionizable sidechains in transmembrane protein domains

The ionization states of individual amino acid residues of membrane proteins in lipid membrane environment are difficult to decipher or assign directly. The effective pK(a) values of protein groups are determined by a complex interplay between local polarity, Coulomb interactions, and structural reorganizations. The analysis is further complicated by the dearth of information about gradients in polarity, electric potentials, and hydration at the protein-membrane interface. In this work we report on a spin-labeling EPR method for assessing effects of membrane surface potential, local environment at the protein-membrane interface, and water penetration along this interface on effective pK(a) of membrane-buried ionizable groups.

Use of Site-Directed Spin Labeling EPR Spectroscopy to Study Protein-LPS Interactions.

Site-directed spin labeling EPR (electron paramagnetic resonance) spectroscopy is a technique used to identify the local conformational changes at a specific residue of interest within a purified protein in response to a ligand. Here, we describe the site-directed spin labeling EPR spectroscopy methodology to monitor changes in the side-chain motion in soluble lipopolysaccharide transport proteins upon the addition of lipopolysaccharide (LPS). A comparison of the spectral overlays of the spin-labeled protein in the absence and presence of LPS provides a qualitative visualization of how LPS binding affects the motion of each spin-labeled site tested within the protein. No change in the spectral lineshapes of a spin-labeled protein in the absence and presence of LPS indicates that the site is not affected by LPS binding, while differences in the spectral lineshapes indicate that LPS does affect the mobility of the spin label side chain within the protein structure. This is a powerful readout of conformational changes at specific residues of interest that can be used to identify a specific site as a reporter of changes induced by ligand binding and to map out the effects of ligand binding through an array of reporter sites within a protein. With the use of AquaStar tubing, protein concentrations as low as 2μM allow for up to a 100-fold excess of LPS. This methodology may also be applied to other protein-ligand or protein-protein interactions with minor adaptations.

Iron chelation therapy with deferiprone improves oxidative status and red blood cell quality and reduces redox-active iron in β-thalassemia/hemoglobin E patients.

The oxidative status of twenty-three β-thalassemia/hemoglobin E patients was evaluated after administration of 75mg/kg deferiprone (GPO-L-ONE®) divided into 3 doses daily for 12 months. Serum ferritin was significantly decreased; the median value at the initial and final assessments was 2842 and 1719ng/mL, respectively. Progressive improvement with significant changes in antioxidant enzyme activity, including plasma paraoxonase (PON) and platelet-activating factor acetylhydrolase (PAF-AH), and in antioxidant enzymes in red blood cells (glutathione peroxidase (GPx), catalase and superoxide dismutase (SOD)) were observed at 3-6months of treatment. The levels of total GSH in red blood cells were significantly increased at the end of the study. Improved red blood cell membrane integrity was also demonstrated using the EPR spin labeling technique. Membrane fluidity at the surface and hydrophobic regions of the red blood cell membrane was significantly changed after 12 months of treatment. In addition, a significant increase in hemoglobin content was observed (6.6±0.7 and 7.5±1.3g/dL at the initial assessment and at 6 months, respectively). Correlations were observed between hemoglobin content, membrane fluidity and antioxidant enzymes in red blood cells. The antioxidant activity of deferiprone may partly be explained by progressive reduction of redox active iron that catalyzes free radical reactions, as demonstrated by the EPR spin trapping technique. In conclusion, iron chelation therapy with deferiprone notably improved the oxidative status in thalassemia, consequently reducing the risk of oxidative-related complications. Furthermore, the improvement in red blood cell quality may improve the anemia situation in patients.

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.

In Situ Continuous Wave Electron Paramagnetic Resonance Investigation of the Amyloid Aggregation of Parkinson’s Protein Alpha-Synuclein—the Second Spin-Label Position

Self-aggregation of amyloid proteins is a crucial step in neurodegenerative disease. The protein alpha-synuclein (αS) is implicated in Parkinson’s disease. In an extension of the demonstration of in situ observation of intermediates in αS-aggregation by continuous wave (cw) EPR at room temperature (Zurlo et al. PLoS One 16: e0245548, 2021) by spin-label EPR, here the spin label is attached to position 90 (R1αS90), rather than at position 56. The aim is to determine, if the spin-label position affects the kinetics of aggregation and if local information on the intermediates is accessible. Probed by the MTSL ((1-Oxyl-2,2,5,5-tetramethylpyrroline-3-methyl) methanethiosulfonate) spin label at position 90, using diamagnetic dilution of 9:1 wild type αS to R1αS90, similar aggregation kinetics are found. Rotation correlation times for the spin label in the oligomer cannot be determined with sufficient accuracy to obtain local information on the oligomer under the conditions used. At the present stage, higher resolution EPR approaches, such as high-field EPR are more promising.

Factors determining barrier properties to oxygen transport across model and cell plasma membranes based on EPR spin-label oximetry.

This review is motivated by the exciting new area of radiation therapy using a phenomenon termed FLASH in which oxygen is thought to have a central role. Well-established principles of radiation biology and physics suggest that if oxygen has a strong role, it should be the level at the DNA. The key aspect discussed is the rate of oxygen diffusion. If oxygen freely diffuses into cells and rapidly equilibrates, then measurements in the extracellular compartment would enable FLASH to be investigated using existing methodologies that can readily measure oxygen in the extracellular compartment. EPR spin-label oximetry allows evaluation of the oxygen permeability coefficient across lipid bilayer membranes. It is established that simple fluid phase lipid bilayers are not barriers to oxygen transport. However, further investigations indicate that many physical and chemical (compositional) factor can significantly decrease this permeation. In biological cell plasma membranes, the lipid bilayer forms the matrix in which integral membrane proteins are immersed, changing organization and properties of the lipid matrix. To evaluate oxygen permeability coefficients across these complex membranes, oxygen permeation across all membrane domains and components must be considered. In this review, we consider many of the factors that affect (decrease) oxygen permeation across cell plasma membranes. Finally, we address the question, can the plasma membrane of the cell form a barrier to the free diffusion of oxygen into the cell interior? If there is a barrier then this must be considered in the investigations of the role of oxygen in FLASH.

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.

Site-directed spin labeling-electron paramagnetic resonance spectroscopy in biocatalysis: Enzyme orientation and dynamics in nanoscale confinement

Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy probes the otherwise inaccessible structural information in complex biological systems. We recently extended SDSL-EPR to reveal the relative orientation and backbone dynamics of enzymes upon encapsulation in mesoporous nanostructures, which set the structural basis underlying the observed biocatalytic activity. Our strategy had generated interest in the biocatalysis community, and thus in this resource article, we contribute an introduction to the principles and experimental procedure that generalize SDSL-EPR to heterogeneous biocatalysis. We will focus on enzymes in mesoporous materials with examples demonstrating the methods and cautions of potential pitfalls. The ultimate goal is to provide the biocatalysis community with a powerful resource to fill in a long-standing knowledge gap in heterogeneous biocatalysis and the structure-function relationship of enzymes at the interface of enzyme-mesoporous materials and utilize the structural insights to guide the rational design of porous platforms for enzyme immobilization. • Enzyme orientation is determined by using EPR to probe its contact with nanoscale pores • Enzyme dynamics in nanoscale pores and associated catalytic activities are revealed • Protocols and procedures are given for experimental data acquisition, analysis, and interpretation • Various EPR techniques and spin labels can be extended to heterogeneous biocatalysis Enzyme immobilization in nanoscale confinement offers enhanced enzyme protection and reusability, yet the structure-function relationship of the entrapped enzymes remains elusive. Site-directed spin labeling in combination with electron paramagnetic resonance (EPR) spectroscopy has primarily been applied in determining protein structural information in complex biological systems. In this resource article, we introduce the extension of the X-band continuous-wave EPR with a methanethiosulfonate spin label to reveal enzyme structural basis upon immobilization into nanoscale confinement. Using our recent works as examples, we demonstrate the method for determining enzyme orientation and backbone dynamics upon immobilization with a special focus on generalizing the method to heterogeneous biocatalysis and cautions in each step. Furthermore, we summarize the recent findings in other EPR techniques and spin labels so that the whole EPR toolbox can be extended to heterogeneous biocatalysis. In this resource article, Yang and colleagues introduce a general procedure that can reveal the orientation and backbone dynamics of enzymes entrapped in mesoporous materials based on metal-organic materials (MOMs) by using X-band SDSL-EPR. They demonstrate the method by using recently published works focusing on enzyme-MOM interfaces, as well as highlight cautions and potential solutions at each step. They then summarize a variety of other EPR techniques and spin labels in order to pinpoint their potential applications in heterogeneous biocatalysis in the near future.