1. Interactions of myristoylated alanine-rich C kinase substrate (MARCKS)-related protein with a novel solid-supported lipid membrane system (TRANSIL).
- Author
-
Schmitz AA, Schleiff E, Röhring C, Loidl-Stahlhofen A, and Vergères G
- Subjects
- Base Sequence, DNA Primers genetics, Escherichia coli genetics, Escherichia coli metabolism, Kinetics, Lipid Bilayers chemistry, Lipid Bilayers metabolism, Membrane Lipids chemistry, Membrane Proteins chemistry, Membrane Proteins genetics, Myristic Acids chemistry, Phospholipids chemistry, Phospholipids metabolism, Protein Binding, Silica Gel, Silicon Dioxide, Temperature, Thermodynamics, Membrane Lipids metabolism, Membrane Proteins metabolism
- Abstract
The determination of partition coefficients is crucial for the biochemical analysis of membrane-based processes, but requires tedious procedures. We have facilitated this analysis using a silica gel coated with a single phospholipid bilayer (TRANSIL) as the membranous phase. We demonstrate the validity of this method using MARCKS-related protein, a 20-kDa member of the MARCKS family (an acronym for myristoylated alanine-rich C kinase substrate). The partition coefficients describing the association of unmyristoylated and myristoylated MARCKS-related protein with membranes of different phospholipid composition are in agreement with previous work with vesicles and show that both the myristoyl moiety and the basic effector domain of MARCKS-related protein mediate the binding. However, no significant cooperativity is observed between these two domains. Interestingly, MARCKS-related protein binds to TRANSIL membranes more strongly at temperatures below their phase-transition temperature. Taking advantage of this property, MARCKS-related protein was purified by phase-transition chromatography, loading Escherichia coli lysates on a TRANSIL column at 4 degrees C and eluting MRP at room temperature. In conclusion, TRANSIL is a versatile tool to determine the affinity of compounds for phospholipid membranes and to purify membrane-bound proteins. TRANSIL should also enable functional studies of protein-ligand and protein-protein interactions at the surface of membranes., (Copyright 1999 Academic Press.)
- Published
- 1999
- Full Text
- View/download PDF