Heterogeneity in the Nitroxide Micro-Environment: Polarity and Proticity Effects in Spin-Labeled Proteins Studied by Multi-Frequency EPR

This study aims to investigate the g _xx heterogeneity of the g-tensor commonly observed in high-field electron paramagnetic resonance (EPR) spectra of nitroxide spin-labeled sites in proteins. This heterogeneity is addressed in terms of spin-label populations characterized by different polarity and H-bonding properties of the nitroxide micro-environment. The g _xx value for each population is determined from the fit of continuous-wave high-field spectra obtained at 95, 275 and 360 GHz with a series of nitroxide spin-labels covalently attached to different sites in both membrane and water-soluble proteins. The spin-labeled proteins investigated include sensory rhodopsin II and its cognate transducer molecule (HtrII) from Natronomonas pharaonis both in micelles and membranes, bacteriorhodopsin from Halobacterium salinarum in native purple membrane lipid bilayers and water-soluble colicin A from Escherichia coli. To avoid contributions to the g _xx spectral features of the nitroxide label due to nuclear quadrupole interactions arising from ¹⁴N nuclei, and to simplify the nitrogen hyperfine pattern, methanethiosulfonate spin labels, containing the ¹⁵N isotope (I = 1/2) in some experiments, were employed. A consistent analysis of all multi-frequency EPR spectra revealed three distinct g _xx values, g _xx ⁱ , for each investigated position of the labeled proteins. In contrast, distinctly different nitrogen hyperfine splittings A _zz of the nitroxides in the various labeled proteins could not be resolved, but rather an average hyperfine splitting $\bar{A}_{{zz}}$ was obtained. The g _xx ⁱ values as well as the fractions of the different nitroxide populations were found to be correlated with the average hyperfine constant $\bar{A}_{{zz}},$ a parameter which likewise is known to be sensitive to the local polarity of the spin-label micro-environment. Plotting the different g _xx ⁱ values obtained for each EPR spectrum versus $\bar{A}_{{zz}}$ of the labeled proteins reveals new interesting aspects of the nitroxide label micro-environment in terms of polarity and H-bonding propensity (proticity). Linear approximations of the different regions of the plot g _xx ⁱ versus $\bar{A}_{{zz}}$ are presented and compared with theoretical and experimental data available from the literature.     

Polarity dependence of EPR parameters for TOAC and MTSSL spin labels: correlation with DOXYL spin labels for membrane studies

TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) is a nitroxyl amino acid that can be incorporated in the backbone of peptides. DOXYL (4,4-dimethyl-oxazolidine-1-oxyl) is a nitroxyl ring that can be attached rigidly at specific C-atom positions in the acyl chains of phospholipids. Spin-labelled phosphatidylcholines of the DOXYL type have been used previously to establish the transmembrane polarity profile in biological lipid bilayers [D. Marsh, Polarity and permeation profiles in lipid membranes, Proc. Natl. Acad. Sci. USA 87 (2001) 7777-7782]. Here, we determine the polarity dependence of the isotropic (14)N-hyperfine couplings, a(o)(N), and g-values, g(o), in a wide range of protic and aprotic media, for a TOAC-containing dipeptide (Fmoc-TOAC-Aib-OMe) and for a DOXYL-containing fatty acid (12-DOXYL-stearic acid). The correlation between datasets for TOAC and DOXYL nitroxides in the various solvents is used to establish the polarity profile for isotropic hyperfine couplings of TOAC in a transmembrane peptide. This calibration can be used to determine the location of TOAC at selected residue positions in a transmembrane or surface-active peptide. A similar calibration procedure is also applied to a(o)(N) and g(o) for the pyrroline methanethiosulphonate nitroxide (MTSSL) that is used in site-directed spin-labelling studies of membrane proteins.     

核磁共振、X射线小角散射以及计算机模拟相结合构建生物大分子复合物的结构模型

近年来,结构生物学研究越来越注重生物大分子复合物的解析,因为许多重要生物学过程都离不开复合物的参与．溶液核磁共振是目前重要的结构解析方法之一．X射线小角散射(SAXS)作为一种新的结构生物学实验手段,近年来发展迅速．SAXS 能提供生物大分子复合物的较低分辨率结构信息,而核磁共振能解析复合物中各个亚基的原子分辨率结构．此外,通过核磁共振还能得到亚基之间的界面、取向以及距离信息．因此近年来通过计算机模拟,整合核磁共振和 SAXS 不同分辨率的结构信息,可以用来搭建生物大分子复合物的结构模型．该综述重点介绍这方面的研究进展．     

Poisson-Nernst-Planck Equations for Simulating Biomolecular Diffusion-Reaction Processes I: Finite Element Solutions

In this paper we developed accurate finite element methods for solving 3-D Poisson-Nernst-Planck (PNP) equations with singular permanent charges for electrodiffusion in solvated biomolecular systems. The electrostatic Poisson equation was defined in the biomolecules and in the solvent, while the Nernst-Planck equation was defined only in the solvent. We applied a stable regularization scheme to remove the singular component of the electrostatic potential induced by the permanent charges inside biomolecules, and formulated regular, well-posed PNP equations. An inexact-Newton method was used to solve the coupled nonlinear elliptic equations for the steady problems; while an Adams-Bashforth-Crank-Nicolson method was devised for time integration for the unsteady electrodiffusion. We numerically investigated the conditioning of the stiffness matrices for the finite element approximations of the two formulations of the Nernst-Planck equation, and theoretically proved that the transformed formulation is always associated with an ill-conditioned stiffness matrix. We also studied the electroneutrality of the solution and its relation with the boundary conditions on the molecular surface, and concluded that a large net charge concentration is always present near the molecular surface due to the presence of multiple species of charged particles in the solution. The numerical methods are shown to be accurate and stable by various test problems, and are applicable to real large-scale biophysical electrodiffusion problems.     

Biomolecular dynamics and binding studies in the living cell

Isolation and preparation of proteins of higher organisms often is a tedious task. In the case of success, the properties of these proteins and their interactions with other proteins can be studied in vitro. If however, these proteins are modified in the cell in order to gain or change function, this is non-trivial to correctly realise in vitro. When, furthermore, the cellular function requires the interplay of more than one or two proteins, in vitro experiments for the analysis of this situation soon become complex. Instead, we thus try to obtain information on the molecular properties of proteins in the living cell. Then, the cell takes care of correct protein folding and modification. A series of molecular techniques are, and new ones become, available which allow for measuring molecular protein properties in the living cell, offering information on concentration (FCS), dynamics (FCS, RICS, FRAP), location (PALM, STED), interactions (F3H, FCCS) and protein proximities (FRET, BRET, FLIM, BiFC). Here, these techniques are presented with their advantages and drawbacks, with examples from our current kinetochore research. The review is supposed to give orientation to researchers planning to enter the field, and inform which techniques help us to gain molecular information on a multi-protein complex. We show that the field of cellular imaging is in a phase of transition: in the future, an increasing amount of physico-chemical data can be determined in the living cell.     

Low-frequency detected EPR: Principles and applications

The experimental works described are performed by the authors over last two decades by means of the LFD EPR technique. The essence of this method is low-frequency detection of the longitudinal spin magnetization while the magnetic resonance is excited by a strong microwave field. The first kind of LFD EPR is the enchanced longitudinal susceptibility effect (ELSE) which has been elaborated and applied to study spin thermodynamics in solids since 1972. Various applications of ELSE are described such as direct measuring of the spin-spin interaction temperatureT _ss in the course of resonance saturation, spin-lattice and cross relaxation, dynamic nuclear polarization etc. Another version of LFD EPR was employed to study electron spin-lattice relaxation of paramagnetic centers in high-temperature superconductors (HTSC). Recent experimental data are presented on the electron spin-lattice relaxation of Cu²⁺ ions in YBa₂Cu₃O_6+x at various temperatures andx values.     

Radio frequencies in EPR: Conventional and advanced use

This mini-review focuses on various aspects of the application of radio frequency (rf) irradiation in electron paramagnetic resonance (EPR). The development of the electron-nuclear double resonance (ENDOR) technique is briefly described, and we highlight the use of circularly polarized rf fields and pulse ENDOR methodology in one- and two-dimensional experiments. The capability of pulse ENDOR at Q-band is illustrated with interesting experimental examples. Electron spin echo envelope modulation effects induced by an rf field in liquid samples demonstrate another role which rf fields can play. Technical achievements in the design of ENDOR resonators are illustrated by the example of a bridged loop-gap resonator. Finally, the influence of longitudinal rf fields on the dynamics of EPR transitions is explained using a dressed spin resonance treatment.     

Protocol for emergency EPR dosimetry in fingernails

There is an increased need for after-the-fact dosimetry because of the high risk of radiation exposures due to terrorism or accidents. In case of such an event, a method is needed to make measurements of dose in a large number of individuals rapidly and with sufficient accuracy to facilitate effective medical triage. Dosimetry based on EPR measurements of fingernails potentially could be an effective tool for this purpose. This paper presents the first operational protocols for EPR fingernail dosimetry, including guidelines for collection and storage of samples, parameters for EPR measurements, and the method of dose assessment. In a blinded test of this protocol application was carried out on nails freshly sampled and irradiated to 4 and 20 Gy; this protocol gave dose estimates with an error of less than 30%.     

Raman Scattering Tensors in Biological Molecules and Their Assemblies

INTRODUCTION

The tensor associated with a Raman band plays an important role in determining the band intensity and its structural significance. Each Raman tensor interrelates two electric vectors, that of the exciting radiation (i.e. laser photon) and that of the Raman scattered radiation (i.e. the inelastically scattered photon which results from the exchange of a vibrational quantum between the exciting photon and the molecule). The Raman tensor is obtained formally as the first derivative of the molecular polarizability tensor, the derivative being taken with respect to the vibrational normal coordinate. In other words, the Raman tensor associated with a vibrational Raman band is an indicator of how the polarizability of the molecule oscillates with the molecular normal mode of vibration.     

Erbium in silicon carbide crystals: EPR and high-temperature luminescence

Erbium ions have been incorporated for the first time in bulk 6H-SiC crystals during growth, and they were unambiguously identified from the ¹⁶⁷Er EPR hyperfine structure. High-temperature luminescence of erbium ions at a wavelength of 1.54 μm has been detected. The observed luminescence exhibits an increase in intensity with increasing temperature. The observation of Er luminescence in 6H-SiC offers a promising potential for development of semiconductor light-emitting devices at a wavelength within the fiber-optics transparency window. Fiz. Tverd. Tela (St. Petersburg) 41, 38–40 (January 1999)     

The chaotic ball: An intuitive analogy for EPR experiments

Actual realisations of EPR experiments donot demonstrate non-locality. A model is presented that should enable non-specialists as well as specialists to understand how easy it is to find realistic explanations for the observations. The model also suggests new areas where realistic (hidden-variable) models can give valid predictions whilst quantum mechanics fails. It offers straightforward explanations for some anomalies that Aspect was unable to account for, providing perhaps the first experimental evidence that a hidden-variable theory can besuperior to quantum mechanics. The apparent success of quantum mechanics in predicting results is shown to be largely due to the use of unjustifiable and biased analysis of the data. Data that has been discarded because it did not lead to a valid Bell's test may give further evidence that hidden variables exist.     

High-frequency EPR: An occasion for revisiting ligand field theory

The use of high-field electron paramagnetic resonance (EPR) made possible for the first time to obtain detailed information on the anisotropy of integer-spin systems, which are usually silent at conventional EPR frequencies. The data obtained so far by different research groups on non-Kramers ions are reviewed within the framework of ligand field theory at different levels of approximations. Special attention is paid to the use of the angular overlap model, which resulted to be very powerful both in terms of qualitative and quantitative explanation of the experimental results.     

Multifrequency Probe for Pulsed EPR and ENDOR Spectroscopy

The design, construction, and performance of a multifrequency pulsed EPR and ENDOR probe for use at cryogenic temperatures are described. Interchangeable resonators based on a folded strip line design allow variation of the resonance frequency over a range of 5-11 GHz. Variable coupling to the resonator is achieved capacitively via a simple mechanical adjustment which is thermally and mechanically stable. The entire assembly is robust and easily fabricated. Common methods of analyzing the resonator parameters such as the Q-factor and coupling coefficient are discussed quantitatively. Probe performance data and multifrequency pulsed ENDOR spectra are presented.     

EPR and Bell's theorem: A critical review

The argument of Einstein, Podolsky, and Rosen is reviewed with attention to logical structure and character of assumptions. Bohr's reply is discussed. Bell's contribution is formulated without use of hidden variables, and efforts to equate hidden variables to realism are critically examined. An alternative derivation of nonlocality that makes no use of hidden variables, microrealism, counterfactual definiteness, or any other assumption alien to orthodox quantum thinking is described in detail, with particular attention to the quartet or broken-square question.     

FMR and EPR in Ni@C nanocomposites: Size and concentration effects

One-domain Ni@C nanoparticles encapsulated in carbon coating have been investigated depending on the size and concentration of Ni in carbon. The nanoparticles of nickel were prepared with the average diameters changing in a broad range of 4–45 nm, and the concentration of Ni in C varies in 2–12 wt%. To prepare the Ni@C nanocomposites the solid solutions of nickel phthalocyanine–metal-free phthalocyanine (NiPc) _x (H₂Pc)_1–x , 0 ≤ x ≤ 1 were synthesized and the solidphase pyrolysis of these compounds was performed. In the case of ultradispersive Ni nanoparticles (the interval of quantum dots is 1–10 nm), a considerable shift of the resonance field and broadening of resonance absorption field were revealed in the spectra of FMR at room temperature. The data were interpreted taking into account the essential contribution of the surface magnetic anisotropy, the magnetic field of which far exceeds the magnetic field of volume anisotropy.     

Implementing EPR Dosimetry for Life-Threatening Incidents: Factors Beyond Technical Performance

Starting with the assumption that a device to detect unplanned radiation exposures is technically superior to current technology, we examine the additional stakeholders and processes that must be considered to move the device from the lab into use. The use is to provide reliable information to triage people for early treatment of exposure to ionizing radiation that could lead to the Acute Radiation Syndrome. The scenario is a major accident or terrorist event that leaves a large number of people potentially exposed, with the resulting need to identify those to treat promptly or not. In vivo EPR dosimetry is the exemplar of such a technique.Three major areas are reviewed: policy considerations, regulatory clearance, and production of the device. Our analysis of policy-making indicates that the current system is very complex, with multiple significant decision-makers who may have conflicting agendas. Adoption of new technologies by policy-makers is further complicated because many sources of expert input already have made public stances or have reasons to prefer current solutions, e.g., some may have conflicts of interest in approving new devices because they are involved with the development or adoption of competing techniques. Regulatory clearance is complicated by not being able to collect evidence via clinical trials of its intended use, but pathways for approval for emergency use are under development by the FDA. The production of the new device could be problematical if the perceived market is too limited, particularly for private manufacturers; for in vivo EPR dosimetry the potential for other uses may be a mitigating factor.Overall we conclude that technical superiority of a technique does not in itself assure its rapid and effective adoption, even where the need is great and the alternatives are not satisfactory for large populations. Many important steps remain to achieve the goals of approval and adoption for use.     

Clinical applications of in vivo EPR: Rationale and initial results

In vivo electron paramagnetic resonance (EPR) has been very useful for studies in animals, and these results suggest that there are some very attractive potential applications in human subjects. In this article, we describe our rationale for the clinical application of in vivo EPR, some of the principal technical challenges, the initial results in human subjects, and our evaluation of the areas where in vivo EPR is likely to play an important clinical role in the near future. The most obvious area of very high potential for clinical applications is tissue oximetry, where in vivo EPR can provide repeated and accurate measurements of tissue pO₂, a type of measurement that cannot be obtained by other techniques. Oximetry is capable of providing clinicians with information that can impact directly on diagnosis and therapy, especially for peripheral vascular disease, oncology, and wound healing. The other area of great immediate importance is the ability of in vivo EPR to measure clinically significant exposures to ionizing radiation after the fact, which may occur due to accidents, terrorist activity, or nuclear war. The results obtained already from human subjects demonstrate the feasibility of the use of in vivo EPR for measurements in human subjects. We anticipate that in vivo EPR will play a vital role in the clinical management of various pathologies in the years to come.     

An in situ electrochemical cell for Q- and W-band EPR spectroscopy

A simple design for an in situ, three-electrode spectroelectrochemical cell is reported that can be used in commercial Q- and W-band (ca. 34 and 94 GHz, respectively) electron paramagnetic resonance (EPR) spectrometers, using standard sample tubing (1.0 and 0.5 mm inner diameter, respectively) and within variable temperature cryostat systems. The use of the cell is demonstrated by the in situ generation of organic free radicals (quinones and diimines) in fluid and frozen media, transition metal ion radical anions, and on the enzyme nitric oxide synthase reductase domain (NOSrd), in which a pair of flavin radicals are generated.