Homepage PD Carsten Kötting

RUB » Faculty of Biology and Biotechnology » X-ray structure analysis of proteins » Projektgruppe Koetting

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We study proteins using spectroscopic methods. Our focus is on infrared spectroscopy. Almost all molecules interact with infrared radiation. IR spectroscopy can therefore, in principle, be performed on any protein without the need for labeling. Information regarding, for example, the secondary structure can be obtained from the protein’s IR spectrum. To investigate the reaction mechanisms of proteins at the atomic level, we use difference spectroscopy. Here, the difference between the spectra of two different protein states is calculated. This can be performed either with immobilized proteins using ATR-FTIR spectroscopy or with the aid of time-resolved IR spectroscopy. For time-resolved spectroscopy, the protein reactions are initiated by a laser pulse.

Surface Chemistry

For ATR-FTIR spectroscopy, proteins must be immobilized on the ATR crystal. We were involved in the development of a patented surface chemistry for this purpose. Proteins can be covalently bound via maleimide coupling to cysteines or via NHS chemistry. Reversible binding is also possible using protein tags, such as E7/Im7.

Model Compounds

To correctly interpret the obtained difference spectra, the bands must be assigned to specific functional groups. For example, this can be done using isotope labeling. Further information can be obtained through quantum chemical calculations. For this purpose, we collaborate with Till Rudack’s group. By combining experiment and theory, biomolecular processes can be understood much more precisely. To improve this interplay, Till Rudack continues to develop the theoretical methods. These must then be validated with experimental data. This is done using optimal model compounds, whose experimental spectra we record.

GTPases

GTPases are molecular switches that regulate numerous processes within cells. The Ras protein is the subject of the most intensive research; it transmits the signal for cell growth to the cell nucleus. Oncogenic point mutations of Ras in the GTP-binding site prevent the hydrolysis of GTP to GDP, resulting in the uncontrolled generation of a permanent growth signal. This contributes to the development of cancer.
Since GTPases do not exhibit a photoreaction, caged GTP is used here for time-resolved measurements; a UV laser pulse releases the GTP, allowing the GTPase reaction to be studied. Alternatively, Ras is immobilized in a flow cell, and a difference spectrum is generated, for example, by ligand exchange.

Channelrhodopsins

In optogenetics, cells such as neurons are activated by light with high temporal and spatial resolution. Microbial rhodopsins are typically used for this purpose. To improve their application, we are investigating the reaction mechanisms of cation- and anion-conducting channel rhodopsins (CCRs and ACRs).