Electron Paramagnetic Resonance Approach Research Articles

Spin labeling and Double Electron-Electron Resonance (DEER) to Deconstruct Conformational Ensembles of HIV Protease.

An understanding of macromolecular conformational equilibrium in biological systems is oftentimes essential to understand function, dysfunction, and disease. For the past few years, our lab has been utilizing site-directed spin labeling (SDSL), coupled with electron paramagnetic resonance (EPR) spectroscopy, to characterize the conformational ensemble and ligand-induced conformational shifts of HIV-1 protease (HIV-1PR). The biomedical importance of characterizing the fractional occupancy of states within the conformational ensemble critically impacts our hypothesis of a conformational selection mechanism of drug-resistance evolution in HIV-1PR. The purpose of the following chapter is to give a timeline perspective of our SDSL EPR approach to characterizing conformational sampling of HIV-1PR. We provide detailed instructions for the procedure utilized in analyzing distance profiles for HIV-1PR obtained from pulsed electron-electron double resonance (PELDOR). Specifically, we employ a version of PELDOR known as double electron-electron resonance (DEER). Data are processed with the software package "DeerAnalysis" (http://www.epr.ethz.ch/software), which implements Tikhonov regularization (TKR), to generate a distance profile from electron spin-echo amplitude modulations. We assign meaning to resultant distance profiles based upon a conformational sampling model, which is described herein. The TKR distance profiles are reconstructed with a linear combination of Gaussian functions, which is then statistically analyzed. In general, DEER has proven powerful for observing structural ensembles in proteins and, more recently, nucleic acids. Our goal is to present our advances in order to aid readers in similar applications.

Structural characterization of quadruplex DNA with in-cell EPR approaches

Guanosine-rich DNA sequences have the potential to adopt four-stranded conformations termed quadruplexes. The chromosomes of higher organisms are capped by so-called telomeres that are composed of repeats of the sequence TTAGGG. Up to 200 nucleotides of the G-rich strand form an overhang that is suspected to fold into intramolecular G-quadruplexes. Since induction of quadruplexes at the telomeres results in anti-proliferative effects, the intracellular structure of G-quadruplexes is of high interest as an anti-cancer drug target. Here we give a perspective on the elucidation of DNA sequence folds by electron paramagnetic resonance (EPR) distance measurements. The technique complements X-ray crystallography and NMR spectroscopy, as it can be applied in noncrystalline states, is not intrinsically limited by the size of the bio-macromolecular complex, and is able to analyze flexible structures or coexisting DNA conformation.

Refined Distances Between Paramagnetic Centers of a Multi‐Copper Nitrite Reductase Determined by Pulsed EPR (iDEER) Spectroscopy

Refined Distances Between Paramagnetic Centers of a Multi‐Copper Nitrite Reductase Determined by Pulsed EPR (<i>i</i>DEER) Spectroscopy

Discriminative EPR detection of NO and HNO by encapsulated nitronyl nitroxides

Nitric oxide, •NO, is one of the most important molecules in the biochemistry of living organisms. By contrast, nitroxyl, NO−, one-electron reduced analog of •NO which exists at physiological conditions in its protonated form, HNO, has been relatively overlooked. Recent data show that HNO might be produced endogenously and display unique biological effects. However, there is a lack of specific and quantitative methods of detection of endogenous HNO production. Here we present a new method for discriminative •NO and HNO detection by nitronyl nitroxides (NNs) using electron paramagnetic resonance (EPR). It was found that NNs react with •NO and HNO with similar rate constants of about 104 M− 1s− 1 but yield different products: imino nitroxides and the hydroxylamine of imino nitroxides, correspondingly. An EPR approach for discriminative •NO and HNO detection using liposome-encapsulated NNs was developed. The membrane barrier of liposomes protects NNs against reduction in biological systems while is permeable to both analytes, •NO and HNO. The sensitivity of this approach for the detection of the rates of •NO/HNO generation is about 1 nM/s. The application of encapsulated NNs for real-time discriminative •NO/HNO detection might become a valuable tool in nitric oxide-related studies.

Using spin-label electron paramagnetic resonance (EPR) to discriminate and characterize the cholesterol bilayer domain

Electron paramagnetic resonance (EPR) spin-labeling methods make it possible not only to discriminate the cholesterol bilayer domain (CBD) but also to obtain information about the organization and dynamics of cholesterol molecules in the CBD. The abilities of spin-label EPR were demonstrated for Chol/POPC (cholesterol/1-palmitoyl-2-oleoylphosphatidylcholine) membranes, with a Chol/POPC mixing ratio that changed from 0 to 3. Using the saturation-recovery (SR) EPR approach with cholesterol analogue spin labels, ASL and CSL, and oxygen or NiEDDA relaxation agents, it was confirmed that the CBD was present in all membrane suspensions when the mixing ratio exceeded the cholesterol solubility threshold (CST). Conventional EPR spectra of ASL and CSL in the CBD were similar to those in the surrounding POPC bilayer (which is saturated with cholesterol), indicating that in both domains, cholesterol exists in the lipid-bilayer-like structures. The behavior of ASL and CSL (and, thus, the behavior of cholesterol molecules) in the CBD was compared with that in the surrounding POPC-cholesterol domain (PCD). In the CBD, ASL and CSL molecules are better ordered than in the surrounding PCD. This difference is small and can be compared to that induced in the surrounding domain by an ∼10°C decrease in temperature. Thus, cholesterol molecules are unexpectedly dynamic in the CBD, which should enhance their interaction with the surrounding domain. The polarity of the water/membrane interface of the CBD is significantly greater than that of the surrounding PCD, which significantly enhances penetration of the water-soluble relaxation agent, NiEDDA, into that region. Hydrophobicity measured in the centers of both domains is similar. The oxygen transport parameter (oxygen diffusion-concentration product) measured in the center of the CBD is about ten times smaller than that measured in the center of the surrounding domain. Thus, the CBD can form a significant barrier to oxygen transport. The results presented here point out similarities between the organization and dynamics of cholesterol molecules in the CBD and in the surrounding PCD, as well as significant differences between CBDs and cholesterol crystals.

Dynamic mixing processes in spin triads of “breathing crystals” Cu(hfac)2LR: a multifrequency EPR study at 34, 122 and 244 GHz

Spin triads of copper(ii) with two nitroxides are responsible for the magnetic anomalies in a new family of molecular-magnetic compounds called "breathing crystals". We have shown previously that electron paramagnetic resonance (EPR) spectroscopy allows one to investigate the peculiarities of these systems and obtain valuable information on exchange interactions governing the magnetic anomalies. One of the key processes revealed is the dynamic mixing between different spin multiplets that leads to a coalescence of individual EPR lines at high temperatures. The rates of the mixing were found to be fast at EPR frequencies between 9 and 94 GHz. In the present work, we expose the spin triads to higher microwave frequencies of up to 244 GHz in order to reach the conditions of intermediate or slow mixing rates. Three representatives of the family of breathing crystals have been studied. Based on the simulations of EPR data at 34, 122 and 244 GHz, the rates of the mixing processes have been estimated and conclusions on their character and temperature dependence have been drawn. The insights from high-field EPR clarify previously obtained results and aid in the further development of EPR approaches for studying these and similar systems. It is suggested that the static and dynamic Jahn-Teller effects may play an important role in the mechanisms governing the observed spin exchange effects.

A Multi-Frequency Pulse EPR and ENDOR Approach to Study Strongly Coupled Nuclei in Frozen Solutions of High-Spin Ferric Heme Proteins

In spite of the tremendous progress in the field of pulse electron paramagnetic resonance (EPR) in recent years, these techniques have been scarcely used to investigate high-spin (HS) ferric heme proteins. Several technical and spin-system-specific reasons can be identified for this. Additional problems arise when no single crystals of the heme protein are available. In this work, we use the example of a frozen solution of aquometmyoglobin (metMb) to show how a multi-frequency pulse EPR approach can overcome these problems. In particular, the performance of the following pulse EPR techniques are tested: Davies electron nuclear double resonance (ENDOR), hyperfine correlated ENDOR (HYEND), electron-electron double resonance (ELDOR)-detected NMR, and several variants of hyperfine sublevel correlation (HYSCORE) spectroscopy including matched and SMART HYSCORE. The pulse EPR experiments are performed at X-, Q- and W-band microwave frequencies. The advantages and drawbacks of the different methods are discussed in relation to the nuclear interaction that they intend to reveal. The analysis of the spectra is supported by several simulation procedures, which are discussed. This work focuses on the analysis of the hyperfine and nuclear-quadrupole tensors of the strongly coupled nuclei of the first coordination sphere, namely, the directly coordinating heme and histidine nitrogens and the 17O nucleus of the distal water ligand. For the latter, 17O-isotope labeling was used. The accuracy of our results and the spectral resolution are compared in detail to an earlier single-crystal continuous-wave ENDOR study on metMb, and it will be shown how additional information can be obtained from the multi-frequency approach. The current work is therefore prone to become a template for future EPR/ENDOR investigations of HS ferric heme proteins for which no single crystals are available.

Structural organization in photosynthetic proteins as studied by high-field EPR of spin-correlated radical pair states

We demonstrate the potential of high-field (HF) time-resolved electron paramagnetic resonance (EPR) spectroscopy to reveal unique information about electron transfer processes and the structure of photosynthetic systems. The lineshapes and electron spin polarization (ESP) of spin-correlated radical pair (SCRP) spectra recorded with HF-EPR are very sensitive to the magnetic parameters, interactions, and geometry of the radicals in the pair. This sensitivity facilitates an analysis of more sophisticated models and methods to reveal the important relationship between structural organization and light-induced electron transfer of the photosynthetic proteins. In this review, we report on a new time-resolved HF and multi-frequency EPR approach developed in the Freiburg laboratory in cooperation with the Argonne Photosynthesis group. The method is designed to probe the geometric structure of charge separated states in the photosynthetic membrane. First, we discuss the magneto-orientation of photosynthetic cyanobacteria as revealed by time-resolved HF-EPR of SCRPs. Then, we demonstrate how the three-dimensional structure of the SCRP P700(+)A1 from photosystem I of oxygenic photosynthesis and its arrangement in the membrane is obtained from application of multi-frequency including time-resolved HF-EPR techniques.

High-frequency EPR approach to the electron spin-polarization effects observed in the photosynthetic reaction centers

Time-resolved high-frequency electron paramagnetic resonance (EPR) spectroscopy was applied to study the structure and dynamics of the electron transfer pathways in the photosynthetic RC proteins. When the spin-polarized EPR spectra are recorded at the high field, the singlet-triplet mixing in the radical pairs becomes faster due to the increase of Zeeman interaction, and a sequential electron transfer polarization model, which includes both the primary and secondary radical pairs, should be considered. Application of the sequential electron transfer polarization model for the interpretation of the bacterial RC proteins with a “slow” electron transfer rate reveals the importance of the protein dynamics. It was shown that the reorganization energy for the electron transfer process between P 865 + H−QA and P 865 + HQ A − , but not the change in the structure of the donor-acceptor complex, is a dominant factor that alters the electron transfer rate. The relaxation data, obtained in the delay after laser flash experiment, have been used to estimate the magnetic interaction in the weakly coupled radical pair. High-frequency spin-polarized EPR spectra allow the quantitative characterization of isotopically labeled quinone exchange in the PS I reaction center proteins.

Correlation between the Mobility of Spin-Labeled Peptide Chains and Resin Solvation: An Approach To Optimize the Synthesis of Aggregating Sequences1

Resin solvation properties affect the efficiency of the coupling reactions in solid-phase peptide synthesis. Here we report a novel approach to evaluate resin solvation properties, making use of spin label electron paramagnetic resonance (EPR) spectroscopy. The aggregating VVLGAAIV and ING sequences were assembled in benzhydrylamine-resin with different amino group contents (up to 2.6 mmol/g) to examine the extent of chain association within the beads. These model peptidyl-resins were first labeled at their N-terminus with the amino acid spin label 2,2,6,6-tetramethylpiperidine-N-oxyl-4-amino-4-carboxylic acid (Toac). Their solvation properties in different solvents were estimated, either by bead swelling measurement or by assessing the dynamics of their polymeric matrixes through the analysis of Toac EPR spectra, and were correlated with the yield of the acylation reaction. In most cases the coupling rate was found to depend on bead swelling. Comparatively, the EPR approach was more effective. Line shape...