Interactions of myristoylated alanine-rich C kinase substrate (MARCKS)-related protein with a novel solid-supported lipid membrane system (TRANSIL)

The determination of partition coefficients is crucial for the biochemical analysis of membrane-based processes, but requires tedious procedures. We h ave facilitated this analysis using a silica gel coated with a single phosp holipid bilayer (TRANSIL) as the membranous phase. We demonstrate the valid ity of this method using MARCKS-related protein, a 20-kDa member of the MAR CKS family (an acronym for myristoylated alanine-rich C kinase substrate). The partition coefficients describing the association of unmyristoylated an d myristoylated MARCKS-related protein with membranes of different phosphol ipid composition are in agreement with previous work with vesicles and show that both the myristoyl moiety and the basic effector domain of MARCKS-rel ated protein mediate the binding. However, no significant cooperativity is observed between these two domains. Interestingly, MARCKS-related protein b inds to TRANSIL membranes more strongly at temperatures below their phase-t ransition temperature. Taking advantage of this property, MARCKS-related pr otein was purified by phase-transition chromatography, loading Escherichia coli lysates on a TRANSLL column at 4 degrees C and eluting MRP at room tem perature. In conclusion, TRANSIL is a versatile tool to determine the affin ity of compounds for phospholipid membranes and to purify membrane-bound pr oteins. TRANSIL should also enable functional studies of protein-ligand and protein-protein interactions at the surface of membranes. (C) 1999 Academi c Press.