ABCMdb

ABCC1 p.Phe93Trp

Predicted by SNAP2:	A: N (66%), C: N (53%), D: D (63%), E: N (53%), G: N (53%), H: N (72%), I: N (78%), K: D (59%), L: N (87%), M: N (66%), N: N (57%), P: D (63%), Q: N (61%), R: D (53%), S: N (61%), T: N (61%), V: N (78%), W: D (75%), Y: N (72%),
Predicted by PROVEAN:	A: D, C: D, D: D, E: D, G: D, H: D, I: N, K: D, L: N, M: N, N: D, P: D, Q: D, R: D, S: D, T: D, V: N, W: D, Y: N,

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[hide] Membrane binding of MARCKS-related protein studied... Arch Biochem Biophys. 2000 Aug 15;380(2):380-6. 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., [PMID:10933895]

Abstract [show]
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.

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No. Sentence Comment
2 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 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 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
93 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
97 (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
100 Circles, free Trp; squares, myr MRP F93W. Login to comment
111 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 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 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
118 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 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 (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
142 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
165 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
89 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
95 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
99 (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
102 Circles, free Trp; squares, myr MRP F93W. Login to comment
113 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
116 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
119 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
121 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
135 (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
143 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
166 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

[hide] Mapping the interface between calmodulin and MARCK... Proc Natl Acad Sci U S A. 2000 May 9;97(10):5191-6. Ulrich A, Schmitz AA, Braun T, Yuan T, Vogel HJ, Vergeres G
Mapping the interface between calmodulin and MARCKS-related protein by fluorescence spectroscopy.
Proc Natl Acad Sci U S A. 2000 May 9;97(10):5191-6., [PMID:10792048]

Abstract [show]
MARCKS-related protein (MRP) is a myristoylated protein kinase C substrate that binds calmodulin (CaM) with nanomolar affinity. To obtain structural information on this protein, we have engineered 10 tryptophan residues between positions 89 and 104 in the effector domain, a 24-residue-long amphipathic segment that mediates binding of MRP to CaM. We show that the effector domain is in a polar environment in free MRP, suggesting exposure to water, in agreement with a rod-shaped structure of the protein. The effector domain participates in the binding of MRP to CaM, as judged by the dramatic changes observed in the fluorescent properties of the mutants on complex formation. Intermolecular quenching of the fluorescence emission of the tryptophan residues in MRP by selenomethionine residues engineered in CaM reveals that the N-terminal side of the effector domain contacts the C-terminal domain of CaM, whereas the C-terminal side of the effector domain contacts the N-terminal domain of CaM. Finally, a comparison of the fluorescent properties of the myristoylated and unmyristoylated forms of a construct in which a tryptophan residue was introduced at position 4 close to the myristoylated N terminus of MRP suggests that the lipid moiety is also involved in the interaction of MRP with CaM.

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Sentences [show]
No. Sentence Comment
123 The successful engineering of tryptophan residues in MRP is illustrated with the unmyristoylated form of F93W, a mutant with a conservative substitution (Fig. 2 Upper). Login to comment
124 F93W (dashed line) exhibits a typical tryptophan emission spectrum with a ␭max at 351 nm. Login to comment
189 Fig. 5 illustrates this approach for myristoylated F93W. Login to comment