Applied and developed methods
FTIR Difference Spectroscopy
This technique enables the observation of protein action at the
level of single functional groups within huge proteins. The combination of chemical specificity of infrared spectroscopy with an electrochemical approach, is applied to monitor protonation reactions coupled to electron transfer providing essential knowledge’s on structure, function and dynamics of proteins. The mid infrared (MIR) spectral range is well established for the study of the protonation state of individual acidic residues, determination of secondary structure elements. The structural changes are involved in the so called amide I band (1700 to 1600 cm-1) and includes the ν(C=O) vibration of the polypeptide backbone. The amide II region (1580 to 1520 cm-1) includes the C-N stretching and N-H bending vibrations of the polypeptide backbone and may provide information on structural rearrangements as well. Specific contributions from individual amino acids may also be observed, such as the acidic residues above 1700 cm-1.
See for instance Hellwig et al., 1998; Friedrich et al., 2010; Lee et al., 2011.
Far Infrared Spectroscopy
Studies at lower frequencies (below 800 cm-1), however, are just starting to evolve. This spectral range includes interesting contributions from metal-ligand vibrations, like the Fe-S vibrations in iron sulfur clusters and the Fe-N vibrations of hemes, protonation dependent vibrations of aromatic rings as well as breathing modes from the overall protein. At lower frequencies, signals from hydrogen-bonding interaction and ‘breathing’ modes can be expected. Infrared spectroscopy with conventional and synchrotron light sources are being elaborated and applied on the characterization of the cofactors in soluble and membrane proteins from the respiratory chain of different organisms. By coupling with electrochemistry, it becomes possible to follow the changes in metal-ligand vibrations and hydrogen-bonding upon electron transfer.
More details in El Khoury et al., 2010; El Khoury et al., 2011.
Raman Spectroscopy
This technique is complementary to infrared spectroscopy. It consists in focusing a monochromatic light on the sample to be studied and analyzing the scattered light. A great advantage of this technique is the low diffusion of water which allows to work in aqueous solution. By judiciously selecting the excitation frequency (resonance Raman), it is also possible to selectively observe the cofactors of the protein, unhindered by the polypeptide backbone. Hemes such as in cytochrome c for example (see figure) exhibit bands in the 1100-1600 cm-1 region, which give insight into the oxidation and spin state of the iron center. The bridging and terminal modes of the Fe-S clusters can be observed between 300 and 500 cm-1, and are dependent on the cluster environment. Coupling with electrochemistry allows to monitor the environmental changes of the cofactors during electron transfer.
Surface-Enhanced Spectroscopies
The intensity of bands in Raman spectroscopy for molecules adsorbed to rough gold or silver surfaces may be exalted by a factor of 106 (SERRS effect). Similarly, but to a lesser extent, the signals in infrared spectroscopy can be exalted as well (SEIRAS effect). These effects are very interesting for the study of biological molecules such as proteins, because they require lower concentration samples, and interference with water molecules is suppressed. Several studies have shown that the magnitude of the exaltation depends on the metal surface topography and the nature of the bonds between molecules and surface. We are thus developing procedures for the adsorption of membrane proteins on metallic surfaces obtained by chemical reduction methods. Our proteins of interest include complex I which can be reconstituted within a lipid bilayer membrane on the metallic surface (see figure). We are also designing measuring cells allowing the coupling of these surface-enhanced vibrational spectroscopies to electrochemistry. Our main objective is to monitor the changes occuring in the polypeptide chains and cofactors environment when oxidizing or reducing the protein.
More details in Kriegel et al., 2014.
Infrared and Raman Microscopies
Infrared and Raman spectroscopy coupled with microscopy are non-destructive methods which allow to obtain information on the composition and molecular structure of a sample. These methods are increasingly used in the field of medical research as they can characterize the physiopathological condition of a biological sample such as a tissue or a cell. Indeed, each biomolecule has spectral properties well defined in both infrared or Raman, and therefore its vibrational spectrum appears as a true molecular fingerprint. Furthermore, the coupling of infrared and Raman spectroscopy with microscopy combines spectral information with spatial information via the accumulation of thousands of spectra. Our goal is to apply these methods to the study of brain tissue with Alzheimer's disease or other forms of dementia to identify spectroscopic markers at different stages of these diseases. They will help us to understand the molecular mechanisms taking place in the brain and to monitor the effects of different treatments. The figure below shows infrared micrographs of band intensity ratios at 2925 and 2958 cm-1 obtained for mouse brain tissue.
Protein Film Voltammetry
This technique has proven to be a powerful tool to study the thermodynamic and kinetic properties of proteins that are electrochemically connected or ’wired’ to the electrode surface. Membrane proteins are more difficult to manipulate experimentally than soluble proteins, however, their electrochemical properties are of significant interest due to their rich redox catalytic properties. We have developed an immobilization method for membrane proteins based on the use of gold nanoparticles modified with mixtures of thiols. These nanoparticles mediate the communication between the electrode and the redox active centers of the protein and their large surface to volume ratio allows the immobilization of a large amount of protein. We are particularly interested in measuring the electrocatalytic activity of membrane proteins immobilized on such supports.
For more details, see Meyer et al., 2011; Melin et al., 2013; Meyer et al., 2014.