Exploring Protein Interactions with 23Na Triple-quantum MRS and 1H Chemical Exchange Saturation Transfer MRI

Nuclear magnetic resonance (NMR) allows the non-invasive investigation of proteins using 23Na triple-quantum (TQ) and 1H chemical exchange saturation transfer (CEST) signals. Interactions of sodium ions with macromolecules yield an intracellular sensitive TQ signal. A TQ signal increase has been shown to correlate with the loss of cell viability. However, a deeper understanding of the TQ signal on a cellular level is necessary to determine its capability to serve as a potential biomarker for cell viability. CEST indirectly detects low concentrated organic compounds by their magnetization transfer with water. Protein-based CEST signals have been demonstrated in vitro to be closely connected to the protein folding state and have great potential as a non-invasive diagnostic tool for diseases, like cancer and neurodegenerative diseases. Nonetheless, the detectability of denaturation processes in living cells by CEST NMR remains to be verified experimentally. In the first part of this thesis, a dependence of the TQ signal on the pH and protein folding state was demonstrated using protein solutions. An increase in the availability of negatively charged groups in proteins caused an increase in the TQ signal during pH variation (224.5 +- 25.1%) or protein unfolding (40.7 +- 2.3%). Second, the cellular response to a Na/K-ATPase inhibition was monitored using improved TQ signal detection. The TQ signal increased by 38.9 +- 4.1% and 83.4 +- 8.9% during perfusion with 1 mM ouabain and 0 mM K+ medium, respectively, which agreed with an influx of sodium ions during the Na/K-ATPase inhibition. Finally, the cellular heat shock response was investigated using protein-based CEST signals. Heat shock application resulted in a substantial signal decrease by 8.0 +- 0.4% followed by a continuous signal recovery, which agreed with chaperone-induced refolding of misfolded proteins. The proposed NMR techniques combined with the bioreactor system are promising research tools to non-invasively investigate cellular processes by NMR

High-field/High-frequency EPR Spectroscopy in Protein Research: Principles and Examples

During the last decades, the combined efforts of biologists, chemists, and physicists in developing high-field/high-frequency EPR techniques and applying them to functional proteins have demonstrated that this type of magnetic resonance spectroscopy is particularly powerful for characterizing the structure and dynamics of stable and transient states of proteins in action on biologically relevant time scales ranging from nanoseconds to hours. The review article describes how high-field EPR methodology, in conjunction with site-specific isotope and spin-labeling strategies, is capable of providing new insights into fundamental biological processes. Specifically, we discuss the theoretical and instrumental background of continuous-wave and pulse high-field EPR and the multiple-resonance extensions EDNMR, ENDOR, TRIPLE, ESEEM, PELDOR, and RIDME. Some emphasis is placed on a balanced description of both the historical spadework and the achieved performance of advanced EPR at 95 GHz and 360 GHz. This culminates in a coherent treatment of state-of-the-art research of high-field EPR in terms of both instrumentation development and application to representative protein complexes such as cofactor binding sites in photosynthesis

A Personal Account

In this minireview, we report on our year-long EPR work, such as electron–nuclear double resonance (ENDOR), pulse electron double resonance (PELDOR) and ELDOR-detected NMR (EDNMR) at X-band and W-band microwave frequencies and magnetic fields. This report is dedicated to James S. Hyde and honors his pioneering contributions to the measurement of spin interactions in large (bio)molecules. From these interactions, detailed information is revealed on structure and dynamics of macromolecules embedded in liquid- solution or solid-state environments. New developments in pulsed microwave and sweepable cryomagnet technology as well as ultra-fast electronics for signal data handling and processing have pushed the limits of EPR spectroscopy and its multi-frequency extensions to new horizons concerning sensitivity of detection, selectivity of molecular interactions and time resolution. Among the most important advances is the upgrading of EPR to high magnetic fields, very much in analogy to what happened in NMR. The ongoing progress in EPR spectroscopy is exemplified by reviewing various multi-frequency electron–nuclear double-resonance experiments on organic radicals, light- generated donor–acceptor radical pairs in photosynthesis, and site- specifically nitroxide spin-labeled bacteriorhodopsin, the light-driven proton pump, as well as EDNMR and ENDOR on nitroxides. Signal and resolution enhancements are particularly spectacular for ENDOR, EDNMR and PELDOR on frozen-solution samples at high Zeeman fields. They provide orientation selection for disordered samples approaching single-crystal resolution at canonical g-tensor orientations—even for molecules with small g-anisotropies. Dramatic improvements of EPR detection sensitivity could be achieved, even for short-lived paramagnetic reaction intermediates. Thus, unique structural and dynamic information is revealed that can hardly be obtained by other analytical techniques. Micromolar concentrations of sample molecules have become sufficient to characterize stable and transient reaction intermediates of complex molecular systems—offering exciting applications for physicists, chemists, biochemists and molecular biologists

Biomolecular EPR Meets NMR at High Magnetic Fields

In this review on advanced biomolecular EPR spectroscopy, which addresses both the EPR and NMR communities, considerable emphasis is put on delineating the complementarity of NMR and EPR regarding the measurement of interactions and dynamics of large molecules embedded in fluid-solution or solid-state environments. Our focus is on the characterization of protein structure, dynamics and interactions, using sophisticated EPR spectroscopy methods. New developments in pulsed microwave and sweepable cryomagnet technology as well as ultrafast electronics for signal data handling and processing have pushed the limits of EPR spectroscopy to new horizons reaching millimeter and sub-millimeter wavelengths and 15 T Zeeman fields. Expanding traditional applications to paramagnetic systems, spin-labeling of biomolecules has become a mainstream multifrequency approach in EPR spectroscopy. In the high-frequency/high-field EPR region, sub-micromolar concentrations of nitroxide spin-labeled molecules are now sufficient to characterize reaction intermediates of complex biomolecular processes. This offers promising analytical applications in biochemistry and molecular biology where sample material is often difficult to prepare in sufficient concentration for NMR characterization. For multifrequency EPR experiments on frozen solutions typical sample volumes are of the order of 250 μL (S-band), 150 μL (X-band), 10 μL (Q-band) and 1 μL (W-band). These are orders of magnitude smaller than the sample volumes required for modern liquid- or solid-state NMR spectroscopy. An important additional advantage of EPR over NMR is the ability to detect and characterize even short-lived paramagnetic reaction intermediates (down to a lifetime of a few ns). Electron–nuclear and electron–electron double-resonance techniques such as electron–nuclear double resonance (ENDOR), ELDOR-detected NMR, PELDOR (DEER) further improve the spectroscopic selectivity for the various magnetic interactions and their evolution in the frequency and time domains. PELDOR techniques applied to frozen-solution samples of doubly spin-labeled proteins allow for molecular distance measurements ranging up to about 100 Å. For disordered frozen-solution samples high-field EPR spectroscopy allows greatly improved orientational selection of the molecules within the laboratory axes reference system by means of the anisotropic electron Zeeman interaction. Single-crystal resolution is approached at the canonical g-tensor orientations—even for molecules with very small g-anisotropies. Unique structural, functional, and dynamic information about molecular systems is thus revealed that can hardly be obtained by other analytical techniques. On the other hand, the limitation to systems with unpaired electrons means that EPR is less widely used than NMR. However, this limitation also means that EPR offers greater specificity, since ordinary chemical solvents and matrices do not give rise to EPR in contrast to NMR spectra. Thus, multifrequency EPR spectroscopy plays an important role in better understanding paramagnetic species such as organic and inorganic radicals, transition metal complexes as found in many catalysts or metalloenzymes, transient species such as light-generated spin-correlated radical pairs and triplets occurring in protein complexes of photosynthetic reaction centers, electron-transfer relays, etc. Special attention is drawn to high-field EPR experiments on photosynthetic reaction centers embedded in specific sugar matrices that enable organisms to survive extreme dryness and heat stress by adopting an anhydrobiotic state. After a more general overview on methods and applications of advanced multifrequency EPR spectroscopy, a few representative examples are reviewed to some detail in two Case Studies: (I) High-field ELDOR-detected NMR (EDNMR) as a general method for electron–nuclear hyperfine spectroscopy of nitroxide radical and transition metal containing systems; (II) High-field ENDOR and EDNMR studies of the Oxygen Evolving Complex (OEC) in Photosystem II, which performs water oxidation in photosynthesis, i.e., the light-driven splitting of water into its elemental constituents, which is one of the most important chemical reactions on Earth. View Full-Tex

37th Rocky Mountain Conference on Analytical Chemistry

Final program, abstracts, and information about the 37th annual meeting of the Rocky Mountain Conference on Analytical Chemistry, co-sponsored by the Colorado Section of the American Chemical Society and the Rocky Mountain Section of the Society for Applied Spectroscopy. Held in Denver, Colorado, July 23-27, 1995

38th Rocky Mountain Conference on Analytical Chemistry

Final program, abstracts, and information about the 38th annual meeting of the Rocky Mountain Conference on Analytical Chemistry, co-sponsored by the Colorado Section of the American Chemical Society and the Rocky Mountain Section of the Society for Applied Spectroscopy. Held in Denver, Colorado, July 21-26, 1996