Abstract

The Cholesterol Recognition/Interaction Amino Acid Consensus (CRAC) motif is a critical structural element in many membrane proteins, mediating specific interactions with cholesterol and influencing cellular functions. This study investigates the biophysical interaction of synthetic peptides containing CRAC motifs with model lipid membranes of varying compositions, a key step in understanding the molecular mechanism of cholesterol-dependent protein activity. Using a combination of biophysical techniques, such as fluorescence spectroscopy and molecular dynamics simulations, we analyzed the peptides' affinity, insertion depth, and effect on membrane fluidity and organization. Our results demonstrate that the interaction is highly dependent on the membrane's lipid composition, particularly the concentration of cholesterol and the presence of specific phospholipids. The CRAC-containing peptides showed a preferential affinity for cholesterol-rich domains, suggesting a mechanism for localizing and modulating protein function within membrane rafts. These findings provide crucial insights into the fundamental principles governing peptide-lipid interactions and the role of CRAC motifs in regulating membrane protein function and cellular signaling pathways.