Contact

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Marina Bennati
Research Group Leader, Professor at the University of Göttingen
Phone:+49 551 201-1911Fax:+49 551 201-1383

Curriculum Vitae

Monika Frömel
Assistant
Phone:+49 551 201-1905

Marina Bennati

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

<span>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.</span> Zoom Image
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

<p>94-GHz [<sup>2</sup>H] ENDOR spectrum of the NH<sub>2</sub>Y<sub>730</sub><sup>·</sup>intermediate trapped on the PCET pathway in Y<sub>730</sub>NH<sub>2</sub>Y-a2b2 <em>E. coli</em> RNR. The exchangeable protons were replaced by deuterons by H<sub>2</sub>O/D<sub>2</sub>O 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 Y<sub>122</sub> in the b2 and C<sub>439</sub> in the a2 of <em>E. coli</em> RNR (2). </p> Zoom Image

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). 

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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.

 
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