Here we present the use of an unnatural amino acid as IR sensor for conformation-specific detection of calmodulin-binding. Calmodulin (CaM) is an eucaryotic protein that binds four Ca2+ ions and acts as a calcium sensor by translating the Ca2+ signal into cellular processes. The binding of calcium leads to conformational changes of CaM which enable Ca2+/CaM to recognize and bind various target proteins. There are diverse binding partners and binding modes of CaM. For the conformation-specific detection of the CaM-binding to its partner, we incorporated the unnatural amino acid p-azidophenylalanine (AzF) in different positions of calmodulin.
AzF contains the azide vibrational reporter that shows a characteristic IR signal in a defined region of the infrared spectrum where the native protein does not display any IR-signals. The unnatural amino acid can be used to detect changes in the chemical environment. We followed the signal of the AzF probe in various CaM/peptide-complexes by FTIR spectroscopy. This system allows conformation-specific detection of CaM binding to its binding partners. With the crystallization of the CaM/peptide-complexes we want to get more insight into the structures with the incorporated unnatural amino acid.