Electron-Spin Resonance Spectroscopy

We are developing and applying electron paramagnetic resonance spectroscopy to study the structure and function of biomolecular systems, particularly at high microwave frequencies. We employ electron-electron and electron-nuclear double resonance techniques to investigate biomolecular structure at the atomic and at the nanometer scale. The latter method allows us to study the assembly of protein complexes, if suited paramagnetic labels can be inserted.

Advanced EPR Methods for Studies of Biomolecules

Overview of different spin-spin interactions in a protein and corresponding EPR techniques used for detection of these interactions, which relate to structural and electronic properties of the investigated system.

Pulsed and high-field magnetic resonance techniques, with their enhanced resolution and sensitivity, have rendered electron paramagnetic resonance (EPR) an essential tool to study the structure and function of large enzymes, proteins and oligonucleotides. Our research is devoted to the development of state-of-the-art EPR methods and instrumentation combined with the investigation of such bio-macromolecules. We routinely employ single and double resonance EPR techniques (electron-nuclear and electron-electron double resonance) at different fields (0.34, 1.2, 3.4 and 9 Tesla) to manipulate interacting spins and to obtain structural information at the atomic and nanometer scale. The methodical aspects of EPR at high microwave frequencies (> 90 GHz) represent our core expertise.

Structural Studies in Biomolecules by double Resonance Techniques

94-GHz [2H] ENDOR spectrum of the NH2Y730·intermediate trapped on the PCET pathway in Y730NH2Y-a2b2 E. coli RNR. The exchangeable protons were replaced by deuterons by H2O/D2O buffer exchange. The spectrum displays the resonances of the deuterons coupled to the radical. The central part of the spectrum (weak coupling region) shows the resonance of a deuteron assigned to a water molecule which is proposed to have mechanistic implications. The structure on the right represents the optimized DFT cluster model with distances and orientations to the exchangeable protons consistent with the ENDOR data. The upper inset shows the current model for the long-range (~ 35 Å), reversible PCET between Y122 in the b2 and C439 in the a2 of E. coli RNR (2). 

Local structure at atomic resolution can be achieved by detecting the interaction of electron spins with surrounding magnetic nuclei in electron-nuclear double resonance (ENDOR). This method permits one to determine the distance as well as the orientation of nuclear spins with respect to a paramagnetic centre. Particularly, hydrogen bond interactions in the active states of an enzyme can be detected that are not easily accessible by other techniques such as X-ray or NMR. The combination of ENDOR with density functional cluster calculations (coll. with Prof. Frank Neese) provides a unique tool to elucidate the structure of complex intermediate states during enzymatic catalysis. A recent example for the application of this technique at 94 GHz is illustrated in the figure below.

Nano-meter distances and orientations in protein and nucleic acids by high-field pulse EPR. The pulse EPR technique, called DEER or PELDOR (electron-electron double resonance), can be used to detect the interaction between two unpaired electron spins and to measure their inter-spin distance. The method applied at high-fields contains also information about the relative orientation of paramagnetic species with a precision of a few degrees. For this aim, we are examining the performance of different paramagnetic probes combined with the home built dual mode resonator and in-house developed analysis routines to reconstruct the local geometry of radicals in a pair. In diamagnetic biomolecules the method requires the introduction of paramagnetic spin labels that are possibly small and non-interfering with the molecular structure. 1-oxy-2,2,5,5-tetra-methylpyrrolynil-3-methyl (MTSL) is the most common spin probe for proteins as it can be easily attached to cysteine residues. For example, we are employing this label for structural studies of alpha-synuclein fibrils. A new more rigid spin label (i.e. the TOPP label) has been recently synthesized and investigated in collaboration with the group of Prof. U. Diederichsen at the University of Göttingen. New labels for RNA structures have been developed in collaboration with the group of Dr. C. Höbartner. The labeling of RNA has permitted to study conformational changes in RNA secondary structures.

Dynamic Nuclear Polarization (DNP)

The large magnetization of an electron spin can be used to polarize nuclei via dynamic nuclear polarization, a method which could potentially enhance the sensitivity of solution NMR on biological systems by orders of magnitude. In the past years, we have defined the physical parameters that govern the polarisation transfer in solution to water molecules with different types of paramagnetic polarizers. We have implemented lowand medium-field DNP spectrometers operating at 0.34 T and 3.4 T, which enabled us to achieve very large DNP enhancements for such fields. Investigations are in progress to use pulse microwave excitation that accelerates the DNP transfer kinetics. The successful low field results have prompted the design of a new shuttle DNP spectrometer, in which the sample is polarized at 0.34 T (X-band) and the NMR is detected at 14 T (collab. with NMR Dept. of the MPIbpc and Bruker BioSpin, EU Design Study Bio-DNP). An optimized spectrometer, which is designed to minimize polarization losses during the sample transfer and to polarize large biomolecules with inherently short relaxation times, is under construction.

Instrumental Development at 94 and 263 GHz

Dual mode resonator for 94 GHz. Recently, we have designed and constructed a new dual mode resonator that can be tuned over a frequency range up to about 1 GHz. The resonator allows us to perform PELDOR/DEER experiments at 94 GHz with high sensitivity and covering the whole spectral range of a nitroxide radical (spin label) in frozen solution. The resonator can also be used for other experiments such as saturation transfer and ELDOR in liquid solution of nitroxide radicals for mechanistic DNP inverstigations.

Quasi-optical pulse EPR at 263 GHz. In December 2011 we have installed the world-wide second commercial 263 GHz pulsed EPR spectrometer that uses a quasi-optical microwave bridge. The instrument will be tested and optimized for studies of biological samples, particularly for two purposes: 1) to record high-resolution EPR spectra of radical intermediates during enzymatic catalysis in combination with rapid freeze quenching techniques and 2) for distance measurements with high sensitivity towards long inter-spin distances (> 5 nm). 

Enzyme catalysis

Long-range electron transfer pathway in class I ribonucleotide reductases.

Ribonucleotide Reductases (RNRs).In the past two decades, it has been recognized that several biological processes, in particular enzymatic reactions, occur over paramagnetic intermediates that are generated via redox reactions or electron/radical transfer. RNRs enzymes, which catalyze the reduction of RNA to DNA building blocks in every living organism, provide a paradigm for enzymatic mechanisms involving radical chemistry. In a long-standing collaboration with the group of J. Stubbe (MIT) we have been employing a wide repertoire of EPR techniques to elucidate several steps in the catalytic cycle. Currently, we are investigating the long-range (3.5 nm) proton-coupled electron transfer (PCET) between the RNR subunits using site-specifically incorporated unnatural amino acids as radical traps. With high field EPR and ENDOR we are mapping the hydrogen bond network proposed to be involved in PCET mechanism. The results will be essential to formulate a mechanism for a long-range PCET.

Heterodisulfide reductase (HDR).HDR is a key enzyme in the energy metabolism of methanogenic archaea. Based on the current knowledge of methanogenesis, it has been recently postulated that HDR-enzyme complexes could play a new role in energy conservation processes of many different organisms. By means of W-band (95 GHz) and X-band (9 GHz) ENDOR spectroscopy we have demonstrated that HDR contains a unique FeS cluster, which functions as a catalytic centre and directly binds substrate to carry out substrate chemistry. In a collaborative effort with several research groups in Germany (DFG network SPP1319), we are currently elucidating the structural details of the interaction between the cluster and the substrate as well as the unusual binding motif of the cluster.

So-called psi factors (fatty acid based substances) regulate the balance between fungal life cycles and are produced by psi-factor producing oxygenases (Ppo). In a new collaboration with the Dept. of Plant Biochemistry (I. Feussner, Univ. Göttingen) and supported by the IRTG 1422, we have characterized two heme domains discovered in the monomeric subunit of the tetrameric PpoA enzyme. Experiments on samples prepared by freeze quenching the enzyme with the substrate, i.e. PpoA with either (8R)-HPODE or linoleic acid, showed the formation of several tyrosyl radical species, presumably formed on different monomeric subunits. The results provide the first evidence for an enzymatic mechanism involving amino acid radicals and further studies are planned to elucidate more intermediate steps.

Press Releases & Research News

A research team headed by Marina Bennati at the MPI for Biophysical Chemistry, together with colleagues at the University of Florence (Italy), has shown that 13C NMR spectroscopy signals can be strongly enhanced in solution by resonant microwave irradiation of a nitroxide organic radical used as polarizer for 13C nuclei. The new method shows up to 1000-fold improvements in sensitivity and promises to study small molecules and metabolites in much greater detail. more

Electron spins as probes for biomolecules
Unpaired electrons possess a magnetic moment, which is about three orders of magnitude larger than the one of a proton. This moment can be employed as a highly sensitive probe in EPR spectroscopic investigations to gain structural information at the atomic up to the nanometer scale. The experiments provide insights into structural changes of biomolecules during their functional states. We have developed and implemented multi-frequency EPR methodologies to investigate enzymatic reactions in proteins and oligonucleotides. (in German)
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