Spectroscopic studies on peptides and proteins with cysteine-containing heme regulatory motifs (HRM)

• Heme binding of peptides containing heme regulatory motifs (HRM) analyzed by EPR spectroscopy
• EPR spectroscopy confirms the importance of the Cys-Pro motif in the HRM
• Spin states of the heme ligand depend on the peptide sequence
• Expression and purification of two proteins (FeoB and GlpF) containing HRM sequences
• Analysis of protein:heme binding by cw-X-Band EPR- and UV-Vis spectroscopy

The role of heme as a cofactor in enzymatic reactions has been studied for a long time and in great detail. Recently it was discovered that heme can also serve as a signalling molecule in cells but so far only few examples of this regulation have been studied. In order to discover new potentially heme-regulated proteins, we screened protein sequence databases for bacterial proteins that contain sequence features like a Cysteine–Proline (CP) motif, which is known for its heme-binding propensity. Based on this search we synthesized a series of these potential heme regulatory motifs (HRMs). We used cw EPR spectroscopy to investigate whether these sequences do indeed bind to heme and if the spin state of heme is changed upon interaction with the peptides. The corresponding proteins of two potential HRMs, FeoB and GlpF, were expressed and purified and their interaction with heme was studied by cw EPR and UV–Visible (UV–Vis) spectroscopy.

Peptides and proteins containing potential heme regulatory motifs (HRMs) are investigated by cw EPR spectroscopy and UV–Vis spectroscopy. The results show that several peptides strongly bind to heme. An interaction with heme is also found for the ferrous iron transport protein FeoB from Escherichia coli.Figure optionsDownload as PowerPoint slide