Membrane binding of MARCKS-related protein studied by tryptophan fluorescence spectroscopy.
MARCKS-related protein (MRP) is a peripheral membrane protein whose binding to membranes is mediated by the N-terminal myristoyl moiety and a central, highly basic effector domain. MRP mediates cross-talk between protein kinase C and calmodulin and is thought to link the actin cytoskeleton to the plasma membrane. Since MRP contains no tryptophan residues, we mutated a phenylalanine in the effector domain to tryptophan (MRP F93W) and used fluorescence spectroscopy to monitor binding of the protein to phospholipid vesicles. We report in detail the evaluation procedure necessary to extract quantitative information from the raw data. The spectra of MRP F93W obtained in the presence of increasing amounts of lipid crossed at an isosbestic point, indicating a simple transition between two states: free and membrane-bound protein. The change in fluorescence toward values typical of a more hydrophobic environment was used to quantify membrane binding. The partition coefficient agreed well with values obtained previously by other methods. To study the interaction of the N-terminus of MRP with membranes, a tryptophan residue was also introduced at position 4 (MRP S4W). Our data suggest that only the myristoylated N-terminus interacted with liposomes. These results demonstrate the versatility of site-directed incorporation of tryptophan residues to study protein-membrane interactions.
Department of Biophysical Chemistry, Biozentrum of the University of Basel, Klingelbergstrasse 70, Basel, CH-4056, Switzerland.
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Schmitz
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Ulrich
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Vergères
D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002462:Cell Membrane / Q000378:metabolism; D066298:In Vitro Techniques; D008081:Liposomes; D008565:Membrane Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D019814:Myristic Acid / Q000378:metabolism; D011485:Protein Binding; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry
D008081:Liposomes; D008565:Membrane Proteins; D011994:Recombinant Proteins; D019814:Myristic Acid; D014364:Tryptophan