Inhibitor and peptide binding to calmodulin characterized by high pressure Fourier transform infrared spectroscopy and F örster resonance energy transfer

Publication date: Available online 17 March 2018 Source:Biochimica et Biophysica Acta (BBA) - Proteins and Proteomics Author(s): Süleyman Cinar, Claus Czeslik We compare the binding of an inhibitor with that of a natural peptide to Ca2+ saturated calmodulin (holo-CaM). As inhibitor we have chosen trifluoperazine (TFP) that is inducing a huge conformational change of holo-CaM from the open dumbbell-shaped to the closed globular conformation upon binding. On the other hand, melittin is used as model peptide, which is a well-known natural binding partner of holo-CaM. The experiments are carried out as a function of pressure to reveal the contribution of volume or packing effects to the stability of the calmodulin-ligand complexes. From high-pressure Fourier transform infrared (FTIR) spectroscopy, we find that the holo-CaM/TFP complex has a much higher pressure stability than the holo-CaM/melittin complex. Although the analysis of the secondary structure of holo-CaM (without and with ligand) indicates no major changes up to several kbar, pressure-induced exposure of α-helices to water is most pronounced for holo-CaM without ligand, followed by holo-CaM/melittin and then holo-CaM/TFP. Moreover, structural pressure resistance of the holo-CaM/TFP complex in comparison with the holo-CaM/melittin complex is also clearly visible by higher Ca2+ affinity. Förster resonance energy transfer (FRET) from the Tyr residues of holo-CaM to the Trp residue of melittin even suggests some pa...

Source: Biochimica et Biophysica Acta (BBA) Proteins and Proteomics - March 19, 2018 Category: Biochemistry Source Type: research