High-Field EPR Spectroscopy on Proteins and their Model Systems: Characterization of Transient Paramagnetic States - Hardcover

Klaus Möbius

Hardcover

ISBN 10: 0854043683 ISBN 13: 9780854043682

Publisher: Royal Society of Chemistry, 2008

Synopsis

High-Field EPR Spectroscopy on Proteins and their Model Systems: characterization of Transient Paramagnetic States offers a comprehensive overview of experimental techniques in, and paradigmatic examples of, the application of high-field EPR spectroscopy in biology and chemistry.

About this title

About the Author

Klaus Mobius has worked in the field of EPR spectroscopy for more than 40 years. During the last 15 years, his research has focussed on high-field EPR and related techniques on biochemical systems. Anton Savitsky has worked in the field of EPR spectroscopy for over a decade. Since 1998, his research has focussed on high-field EPR instrumentation development and application to biochemical systems.

From the Back Cover

Understanding the major factors determining the specificity of transmembrane transfer processes in proteins is now a hot topic in molecular bio-science. Advanced electron paramagnetic resonance (EPR) at high magnetic fields is a powerful technique for characterizing the transient states of proteins in action. High-Field EPR Spectroscopy on Proteins and their Model Systems: characterization of Transient Paramagnetic States offers a comprehensive overview of experimental techniques in, and paradigmatic examples of, the application of high-field EPR spectroscopy in biology and chemistry. The book's focus is on the use of the technique in conjunction with site-specific mutation strategies and advanced quantum-chemical computation methods to reveal protein structure and dynamics. This yields new insights into biological processes at the atomic and molecular level. The theoretical and instrumental background of high-field EPR is described and examples of paradigmatic protein systems, such as photosynthetic reaction centres, are discussed in the light of recent investigations. Aspects of structure dynamics-function relations that are revealed by studying site-specific mutants are highlighted, thereby combining high-field EPR with genetic engineering techniques. The information obtained complements that obtained from protein crystallography, solid-state NMR, infrared and optical spectroscopy. The book documents both background knowledge and results of the latest research in the field. Unique features include comparisons of information content of EPR, ENDOR, Triple resonance, ESEEM and PELDOR taken at different microwave frequencies and magnetic fields. Coherent treatment of the subject by the leading Berlin high-field EPR laboratory covers the theoretical background as well as state-of-art research both in terms of instrumentation and application to biological systems. The book provides an outlook to future developments and references for further reading and is essential reading for postdoctoral scientists, professionals, academics and graduate students working in this field.

Excerpt. © Reprinted by permission. All rights reserved.

High-Field EPR Spectroscopy on Proteins and Their Model Systems

Characterization of Transient Paramagnetic States

By Klaus Möbius, Anton Savitsky

The Royal Society of Chemistry

Copyright © 2009 Klaus Möbius and Anton Savitsky
All rights reserved.

ISBN: 978-0-85404-368-2

Contents

Summary, xiii, 
Acknowledgements, xv, 
Chapter 1 Introduction, 
Chapter 2 Principles and Illustrative Examples of High-Field/High-Frequency EPR, 
Chapter 3 Instrumentation, 
Chapter 4 Computational Methods for Data Interpretation, 
Chapter 5 Applications of High-Field EPR on Selected Proteins and their Model Systems, 
Chapter 6 Conclusions and Perspectives, 
Subject Index, 366, 

CHAPTER 1

Introduction

During the last decade, the chemistry, biology and physics communities have apparently witnessed a boost of new EPR (electron paramagnetic resonance) applications. This is largely due to technological breakthroughs in the development of pulsed microwave sources and components, sweepable cryomagnet design and fast data-acquisition instrumentation. They enable the EPR spectroscopists to introduce multiple-pulse microwave irradiation schemes, very much in analogy to what is common practice in modern NMR (nuclear magnetic resonance), and to apply advanced multifrequency high-field EPR techniques as powerful spectroscopic tools with unique potential for the elucidation of structure and dynamics of complex systems, for example membrane proteins in biological action.

This assessment is corroborated by the substantial increase of publications related to high-field/high-frequency EPR since the last 15 years (see Figure 1.1). The growing appreciation is mirrored also by the rising number of research groups in Europe, the US and Japan dedicated to the development and/or application of high-field EPR spectroscopy. This was made possible by increased financial support from national and international funding agencies. The European Union, for example, supported the Human Capital and Mobility (HCM) project "High-Field EPR: Technology and Applications" (coordinator J. Schmidt, Leiden, 1993–1996) and the EU network project "SENTINEL" ("Service Enhancement through Infrastructure Networking for Electron Paramagnetic Resonance Spectroscopy with Large Fields", coordinator M. Martinelli, Pisa, 2001–2005). Exceptionally strong support was granted by the DFG (Deutsche Forschungsgemeinschaft) through the Priority Program "High-Field EPR in Biology, Chemistry and Physics" (coordinator K. Möbius, Berlin, 1998–2004). These initiatives acted like seeding programs for the rapid development of high-field EPR spectroscopy in Europe, including Israel and Russia. In Figure 1.2 the present distribution of high-field EPR groups in Europe is shown. There is a noticeable congestion of such groups in Germany, apparently as a benefit from the sustaining support by the DFG.

In the US, there is a strong representation of high-field EPR spectroscopy with about ten research groups throughout the country. Particularly renowned high-field EPR groups are concentrated in dedicated national facilities located in Ithaca, NY, at Cornell University (ACERT, the "National Biomedical Center for Advanced ESR Technology", headed by J.H. Freed,) in Tallahassee, FL, (National High Magnetic Field Laboratory, the EPR group headed until recently by L.-C. Brunel), in Milwaukee, WI, at the Medical College of Wisconsin (National Biomedical EPR Center, headed by J.S. Hyde) and in Cambridge, MA, at MIT (Francis Bitter Magnet Laboratory, headed by R.G. Griffin).

In Japan, high-field EPR research is traditionally devoted to physics with the focus on novel magnetic materials. There exist about ten mm and sub-mm high-field EPR facilities in Japanese universities and national institutes working in the field of physical sciences. They cover broad field ranges up to 150 T using superconducting and hybrid magnets, either in constant-field or repetitive pulse-field mode of operation. But also in chemical and biological sciences a growing interest is noticeable in Japan, both in terms of instrument developments and scientific applications. For example, in Sendai at the Tohoku University, S. Yamauchi and coworkers have demonstrated the advantages of W-band high-field EPR for their studies of organic excited multiplet states in fluid solution over recent years.

1.1 Why EPR at High Magnetic Fields?

Induced chemical reactions in condensed phases often proceed via the formation of radical pairs as reaction intermediates. Radical pairs are formed, for example, after appropriate excitation of donor molecules, by one-electron transfer to acceptor molecules leading to ionic radical pairs. Such electron-transfer reactions are found in many fields of chemistry and biology, for example in tribochemistry, i.e. during chemical reactions initiated by mechanical activation of solid mixtures of compounds by pressure, in photochemistry, i.e. during chemical reactions initiated by the absorption of infrared, visible and ultraviolet light, and in photosynthesis, i.e. during photoinduced chemical reactions in green plants, algae and certain bacteria by which carbon dioxide and water are converted to carbohydrates and oxygen.

In principle, EPR appears to be a promising spectroscopic technique to study both stable and transient radical-pair intermediates. In practice, however, for large spin systems in solid-state reactions, standard EPR – similar to other types of spectroscopy – soon reaches its limits of useful information content, unless single-crystal samples are available. Unfortunately, large molecular complexes are often available only as disordered samples. Their standard X- band (9.5 GHz) EPR spectra are poorly resolved, and the information on magnetic parameters and molecular orientations is hidden under the broad lines. By going to higher and higher magnetic fields and microwave frequencies, for example to EPR at W-band (95 GHz) or even at 360 GHz, at least five important features, (i)–(v), are emerging from the EPR spectra: (i) enhanced spectral resolution; (ii) enhanced orientational selectivity in disordered samples; (iii) enhanced low-temperature electron-spin polarization; (iv) enhanced detection sensitivity for restricted-volume samples such as small single crystals of proteins or fullerenes, and (v) last but not least enhanced sensitivity for probing fast motional dynamics, i.e. high-frequency EPR acts as a faster "snapshot" for molecular motion.

Ad (i): The strategy for spectral resolution enhancement is similar in EPR and NMR: With increasing external Zeeman field the field-dependent spin interactions in the spin Hamiltonian are separated from the field-independent ones (see Figure 1.3). In high-field EPR, the g-factor resolution is increased in relation to the hyperfine couplings, in high-field NMR the chemical-shift resolution is increased in relation to the spin–spin couplings.

Ad (ii): The important feature of enhanced orientation selectivity by high-field EPR on randomly oriented spin systems becomes essential for organic radicals with only small g-anisotropy (see Figure 1.4) Well below room temperature, the overall rotation of, for example, a protein complex becomes so slow that powder-type EPR spectra are obtained. If the anisotropy of the leading interaction in the spin Hamiltonian is larger than the inhomogeneous linewidth, even from disordered powder-type EPR spectra the canonical orientations of the dominating interaction tensor can be resolved. As a con- sequence, single-crystal-like information on the hyperfine interactions can be extracted by performing orientation-selective ENDOR at the field values of resolved spectral features. In the case of transition-metal complexes the hyperfine anisotropy of the metal ion may provide this orientation selectivity from the entire orientational distribution of the molecules. Often, their g-anisotropy is large enough to allow for distinct orientational selectivity already in X-band EPR allowing for single-crystal-like ENDOR.5–7 The best approach for elucidating molecular structure and orientation in detail is, of course, to study single-crystal samples. Unfortunately, to prepare them for large biological complexes like membrane proteins is often difficult or even impossible.

Ad (iii): The enhanced low-temperature electron-spin polarization at high Zeeman fields allows to extract the absolute sign of the zero-field splitting parameter, D, of a two-spin system like a biradical or triplet state. At high fields, considerable thermal spin polarization can be achieved already well above helium temperature, provided that the sample temperature becomes comparable with the Zeeman temperature, [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII] (g: electron g-factor, μB: Bohr magneton, B0: Zeeman field; kB: Boltzmann constant). At T [much greater than] TZ, the characteristic triplet powder EPR spectrum (see Figure 1.5) is symmetric at its low- and high-field sides and, hence, contains no information of the sign of D. At T< TZ, the Boltzmann distribution leads to increased populations of the low-energy levels, resulting in asymmetric lineshapes from which the absolute sign of D can be directly read off. Thermal spin polarization as a means to determine the absolute sign of D in high-spin systems has been used at a variety of EPR frequencies, for example at 9.5 GHz (TZ ≈ 0.4 K), at 95 GHz (TZ ≈ 4 K), 140 GHz (TZ ≈ 6.5 K), 360 GHz (TZ ≈ 15.5 K).

Ad (iv): With respect to detection sensitivity and its enhancement with increasing microwave frequencies, one has to distinguish between the absolute and relative (concentration) sensitivities. The absolute sensitivity is defined by the minimum detectable number of spins in the sample, Nmin, the relative (or concentration) sensitivity is given by Nmin/VS, i.e. is scaled by sample volume, VS. This is limited by the amount of sample that can be introduced into the cavity of high-field EPR spectrometers that, of course, is usually significantly smaller than standard X-band cavities. Consequently, if the amount of sample available is limited, as in single crystals of proteins, the sensitivity of high-field EPR can be superior by orders of magnitude because the absolute sensitivity grows with increasing frequencies much more strongly than the relative sensitivity does. Under certain experimental conditions, for constant incident microwave power and unsaturated EPR lines, one obtains theoretical expressions for the absolute sensitivity, [MATHEMATICAL EXPRESSION NOT REPRODUCIBLE IN ASCII], and for the relative sensitivity, ≈ (see detailed sensitivity discussions in Chapters 2 and 3, which are based on refs. [12,13]).

Ad (v): The faster "snapshot" capability for complex motional dynamics with increasing EPR frequency can be used in a multifrequency continuous- wave (cw) EPR approach at the same temperature to probe fast internal modes of motion and to discriminate them from the slow restricted motion of a macromolecule in solution. In high-frequency cw EPR spectra, slow motions appear to be frozen out, whereas fast motions dominate the observed spectral lineshape.

In many fields of chemistry the precise nature of primary reactions with an involvement of paramagnetic intermediates is not well understood. This situation motivated us, when writing this book, to put particular emphasis on high-field/high-frequency EPR techniques used in (bio)chemistry as a means to elucidate the structural and dynamical details of short-lived paramagnetic intermediates involved in primary reactions.

1.2 NMR versus EPR

Up to 15 years ago, when asking scientists from outside the magnetic resonance community the question "what is magnetic resonance?" most of them, no matter which specific discipline they were working in, probably would have responded positively, but only with reference to NMR. When asking biologists, chemists and physicists the specific question "what is EPR?", probably only a small minority would have responded positively, and this only with reference to conventional cw X–band EPR spectroscopy (operating at a microwave frequency of around 9 GHz). Admittedly, such an imbalance of appreciation of the two magnetic resonance sisters was totally justified, because over the last decades NMR, in contrast to EPR, had grown up much faster and had become well established in the material sciences, in the life sciences and even in medical diagnostics as a unique tool for obtaining details of information inaccessible so far on molecular structure, chemical kinetics and medical imaging. Not surprisingly, therefore, that as many as four Nobel prizes for NMR methodology and applications have been awarded within the last 15 years (to R.R. Ernst in Chemistry in 1991, to K. Wüthrich in Chemistry in 2002, to P.C. Lauterbur and to P. Mansfield in Physiology and Medicine in 2003). But none, so far, for EPR!

EPR seemed to be hopelessly lagging behind its famous (though younger) sister NMR, and it is only during the last decade that the chemistry, biology and physics communities have started to appreciate the dramatic catching up of EPR in modern molecular spectroscopy. The reasons for EPR's broad jump ahead are to be found in the remarkable technological breakthroughs in pulsed microwave technology, sweepable cryomagnet fabrication and fast data- acquisition and -handling instrumentation. Indeed, modern EPR is apparently booming now, rather similar to what had happened with NMR 15–20 years earlier. And when nowadays the question "what is magnetic resonance?" is posed again, many chemists, biologists and physicists would probably respond differently from what they had said 20 years ago: They would agree that EPR spectroscopy matured to an attractive sister of NMR spectroscopy, both exhibiting unique and complementary capabilities in elucidating structure and dynamics of complex (bio)chemical systems in the fluid, glassy or solid state.

Why is there such a discrepancy between the technical requirements for pulsed NMR and pulsed EPR? The answer is related to the different time scales of the NMR and EPR phenomena that, in turn, are a consequence of the vastly different magnetic moments of nuclei and electrons (for example for 1H the magnetic moment ratio is 1.5 x 10-3, for 14N it is 1.1 x 10-4). Thus, the time scales are determined by the nuclear and electron resonance frequencies (in the radiofrequency (rf) and microwave (mw) domains, respectively), and the characteristic frequency separations in the respective spectra (Hz versus MHz) and the relaxation times T1, T2 (ms versus ns) are vastly different. Because of the long nuclear T1 and T2 times in diamagnetic molecules, NMR pulses need not be shorter than 10 ms, which do not pose technical problems to generate and detect coherently. The electronic transverse relaxation times (T2), however, are typically in the 100 ns range and, consequently, in EPR the mw pulses have to be as short as a few ns. To generate and detect them coherently poses great technical problems even today. This holds, for example, for the mw sources with adequate output power, for fast mw switches and mixers as well as for fast electronic semiconductor components and computers for controlling the pulse trains, likewise for detecting and handling the transient signals in the ns time scale.

Nowadays, pulse NMR has completely replaced cw NMR, culminating in multidimensional spectroscopy. High-field steady-state cryomagnets and nuclear resonance frequencies up to 1 GHz (for protons) have dramatically improved the detection sensitivity and chemical-shift separations. And even formerly exotic nuclei have now become routinely observable. Despite the spectacular breakthroughs in mm and sub-mm microwave technologies in the last decade, in EPR the cw versus pulse situation is still very different from that in NMR. In fact, the general prognosis is that coexistence between cw and pulse EPR will continue to persist. Which option to choose will be determined entirely by the sample under study. The specific sample properties and relaxation times ultimately dictate the preference for either a cw or pulse experiment to be performed.

Regarding spectral resolution and detection sensitivity, modern EPR and NMR spectroscopies both follow similar strategies: to apply higher and higher static Zeeman fields to separate field-dependent from field-independent spin interactions in the molecular system. By this strategy not only can otherwise overlapping lines be disentangled, but also the population difference and quantum energy of the driven transitions between electron and nuclear spin energy levels will be increased, allowing for the detection of fewer and fewer spins.

1.3 From Basic to Advanced Multifrequency EPR, a Chronological Account

EPR and NMR phenomena were originally observed in radiofrequency spectroscopy experiments employing cw electromagnetic fields, EPR in 1944 by E.K. Zavoisky at Kazan University, NMR in 1946 by E.M. Purcell, H.G. Torrey and R.V. Pound at Harvard and, independently, by F. Bloch, W. Hansen and M.E. Packard at Stanford. These classic NMR experiments were honoured as early as 1952 by the Nobel Prize in Physics to Bloch and Purcell. Zavoisky's discovery of EPR, on the other hand, was only inadequately recognized on the western side of the Iron Curtain – in contrast to the eastern side: In 1957, Zavoisky was awarded the Lenin Prize, the highest sign of recognition in the former USSR for his discovery of electron paramagnetic resonance; and in 1970 the State Committee on Inventions and Discoveries enlisted "The Electron Paramagnetic Resonance Phenomenon" into the State Register of the USSR. It was as late as 1977, when finally Zavoisky was honoured also internationally. Not by a Nobel Prize, though, but at least by the prestigious ISMAR Award of the International Society of Magnetic Resonance, presented at the ISMAR Conference in Banff, Canada, on May 21, 1977 – alas posthumously as he had died October 9, 1976, in Moscow just after having been informed about the decision of the ISMAR Prize Committee (see Figure 1.6).

(Continues...)

Excerpted from High-Field EPR Spectroscopy on Proteins and Their Model Systems by Klaus Möbius, Anton Savitsky. Copyright © 2009 Klaus Möbius and Anton Savitsky. Excerpted by permission of The Royal Society of Chemistry.
All rights reserved. No part of this excerpt may be reproduced or reprinted without permission in writing from the publisher.
Excerpts are provided by Dial-A-Book Inc. solely for the personal use of visitors to this web site.

Edition details

• Publisher: Royal Society of Chemistry
• Publication date: 2008
• Language: English
• ISBN 10: 0854043683
• ISBN 13: 9780854043682
• Binding: Hardcover
• Number of pages: 392