Cysteine-Targeting Gd-Based Spin Label and Its Application in Electron Paramagnetic Resonance Spectroscopy.

Bax and Bid are pro-apoptotic members of the Bcl-2 protein family. Upon cleavage by caspase-8, Bid activates Bax. Activated Bax inserts into the mitochondrial outer membrane forming oligomers which lead to membrane poration, release of cytochrome c, and apoptosis. The detailed mechanism of Bax activation and the topology and composition of the oligomers are still under debate. Here molecular details of Bax activation and oligomerization were obtained by application of several biophysical techniques, including atomic force microscopy, cryoelectron microscopy, and particularly electron paramagnetic resonance (EPR) spectroscopy performed on spin-labeled Bax. Incubation with detergents, reconstitution, and Bid-triggered insertion into liposomes were found to be effective in inducing Bax oligomerization. Bid was shown to activate Bax independently of the stoichiometric ratio, suggesting that Bid has a catalytic function and that the interaction with Bax is transient. The formation of a stable dimerization interface involving two Bcl-2 homology 3 (BH3) domains was found to be the nucleation event for Bax homo-oligomerization. Based on intermolecular distance determined by EPR, a model of six adjacent Bax molecules in the oligomer is presented where the hydrophobic hairpins (helices alpha5 and alpha6) are equally spaced in the membrane and the two BH3 domains are in close vicinity in the dimer interface, separated by >5 nm from the next BH3 pairs.

Binding of ligands to the maltose-binding protein (MBP) of Escherichia coli often causes a global conformational change involving the closure of its two lobes. We have introduced a cysteine residue onto each of these lobes by site-directed mutagenesis and modified these residues with spin labels. Using EPR spectroscopy, we examined the changes, caused by the ligand binding, in distance between the two spin labels, hence between the two lobes. The binding of both maltose and maltotetraose induced a considerable closure of the N- and C-terminal lobes of MBP. Little closure occurred upon the binding of maltotetraitol or beta-cyclodextrin. Previous study by fluorescence and UV differential absorbance spectroscopy (Hall, J. A., Gehring, K., and Nikaido, H. (1997) J. Biol. Chem. 272, 17605-17609) showed that maltose and a large portion of maltotetraose bound to MBP via one mode (R mode or "end-on" mode), which is physiologically active and leads to the subsequent transport of the ligands across the cytoplasmic membrane. In contrast, maltotetraitol and beta-cyclodextrin bound to MBP via a different mode (B mode or "middle" mode), which is physiologically inactive. The present work suggests that the B mode is nonproductive because ligands binding in this manner prevent the closure of the two domains of MBP, and, as a result, the resulting ligand-MBP complex is incapable of interacting properly with the inner membrane-associated transporter complex.

Lipid-peptide interactions are involved in many essential processes of living organisms, such as cell fusion or transport processes and are therefore, in the center of research interest. One efficient technique to analyze such interactions is the electron paramagnetic resonance (EPR) spectroscopy, in particular the double electron electron resonance (DEER) and pulsed electron double resonance (PELDOR) experiments, which allow to determine intramolecular distances within peptides and to investigate the orientation of peptides incorporated into membranes. One requirement to use this analytical technique is the labeling of the peptides with molecules that bear a paramagnetic center such as a nitroxide radical. Thereby, the peptides can be labeled either by direct incorporation of the spin label into the peptide backbone during the peptide synthesis or by side-directed spin labeling (SDSL). To improve the accuracy of distance and orientation-selection measurements by EPR spectroscopy the aim of this work was to design and synthesize a new spin label with enhanced rigidity for direct incorporation into peptides. The design of the new and rigid spin label was based on the approach to stabilize the peptide conformation by direct incorporation of the spin label. In the course of this work, a new spin label that is capable to induce ß-turn conformations by geometrical preorganization was designed and its synthesis tested. In addition to that, one goal was the investigation of more complex protein systems using the the semi-rigid 4-(3,3,5,5-tetra-methyl-2,6-dioxo-4-oxylpiperazin-1-yl)-l-phenylglycine (TOPP) spin label, which requires a reliable and general method for incorporation of this spin label into large peptide systems. One established method to construct large peptide systems is the native chemical ligation (NCL). This method enables the synthesis of large peptide systems from two or more peptide fragments and allows the incorporation of modified peptide fragments. Thus, the NCL was considered as a promising approach to synthesize complex TOPP spin labeled peptides. The introduction of the TOPP spin label in large peptides constructed by NCL would allow the investigation of large peptide systems by EPR spectroscopy using the semi-rigid TOPP spin label. Therefore, one goal of this work was to synthesize a TOPP spin labeled peptide by NCL.

Spin Labeling Analysis of Amyloids and Other Protein Aggregates

Electron paramagnetic resonance (EPR) spectroscopy has increasingly been applied for the study of nucleic acid structure and dynamics. Such studies require incorporation of free radicals (spin labels) into the biopolymer. The labels can be incorporated during chemical synthesis of the oligomer (phosphoramidite approach) or postsynthetically, by reaction of a spin-labeling reagent with a reactive functional group on the oligonucleotide. Incorporation of the rigid nitroxide spin label Ç is an example of the phosphoramidite method, and reaction of a spin-labeled azide with an alkyne-modified oligomer to yield a triazole-derived, spin-labeled nucleotide illustrates the postsynthetic spin-labeling strategy. Characterization and application of these labels to study structural features of DNA by EPR spectroscopy is discussed. Finally, a new spin-labeling strategy is described for nucleic acids that relies on noncovalent interactions between a spin-labeled nucleobase and an abasic site in duplex DNA.

Decision letter: Structural intermediates observed only in intact Escherichia coli indicate a mechanism for TonB-dependent transport

Spectroscopy and imaging are widely used to characterise systems structurally and functionally, and this information allows for the rational development of novel drugs and theranostics in many diverse areas of medicine. This thesis focuses on applying Electron Paramagnetic Resonance (EPR) techniques to determine the structure and function of protein molecules. A feasibility study to develop paramagnetic probes for EPR imaging is also presented.In EPR the probe is a paramagnetic centre and information is obtained by measuring interactions of this probe with its environment. In this thesis continuous wave (CW) EPR and Double Electron Electron Resonance (DEER) in conjunction with a site-directed spin labelling (SDSL) were employed to study structure and function of spin-labelled protein molecules. The techniques allow examination of molecular systems that cannot be crystallised or are too big for efficient NMR investigation. DEER provides information on distance distributions between two spin labels located on Cys-mutated residues of the protein molecule and can access longer distances in comparison with NMR. CW EPR provides information about the mobility of a single spin label which can be used to characterise protein dynamics. Structural modelling was used to combine the crystal structures and Molecular Dynamics (MD) simulations with DEER distance constraints to present a DEER-based structural model of the conformational variability of the protein molecule.Chapters 2 and 3 focus on characterising two metal ion substrate binding proteins (SBP), Zn2+-binding AdcA and Mn2+-binding PsaA, using DEER and CW EPR. The ATP-Binding Cassette (ABC) permeases, with which the SBPs are associated, are a primary importer used by bacteria to scavenge the essential first-row metal ions (e.g. iron, zinc, manganese) from a host environment. The process is essential for bacterial survival and propagation. Understanding the metal ion acquisition mechanisms by these SBPs can provide new opportunities for targeted drug development. Collectively, the CW EPR and DEER data along with crystal structures, differential scanning fluorimetry (DSF), Molecular Dynamics simulation, and smFRET microscopy, were able to determine a structural model for Zn2+-binding in the AdcA protein referred to as “trap-door” mechanism. Our data show that the “trap-door” mechanism employed by AdcA is different from the “spring-hammer” mechanism employed by PsaA.Non-Ribosomal Peptide Synthetase (NRPS) are the topic of chapter 4. NRPS’s are a family of mega enzymes that produce a diverse range of pharmaceuticals, yet how these molecular machines operate to produce such complex chemicals is very poorly understood. This project will investigate module 7 of the teicoplanin NRPS, a 200 kDa protein, which is the last step in the production of the glycopeptide antibiotics (GPA). A comprehensive structural and functional understanding of the teicoplanin NRPS machinery will provide a paradigm for this enzyme family, enabling the tailored re-engineering of the in vivo NRPS biosynthesis for the development of new antibiotics and pharmaceuticals. This thesis concentrates on a di-domain complex PCP-X from module 7 (Tcp12) of teicoplanin NRPS which contains the peptidyl carrier protein (PCP) domain and the X-domain. These two domains are joined by a long linker which is thought to be flexible. The goal is to investigate conformational dynamics of the PCP-X construct in different states. This includes the PCP domain in loaded and unloaded states, and how the PCP-X construct interacts with an OxyB (P450) protein. Using DEER data from a large number of double spin-labelled PCP-X mutants, rigid-body structural models based on the domain’s crystal structures were investigated to characterise the mobility of the PCP-X di-domain and compare it to very limited crystal structure data of the larger domain construct.Chapter 5 describes the application of the Maximum Entropy (MaxEnt) approach to 2D HYSCORE image reconstruction and its comparison with the conventionally utilised Discrete Fourier Transformation (DFT). HYSCORE is a widely used electron spin echo envelope (ESEEM) experiment used for measuring the nuclear quadrupole and hyperfine couplings. While EPR experiments are generally very sensitive, the time required for two and higher dimensional techniques are a significant limitation. The results show that the MaxEnt algorithm allows sampling of data non-uniformity, provides better sensitivity and an increase in resolution compared to DFT.Lastly, chapter 6 describes the development of PEG-based hyperbranched polymers and pPEGMA-coated gold nanoparticles as EPR imaging agents and sensors. Both nanoparticles have been shown as promising platforms that can be functionalized with targeting ligands and various imaging probes (i.e. a paramagnetic spin label) for the purpose of using multiple molecular imaging modalities for in vivo theranostics. The choice of a spin label dictates the possible application of the final agent, either for EPR imaging or EPR spectroscopy. The preliminary study assessed the sensitivity of commercially available EPR equipment and showed that nitroxides are more suitable for use as a sensor rather than an imaging probe. Future work based on the results in the thesis are discussed at the end of this chapter.

The determination of distances between specific points in nucleic acids is essential to understanding their behaviour at the molecular level. The ability to measure distances of 2–10 nm is particularly important: deformations arising from protein binding commonly fall within this range, but the reliable measurement of such distances for a conformational ensemble remains a significant challenge. Using several techniques, we show that electron paramagnetic resonance (EPR) spectroscopy of oligonucleotides spin-labelled with triazole-appended nitroxides at the 2′ position offers a robust and minimally perturbing tool for obtaining such measurements. For two nitroxides, we present results from EPR spectroscopy, X-ray crystal structures of B-form spin-labelled DNA duplexes, molecular dynamics simulations and nuclear magnetic resonance spectroscopy. These four methods are mutually supportive, and pinpoint the locations of the spin labels on the duplexes. In doing so, this work establishes 2′-alkynyl nitroxide spin-labelling as a minimally perturbing method for probing DNA conformation.

Probing the Structural Topology of a Membrane Peptide in Mechcanically Aligned Lipid Bilayers using Bifunctional Spin Labeling EPR Spectroscopy

Evaluation of preservation solutions by ESR-spectroscopy: Superior effects of University of Wisconsin over Histidine–Tryptophan–Ketoglutarate in reducing renal reactive oxygen species

Apolipoprotein A-I (apoA-I) is the major protein component of high density lipoproteins (HDL) and a critical element of cholesterol metabolism. To better elucidate the role of the apoA-I structure-function in cholesterol metabolism, the conformation of the apoA-I N terminus (residues 6-98) on nascent HDL was examined by electron paramagnetic resonance (EPR) spectroscopic analysis. A series of 93 apoA-I variants bearing single nitroxide spin label at positions 6-98 was reconstituted onto 9.6-nm HDL particles (rHDL). These particles were subjected to EPR spectral analysis, measuring regional flexibility and side chain solvent accessibility. Secondary structure was elucidated from side-chain mobility and molecular accessibility, wherein two major α-helical domains were localized to residues 6-34 and 50-98. We identified an unstructured segment (residues 35-39) and a β-strand (residues 40-49) between the two helices. Residues 14, 19, 34, 37, 41, and 58 were examined by EPR on 7.8, 8.4, and 9.6 nm rHDL to assess the effect of particle size on the N-terminal structure. Residues 14, 19, and 58 showed no significant rHDL size-dependent spectral or accessibility differences, whereas residues 34, 37, and 41 displayed moderate spectral changes along with substantial rHDL size-dependent differences in molecular accessibility. We have elucidated the secondary structure of the N-terminal domain of apoA-I on 9.6 nm rHDL (residues 6-98) and identified residues in this region that are affected by particle size. We conclude that the inter-helical segment (residues 35-49) plays a role in the adaptation of apoA-I to the particle size of HDL.

Location of the TEMPO Moiety of TEMPO-PC in Lipid Bilayers

The major yeast phosphatidylinositol/phosphatidylcholine transfer protein Sec14p is the founding member of a large eukaryotic protein superfamily. Functional analyses indicate Sec14p integrates phospholipid metabolism with the membrane trafficking activity of yeast Golgi membranes. In this regard, the ability of Sec14p to rapidly exchange bound phospholipid with phospholipid monomers that reside in stable membrane bilayers is considered to be important for Sec14p function in cells. How Sec14p-like proteins bind phospholipids remains unclear. Herein, we describe the application of EPR spectroscopy to probe the local dynamics and the electrostatic microenvironment of phosphatidylcholine (PtdCho) bound by Sec14p in a soluble protein-PtdCho complex. We demonstrate that PtdCho movement within the Sec14p binding pocket is both anisotropic and highly restricted and that the C5 region of the sn-2 acyl chain of bound PtdCho is highly shielded from solvent, whereas the distal region of that same acyl chain is more accessible. Finally, high field EPR reports on a heterogeneous polarity profile experienced by a phospholipid bound to Sec14p. Taken together, the data suggest a headgroup-out orientation of Sec14p-bound PtdCho. The data further suggest that the Sec14p phospholipid binding pocket provides a polarity gradient that we propose is a primary thermodynamic factor that powers the ability of Sec14p to abstract a phospholipid from a membrane bilayer.

Electron paramagnetic resonance (EPR) spectroscopy of spin-labeled membrane proteins is a valuable biophysical technique to study structural details and conformational transitions of proteins close to their physiological environment, for example, in liposomes, membrane bilayers, and nanodiscs. Unlike in nuclear magnetic resonance (NMR) spectroscopy, having only one or few specific side chains labeled at a time with paramagnetic probes makes the size of the object under investigation irrelevant in terms of technique sensitivity. As a drawback, extensive site-directed mutagenesis is required in order to analyze the properties of the protein under investigation. EPR can provide detailed information on side chain dynamics of large membrane proteins or protein complexes embedded in membranes with an exquisite sensitivity for flexible regions and on water accessibility profiles across the membrane bilayer. Moreover, distances between the two spin-labeled side chains in membrane proteins can be detected with high precision at cryogenic temperatures. The application of EPR to membrane proteins still presents some challenges in terms of sample preparation, sensitivity and data interpretation, thus it is difficult to give ready-to-go methodological recipes. However, new technological developments (arbitrary waveform generators) and new spin labels spectroscopically orthogonal to nitroxides increased the range of applicability from in vitro toward in-cell EPR experiments. This chapter is an updated version of the one published in the first edition of the book and describes the state of the art in the application of nitroxide-based site-directed spin labeling EPR to membrane proteins, addressing new tools such as arbitrary waveform generators and spectroscopically orthogonal labels, such as Gd(III)-based labels. We will present challenges in sample preparation and data analysis for functional and structural membrane protein studies using site-directed spin labeling techniques and give experimental details on EPR techniques providing information on side chain dynamics and water accessibility using nitroxide probes. An updated optimal Q-band DEER setup for nitroxide probes will be described, and its extension to gadolinium-containing samples will be addressed.

Electron paramagnetic resonance (EPR) detection of free nitric oxide (NO) by spin-trapping is now widely applied in model and biological systems (Archer, 1993; Henry et al., 1993). This method involves chemical interaction of NO with hemoglobin, nitroso-compounds, or other spin-traps during which a stable visible EPR adduct is formed, but NO is “trapped” and removed from the system. The other method involving EPR spectroscopy is called spin-label NO-metry and can be defined as the use of nitroxide radical spin labels to monitor the NO diffusion-concentration product. Here during bimolecular collisions of NO (fast relaxing, unseen paramagnetic species) with nitroxide radical spin labels (slow relaxing, visible EPR species), physical interaction between molecules occurs that involves Heisenberg exchange and/or dipole-dipole interaction (Molin et al., 1980). This method does not disturb the concentrations of colliding species. As was shown qualitatively by Singh et al. (1994) during collisions of NO with spin labels located in water or in membranes, both the linewidth and the spin-lattice relaxation time of spin labels are altered. In our previous paper (Lomnicka & Subczynski, 1996), we demonstrated that spin-label NO-metry is also a quantitative method because every collision of NO with spin label leads to an observable event — EPR line broadening. These results allow us to connect the Smoluchowski equation for colliding molecules, with NO-induced line broadening of the EPR spin label spectrum, expressed in frequency units. Here R is an interaction distance for a collision (4.5 A as was shown by Lomnicka and Subczynski, 1996, and p is the probability that a spectroscopically observable event occurs when a collision occurs. It is also assumed that the diffusion coefficient of NO, D NO, is much higher than the diffusion coefficient of the spin label. This assumption should always be considered critically, but if it can be made, then an experimental observable that depends on w yields the so-called diffusion-concentration product of NO, D NO [NO]. Equation (5.2) is appropriate in principle if the line shape in the absence of NO is Lorentzian. Here DH pp is the NO-induced peak-to-peak line broadening, and g is the magnetogyric ratio of the electron.