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Expertise in Structural Biology

EPR spectroscopy

Is the method of choice to study paramagnetic molecules and complexes with one or more unpaired electrons, like radicals, radical pairs, triplet states and transition metal complexes. These species are often reactive and have a short lifetime, they occur as intermediates in many (bio)chemical reactions. Modern EPR uses pulsed microwave radiation and variable magnetic fields. The method is very sensitive and has a time resolution in the nanosecond range. Besides the detection of paramagnetic species, it allows a detailed characterization of the electronic structure of the species studies

ENDOR spectroscopy

This technique uses both microwave (EPR) and radio frequency (NMR) radiation to enable a higher resolution than in conventional EPR experiments and a much higher sensitivity than in NMR performed at paramagnetic systems. ENDOR allows the sensitive detection of the nuclear interactions (hyperfine and quadrupole couplings) in such species and is able to deliver information about the electron spin distribution and the wavefunctions of the system (electronic structure).

PELDOR

This is a pulsed electron-electron double resonance method by which distances of spin centers and dynamical properties of the molecules can be obtained. When performed at high enough magnetic fields orientational information is also accessible. Through the method of (electron) spin labelling of molecules (e.g. proteins) PELDOR can also be applied to diamagnetic systems. Multiple labelling can, in principle, be used to determine the 3-dimensional structure of biological macromolecules.

MCD spectroscopy

In magnetic circular dichroism (MCD) spectroscopy, a sample is inserted into a strong magnetic field which causes the splitting of certain degenerate electronic states according to the Zeeman effect. The resulting transitions absorb left and right circular polarized light differently giving insight into the electronic states existing in the molecule. Variation of the magnetic field allows the determination of the magnetic properties of the chromophore. In cases where the ground state is splitted by the Zeeman effect (so called C-terms), decreased temperatures will populate the lower state which allows the assignment of the C-term. In contrast to EPR spectroscopy, MCD is also applicable to systems without unpaired electrons (diamagnetic systems) and helps to assess their electronic properties.

Microcalorimetry

In a microcalorimeter, a titration of one molecule, for example a protein in solution, is performed with a second molecule that reacts with the first protein at a given temperature. The reaction generates or consumes heat (exothermic or endothermic reaction), which can be monitored directly. Microcalorimetry is a very powerful method because it can provide a number of parameters in parallel, i.e., the enthalpy change ΔH, the binding affinity, and the stoichiometry. From these parameters, the entropy change ΔS, and the change in Gibbs energy ΔG can be directly calculated.

Mößbauer spectroscopy

is used in bioinorganic chemistry to probe the charge and spin distribution around iron centers. It is a nuclear method which is highly selective for ⁵⁷Fe, a stabile isotope that is 2% naturally abundant. The light of a radioactive source (⁵⁷Co, 14 keV γ-rays) is resonantly absorbed by the iron nuclei of the sample. In zero applied field the Mößbauer spectrum consists of a single pair of lines which is called 'quadrupole doublet'. The center of the doublet is referred to as 'isomer shift' and the splitting is known a 'quadrupole splitting'. The isomer shift is the key parameter for the assignment of the valence state, because it senses the electric charge density at the nucleus. The quadrupole indicates details of the electron configuration, i. e. the population of valence orbitals, and the type of covalent chemical bonds, because it senses the charge asymmetry. Applied field measurements yield complex magnetic hyperfine spectra which probe the paramagnetic properties of the samples. The information is complementary to that of EPR spectroscopy, because Mößbauer spectroscopy is not focussed on half-integer spin states but senses any spin and valence state of iron. Biological samples usually have to be enriched to about 30% of ⁵⁷Fe and the sample should be at least 0.3 mM, about 500 µl.

Vibrational spectroscopy

In this technique, light is absorbed by the sample molecule which excites the vibrations of the protein. In case of FTIR spectroscopy, the energy of the light (infra red light, IR) can be absorbed by vibrations that are possible to carry out by the bonds in the molecule of interest. This provides specific information on the presence and the energy of particular bonds. Fourier transformation (FT) allows the rapid pulsing of the full IR spectrum and subsequent deconvolution of the vibrations, thus allowing the observation of dynamic processes. In Raman spectroscopy the inelastic scattering of a molecule is observed upon perpendicular irradiation of the sample by laser light (Raman effect). Both methods provide complementary information.

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