Site-directed Spin Labeling Electron Paramagnetic Resonance Spectroscopy Research Articles

Site-Directed Spin Labeling EPR Spectroscopy for Determination of Albumin Structural Damage and Hypoalbuminemia in Critical COVID-19.

The main factors in the COVID-19 pathology, which can initiate extensive structural changes at the cellular and molecular levels, are the generation of free radicals in abnormal amounts, and oxidative stress. Under "oxidative shock" conditions, the proteins undergo various modifications that affect their function and activity, and as a result distribute malfunctioning protein derivatives in the body. Human serum albumin is a small globular protein characterized by a high overall binding capacity for neutral lipophilic and acidic dosage forms. The albumin concentration is crucial for the maintenance of plasma oncotic pressure, the transport of nutrients, amino acids, and drugs, the effectiveness of drug therapy, and the prevention of drug toxicity. Hypoalbuminemia and structural defects molecule in the protein suggest a risk of changed metabolism and increased plasma concentration of unbound drugs. Therefore, the albumin structural and functional changes accompanied by low protein levels can be a serious prerequisite for ineffective therapy, frequent complications, and high mortality in patients with SARS-CoV-2 infection. The current opinion aims the research community the application of Site-Directed Spin Labeling Electron Paramagnetic Resonance spectroscopy (SDSL-EPR) and 3-Maleimido-PROXYL radical in determining abnormalities of the albumin dynamics and protein concentrations in COVID-19 critical patients.

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.

Characterization of the Human KCNQ1 Voltage Sensing Domain (VSD) in Lipodisq Nanoparticles for Electron Paramagnetic Resonance (EPR) Spectroscopic Studies of Membrane Proteins.

Membrane proteins are responsible for conducting essential biological functions that are necessary for the survival of living organisms. In spite of their physiological importance, limited structural information is currently available as a result of challenges in applying biophysical techniques for studying these protein systems. Electron paramagnetic resonance (EPR) spectroscopy is a very powerful technique to study the structural and dynamic properties of membrane proteins. However, the application of EPR spectroscopy to membrane proteins in a native membrane-bound state is extremely challenging due to the complexity observed in inhomogeneity sample preparation and the dynamic motion of the spin label. Detergent micelles are very popular membrane mimetics for membrane proteins due to their smaller size and homogeneity, providing high-resolution structure analysis by solution NMR spectroscopy. However, it is important to test whether the protein structure in a micelle environment is the same as that of its membrane-bound state. Lipodisq nanoparticles or styrene-maleic acid copolymer-lipid nanoparticles (SMALPs) have been introduced as a potentially good membrane-mimetic system for structural studies of membrane proteins. Recently, we reported on the EPR characterization of the KCNE1 membrane protein having a single transmembrane incorporated into lipodisq nanoparticles. In this work, lipodisq nanoparticles were used as a membrane mimic system for probing the structural and dynamic properties of the more complicated membrane protein system human KCNQ1 voltage sensing domain (Q1-VSD) having four transmembrane helices using site-directed spin-labeling EPR spectroscopy. Characterization of spin-labeled Q1-VSD incorporated into lipodisq nanoparticles was carried out using CW-EPR spectral line shape analysis and pulsed EPR double-electron electron resonance (DEER) measurements. The CW-EPR spectra indicate an increase in spectral line broadening 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 a superior quality of distance measurement with an increase in the phase memory time (Tm) values upon incorporation of the sample into lipodisq nanoparticles when compared to proteoliposomes. These results are consistent with the solution NMR structural studies on the Q1-VSD. This study will be beneficial for researchers working on investigating the structural and dynamic properties of more complicated membrane protein systems using lipodisq nanoparticles.

A Budding-Defective M2 Mutant Exhibits Reduced Membrane Interaction, Insensitivity to Cholesterol, and Perturbed Interdomain Coupling.

Influenza A M2 is a membrane-associated protein with a C-terminal amphipathic helix that plays a cholesterol-dependent role in viral budding. An M2 mutant with alanine substitutions in the C-terminal amphipathic helix is deficient in viral scission. With the goal of providing atomic-level understanding of how the wild-type protein functions, we used a multipronged site-directed spin labeling electron paramagnetic resonance spectroscopy (SDSL-EPR) approach to characterize the conformational properties of the alanine mutant. We spin-labeled sites in the transmembrane (TM) domain and the C-terminal amphipathic helix (AH) of wild-type (WT) and mutant M2, and collected information on line shapes, relaxation rates, membrane topology, and distances within the homotetramer in membranes with and without cholesterol. Our results identify marked differences in the conformation and dynamics between the WT and the alanine mutant. Compared to WT, the dominant population of the mutant AH is more dynamic, shallower in the membrane, and has altered quaternary arrangement of the C-terminal domain. While the AH becomes more dynamic, the dominant population of the TM domain of the mutant is immobilized. The presence of cholesterol changes the conformation and dynamics of the WT protein, while the alanine mutant is insensitive to cholesterol. These findings provide new insight into how M2 may facilitate budding. We propose the AH-membrane interaction modulates the arrangement of the TM helices, effectively stabilizing a conformational state that enables M2 to facilitate viral budding. Antagonizing the properties of the AH that enable interdomain coupling within M2 may therefore present a novel strategy for anti-influenza drug design.

Dimerization interface and dynamic properties of yeast IF1 revealed by Site-Directed Spin Labeling EPR spectroscopy

The mitochondrial ATPase inhibitor, IF1, regulates the activity of the mitochondrial ATP synthase. The oligomeric state of IF1 related to pH is crucial for its inhibitory activity. Although extensive structural studies have been performed to characterize the oligomeric states of bovine IF1, only little is known concerning those of yeast IF1. While bovine IF1 can be found as an inhibitory dimer at low pH and a non-inhibitory tetramer at high pH, a monomer/dimer equilibrium has been described for yeast IF1, high pH values favoring the monomeric state. Combining different strategies involving the grafting of nitroxide spin labels combined with Electron Paramagnetic Resonance (EPR) spectroscopy, the present study brings the first structural characterization, at the residue level, of yeast IF1 in its dimeric form. The results show that the dimerization interface involves the central region of the peptide revealing that the dimer corresponds to a non-inhibitory state. Moreover, we demonstrate that the C-terminal region of the peptide is highly dynamic and that this segment is probably folded back onto the central region. Finally, the pH-dependence of the inter-label distance distribution has been observed indicating a conformational change between two structural states in the dimer.

Site Directed Spin Label EPR Spectroscopy of Influenza a M2 Protein

M2 is a membrane protein critical to the life cycle of influenza A. We have capitalized on the expanding body of biochemical and high-resolution structural data available for the M2 protein to design and interpret site-directed spin labeling electron paramagnetic resonance spectroscopy (SDSL-EPR) experiments on the conformation and dynamics of the homotetrameric M2 protein embedded in lipid bilayers. We have obtained data for three different M2 constructs (M2TM 22-46, M2TMC 23-60 and full length M2 protein) spin-labeled at multiple sites within the transmembrane and C-terminal domains. CW and pulsed EPR spectra show evidence that M2 adopts multiple conformational states in bilayers, and that cholesterol content dictates the relative populations of the states. Furthermore comparison of full-length M2 protein and a M2TMC 23-60 peptide demonstrates that the C-terminal juxtamembrane region in both constructs forms an amphipathic membrane surface helix.

Conformational Changes and Complex Formation of the Non-Canonical Ribose ABC Transporter

Bacterial ATP-Binding Cassette (ABC) transporters are vital for nutrient uptake. Structural and functional studies have revealed two distinct types (I and II) of importers based primarily upon mechanistic differences. The ribose transporter (RbsABC) in E. coli is a tripartite ABC importer. RbsABC consists of a cytoplasmic ATP-binding cassette, RbsA, with dual fused nucleotide-binding domains (NBD), a homodimeric transmembrane domain (TMD), RbsC, and a periplasmic substrate binding protein (SBP), RbsB. This study, using Electron Paramagnetic Resonance (EPR) spectroscopy and biochemical methods, demonstrates that RbsABC is a non-canonical ABC transporter that shares some structural and functional features with both type I and type II importers. ATP hydrolysis in the NBD is stimulated by the presence of substrate-loaded SBP, leading to ribose release, which is typical of type I importers. Quantitative measurements obtained from site-directed spin-labeling EPR spectroscopy reveal that hydrolysis promotes the opening of RbsB to release ribose and subsequently RbsB. Post-hydrolysis, both nucleotide and magnesium are required to maintain the complex interactions of RbsC and RbsA, based upon co-purification studies and EPR experiments, which is atypical for ABC importers of either type, where the high-affinity TMD-NBD association is maintained perpetually. In addition, the presence of ribose has a destabilizing effect on RbsB-RbsC interactions, a type II importer behavior. Further, RbsB and RbsC interact in an unusual apo-complex consisting only of the SBP-TMD. Finally, substrate transport is accomplished by hydrolysis at a single consensus site in RbsA, with the second site rendered degenerate by mutations of conserved amino acids. This feature is not uncommon to ABC exporters, but heretofore unobserved in importers. Taking these observations together, RbsABC appears to transport substrate by a distinct mechanism, and offers an opportunity to gain insight into the function of non-canonical systems.

Peptide-Membrane Interactions by Spin-Labeling EPR.

Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy is a well-established method that has recently grown in popularity as an experimental technique, with multiple applications in protein and peptide science. The growth is driven by development of labeling strategies, as well as by considerable technical advances in the field, that are paralleled by an increased availability of EPR instrumentation. While the method requires an introduction of a paramagnetic probe at a well-defined position in a peptide sequence, it has been shown to be minimally destructive to the peptide structure and energetics of the peptide-membrane interactions. In this chapter, we describe basic approaches for using SDSL EPR spectroscopy to study interactions between small peptides and biological membranes or membrane mimetic systems. We focus on experimental approaches to quantify peptide-membrane binding, topology of bound peptides, and characterize peptide aggregation. Sample preparation protocols including spin-labeling methods and preparation of membrane mimetic systems are also described.

Investigating Influenza a M2-Cholesterol Interactions by Oriented Sample Magnetic Resonance

The influenza A M2 protein is a homotetrameric membrane protein that is vital to the viral life cycle. M2 has been shown to interact with cholesterol to induce cell membrane curvature and scission in the production of new virus particles. Previous work from our group used site-directed spin labeling electron paramagnetic resonance spectroscopy (SDSL-EPR) to probe the conformation and dynamics of M2 constructs consisting of the transmembrane and C-terminal domains, revealing multiple conformational states in a cholesterol-modulated equilibrium. The observed conformational changes were proposed to arise from both a direct M2-cholesterol interaction through an amino acid consensus region in the C-terminal domain and indirectly through cholesterol-induced changes to the bilayer environment.We present work using lipid bicelles, which spontaneously orient in a magnetic field, as a membrane alternative to detergent micelles and bilayered vesicles. Nuclear magnetic resonance (NMR) and EPR were used to obtain new information describing the specific M2-cholesterol interaction.

Site-directed spin labeling EPR spectroscopy in protein research

Site-directed spin labeling (SDSL) in combination with electron paramagnetic resonance (EPR) spectroscopy has emerged as an efficient tool to elucidate the structure and the conformational dynamics of proteins under conditions close to the native state. This review article summarizes the basics as well as the recent progress in SDSL and EPR methods, especially for investigations on protein structure, protein function, and interaction of proteins with other proteins or nucleic acids. Labeling techniques as well as EPR methods are introduced and exemplified with applications to systems that have been studied in the author's laboratory in the past 15 years, headmost the sensory rhodopsin-transducer complex mediating the photophobic response of the halophilic archaeum Natronomonas pharaonis. Further examples underline the application of SDSL EPR spectroscopy to answer specific questions about the system under investigation, such as the nature and influence of interactions of proteins with other proteins or nucleic acids. Finally, it is discussed how SDSL EPR can be combined with other biophysical techniques to combine the strengths of the different methodologies.

Evidence against a Permanently Folded Nucleation Sequence in the Intrinsically Disordered Protein Stathmin Measured by Site-Directed Spin Labeling EPR Spectroscopy

After their relatively recent discovery and classification, it has been shown that intrinsically disordered proteins (IDPs) play essential roles in cellular regulation and signal transduction. Stathmin is a eukaryotic IDP responsible for regulating the polymerization equilibrium between the tubulin heterodimer and microtubules, and is also highly expressed in cancerous cells. Stathmin exists in a highly-ordered folded state when bound to its binding partner tubulin; however, alone in solution, as an IDP lacking those stabilizing binding interactions, stathmin is likely to explore conformational space in various equilibria between partially and completely folded states. Interestingly, it has been suggested that soluble stathmin has a short helical region (Glu55-Ala73) of persistent foldedness, and that this region might act as an α-helical ‘nucleation sequence' necessary for further folding in the C-terminal direction. Alternatively, we believe that the complete length of stathmin exists in a folding-unfolding equilibrium, and here we report data supporting this hypothesis. Nitroxide spin labeled mutants of stathmin throughout the Glu55-Ala73 region - rotationally immobilized through coupling to CNBr-activated sepharose beads - were studied by electron paramagnetic resonance (EPR) spectroscopy. The continuous wave EPR spectra for all 19 spin labeled mutants unambiguously indicate that this region of stathmin exists in a dynamic equilibrium between folded (ordered) and unfolded (disordered) states, and that no such helical nucleation sequence exists. We also show that this folding equilibrium in stathmin is shifted by the common biochemical viscosity modifiers sucrose, glycerol, and Ficoll-70.

Probing the Conformation and Dynamics of Influenza a M2 Protein using Site-Directed Spin-Label EPR Spectroscopy

M2 is a membrane protein critical to the life cycle of influenza A. We have capitalized on the expanding body of high-resolution structural data available for the 97 amino acid M2 protein to design and interpret site-directed spin labeling electron paramagnetic resonance spectroscopy (SDSL-EPR) experiments on the conformation and dynamics of the homotetrameric M2 protein embedded in lipid bilayers. We have obtained data for three different M2 constructs (M2TM 22-46, M2TMC 23-60 and full length M2 protein) spin-labeled at multiple sites within the transmembrane and C-terminal domains. CW and pulsed EPR spectra show evidence that M2 adopts multiple conformational states in bilayers, and that cholesterol content dictates the relative populations of the states.

Assessing Induced Folding of an Intrinsically Disordered Protein by Site-Directed Spin-Labeling Electron Paramagnetic Resonance Spectroscopy

We used site-directed spin-labeling electron paramagnetic resonance (EPR) spectroscopy to study the induced folding of the intrinsically disordered C-terminal domain of measles virus nucleoprotein (N(TAIL)). Four single-site N(TAIL) mutants (S407C, S488C, L496C, and V517C), located in three conserved regions, were prepared and labeled with a nitroxide paramagnetic probe. We could monitor the gain of rigidity that N(TAIL) undergoes in the presence of either the secondary structure stabilizer 2,2,2-trifluoroethanol (TFE) or one of its physiological partners, namely, the C-terminal domain (XD) of the viral phosphoprotein. The mobility of the spin label grafted at positions 488, 496, and 517 was significantly reduced upon addition of XD, contrary to that of the spin label bound to position 407, which was unaffected. Furthermore, the EPR spectra of spin-labeled S488C and L496C bound to XD in the presence of 30% sucrose are indicative of the formation of an alpha-helix in the proximity of the spin labels. Such an alpha-helix had been already identified by previous biochemical and structural studies. Using TFE we unveiled a previously undetected structural propensity within the N-terminal region of N(TAIL) and showed that its C-terminal region "resists" gaining structure even at high TFE concentrations. Finally, we for the first time showed the reversibility of the induced folding process that N(TAIL) undergoes in the presence of XD. These results highlight the suitability of site-directed spin-labeling EPR spectroscopy to identify protein regions involved in binding and folding events, while providing insights at the residue level.

Characterization of the Walker A Motif of MsbA Using Site-Directed Spin Labeling Electron Paramagnetic Resonance Spectroscopy

MsbA is an ABC transporter that transports lipid A across the inner membrane of Gram-negative bacteria such as Escherichia coli. Without functional MsbA present, bacterial cells accumulate a toxic amount of lipid A within their inner membranes. A crystal structure of MsbA was recently obtained that provides an excellent starting point for functional dynamics studies in membranes [Chang and Roth (2001) Science 293, 1793-1800]. Although a structure of MsbA is now available, several functionally important motifs common to ABC transporters are unresolved in the crystal structure. The Walker A domain, one of the ABC transporter consensus motifs that is directly involved in ATP binding, is located within a large unresolved region of the MsbA ATPase domain. Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy is a powerful technique for characterizing local areas within a large protein structure in addition to detecting and following changes in local structure due to dynamic interactions. MsbA reconstituted into lipid membranes has been evaluated by EPR spectroscopy, and it has been determined that the Walker A domain forms an alpha-helical structure, which is consistent with the structure of this motif observed in other crystallized ABC transporters. In addition, the interaction of the Walker A residues with ATP before, during, and after hydrolysis was followed using SDSL EPR spectroscopy in order to identify the residues directly involved in substrate binding and hydrolysis.

Resting state conformation of the MsbA homodimer as studied by site-directed spin labeling.

MsbA is the ABC transporter for lipid A and is found in the inner membranes of Gram-negative bacteria such as Escherichia coli. Without MsbA present, bacterial cells accumulate a toxic amount of lipid A within their inner membranes. A crystal structure of MsbA was recently obtained that provides an excellent starting point for functional dynamics studies in membranes [Chang, and Roth (2001) Science 293, 1793-1800]. Although a structure of MsbA is now available, many questions remain concerning its mechanism of transport. Site-directed spin labeling (SDSL) electron paramagnetic resonance (EPR) spectroscopy is a powerful approach for characterizing local areas within a large protein structure in addition to detecting and following changes in local structure due to dynamic interactions within a protein. The quaternary structure of the resting state of the MsbA homodimer reconstituted into lipid membranes has been evaluated by SDSL EPR spectroscopy and chemical cross-linking techniques. SDSL and cross-linking results are consistent with the controversial resting state conformation of the MsbA homodimer found in the crystal structure, with the tips of the transmembrane helices forming a dimer interface. The position of MsbA in the membrane bilayer along with the relative orientation of the transmembrane helical bundles with respect to one another has been determined. Characterization of the resting state of the MsbA homodimer is essential for future studies on the functional dynamics of this membrane transporter.

PH-induced Conformational Changes of AcrA, the Membrane Fusion Protein of Escherichia coli Multidrug Efflux System

The multidrug efflux system AcrA-AcrB-TolC of Escherichia coli expels a wide range of drugs directly into the external medium from the bacterial cell. The mechanism of the efflux process is not fully understood. Of an elongated shape, AcrA is thought to span the periplasmic space coordinating the concerted operation of the inner and outer membrane proteins AcrB and TolC. In this study, we used site-directed spin labeling (SDSL) EPR (electron paramagnetic resonance) spectroscopy to investigate the molecular conformations of AcrA in solution. Ten AcrA mutants, each with an alanine to cysteine substitution, were engineered, purified, and labeled with a nitroxide spin label. EPR analysis of spin-labeled AcrA variants indicates that the side chain mobilities are consistent with the predicted secondary structure of AcrA. We further demonstrated that acidic pH induces oligomerization and conformational change of AcrA, and that the structural changes are reversible. These results suggest that the mechanism of action of AcrA in drug efflux is similar to the viral membrane fusion proteins, and that AcrA actively mediates the efflux of substrates.