ABCMdb

PMID: 10933895

Schmitz AA, Ulrich A, Vergeres G
Membrane binding of MARCKS-related protein studied by tryptophan fluorescence spectroscopy.
Arch Biochem Biophys. 2000 Aug 15;380(2):380-6., [PubMed]

Sentences

No. Mutations Sentence Comment

2 ABCC1 p.Phe93Trp 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. Login to comment 4 ABCC1 p.Phe93Trp 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. Login to comment 87 ABCC1 p.Phe93Trp The emission of a 1.25 ␮M solution of a mutant MRP containing a single tryptophan residue (myr MRP F93W) can be detected well with a ␭max at 350 nm (Fig. 1A, squares). Login to comment 89 ABCC1 p.Phe93Trp The emission of a 1.25 òe;M solution of a mutant MRP containing a single tryptophan residue (myr MRP F93W) can be detected well with a òd;max at 350 nm (Fig. 1A, squares). Login to comment 93 ABCC1 p.Phe93Trp ABCC1 p.Phe93Trp ABCC1 p.Phe93Trp Squares, 1.25 ␮M myr MRP F93W; open triangles, 1.25 ␮M myr MRP F93W ϩ 4:1 egg-PC/POPS 100 nm LUVs (1 mM total lipid); filled triangles, 1.25 ␮M myr MRP F93W ϩ 2.5 mM total lipid; open circles, background due to 2.5 mM total lipid; filled circles, buffer. Login to comment 95 ABCC1 p.Phe93Trp ABCC1 p.Phe93Trp ABCC1 p.Phe93Trp Squares, 1.25 òe;M myr MRP F93W; open triangles, 1.25 òe;M myr MRP F93W af9; 4:1 egg-PC/POPS 100 nm LUVs (1 mM total lipid); filled triangles, 1.25 òe;M myr MRP F93W af9; 2.5 mM total lipid; open circles, background due to 2.5 mM total lipid; filled circles, buffer. Login to comment 97 ABCC1 p.Phe93Trp (C) Emission spectra of 1.25 ␮M myr MRP F93W in the presence of increasing concentrations of egg-PC/POPS (4/1) 100 nm LUVs (0-2.5 mM total lipid). Login to comment 99 ABCC1 p.Phe93Trp (C) Emission spectra of 1.25 òe;M myr MRP F93W in the presence of increasing concentrations of egg-PC/POPS (4/1) 100 nm LUVs (0-2.5 mM total lipid). Login to comment 100 ABCC1 p.Phe93Trp Circles, free Trp; squares, myr MRP F93W. Login to comment 102 ABCC1 p.Phe93Trp Circles, free Trp; squares, myr MRP F93W. Login to comment 111 ABCC1 p.Phe93Trp To account for the inner filter effect, we used a parallel titration with the free amino acid tryptophan to correct the spectra of myr MRP F93W. Login to comment 112 ABCC1 p.Phe93Trp ABCC1 p.Phe93Trp Specifically, pure buffer, free Trp (Fig. 1B), and myr MRP F93W (Fig. 1C) were titrated with liposomes using the same titration protocol. Login to comment 113 ABCC1 p.Phe93Trp Specifically, pure buffer, free Trp (Fig. 1B), and myr MRP F93W (Fig. 1C) were titrated with liposomes using the same titration protocol. Login to comment 115 ABCC1 p.Phe93Trp In contrast, the ␭max as well as the intensity of the fluorescence emission of myr MRP F93W change as shown in Fig. 1C and already reported for Fig. 1A: initially, the ␭max changes to lower wavelengths while the intensity increases. Login to comment 116 ABCC1 p.Phe93Trp In contrast, the òd;max as well as the intensity of the fluorescence emission of myr MRP F93W change as shown in Fig. 1C and already reported for Fig. 1A: initially, the òd;max changes to lower wavelengths while the intensity increases. Login to comment 118 ABCC1 p.Phe93Trp Quantification of Membrane Binding Using Trp Fluorescence The data set obtained with free Trp was used to account for the inner filter effect in the data set of myr MRP F93W (Fig. 1B,C). Login to comment 119 ABCC1 p.Phe93Trp Quantification of Membrane Binding Using Trp Fluorescence The data set obtained with free Trp was used to account for the inner filter effect in the data set of myr MRP F93W (Fig. 1B,C). Login to comment 120 ABCC1 p.Phe93Trp These ratios were applied as correction factors on the spectra of myr MRP F93W (Eq. [2]), resulting in the spectra shown in Fig. 2A. Login to comment 121 ABCC1 p.Phe93Trp These ratios were applied as correction factors on the spectra of myr MRP F93W (Eq. [2]), resulting in the spectra shown in Fig. 2A. Login to comment 134 ABCC1 p.Phe93Trp (A) Corrected spectra of 1.25 ␮M myr MRP F93W in the presence of increasing lipid concentrations (0-2.5 mM 4:1 POPC/ POPS LUVs). Login to comment 135 ABCC1 p.Phe93Trp (A) Corrected spectra of 1.25 òe;M myr MRP F93W in the presence of increasing lipid concentrations (0-2.5 mM 4:1 POPC/ POPS LUVs). Login to comment 142 ABCC1 p.Phe93Trp The KP of about 7000 M-1 obtained in this study agree well with the KP for myr MRP F93W determined using lipid bilayer coated silica gel [data not shown; cf. (13)] and with the values of KP determined for wild-type and His-tagged myr MRP using lipid bilayer coated silica gel or sucrose-loaded LUVs [KP ϭ 5000- 8000 M-1 (9); KP ϭ 7300 M-1 (10); KP ϭ 4700 M-1 (13)]. Login to comment 143 ABCC1 p.Phe93Trp The KP of about 7000 Mafa;1 obtained in this study agree well with the KP for myr MRP F93W determined using lipid bilayer coated silica gel [data not shown; cf. (13)] and with the values of KP determined for wild-type and His-tagged myr MRP using lipid bilayer coated silica gel or sucrose-loaded LUVs [KP afd; 5000- 8000 Mafa;1 (9); KP afd; 7300 Mafa;1 (10); KP afd; 4700 Mafa;1 (13)]. Login to comment 165 ABCC1 p.Phe93Trp In contrast, if the fluorophore is situated in the basic effector domain (MRP F93W), binding to charged vesicles (data not shown) or to calmodulin (26) can be detected by changes in ␭max, irrespective of the myristoylation state. Login to comment 166 ABCC1 p.Phe93Trp In contrast, if the fluorophore is situated in the basic effector domain (MRP F93W), binding to charged vesicles (data not shown) or to calmodulin (26) can be detected by changes in òd;max, irrespective of the myristoylation state. Login to comment