ABCA4 p.Lys429Arg
| Predicted by SNAP2: | A: D (53%), C: D (53%), D: D (53%), E: N (57%), F: D (59%), G: N (53%), H: N (61%), I: N (57%), L: N (53%), M: N (53%), N: N (72%), P: D (53%), Q: N (66%), R: N (82%), S: N (61%), T: N (72%), V: N (53%), W: D (75%), Y: D (53%), |
| Predicted by PROVEAN: | A: D, C: D, D: N, E: N, F: D, G: N, H: N, I: D, L: D, M: D, N: N, P: D, Q: N, R: N, S: N, T: D, V: D, W: D, Y: D, |
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[hide] Overexpression, purification, and functional chara... Biochim Biophys Acta. 2003 Feb 17;1610(1):63-76. Cai J, Gros P
Overexpression, purification, and functional characterization of ATP-binding cassette transporters in the yeast, Pichia pastoris.
Biochim Biophys Acta. 2003 Feb 17;1610(1):63-76., 2003-02-17 [PMID:12586381]
Abstract [show]
The ATP-binding cassette (ABC) transporter superfamily is a large gene family that has been highly conserved throughout evolution. The physiological importance of these membrane transporters is highlighted by the large variety of substrates they transport, and by the observation that mutations in many of them cause heritable diseases in human. Likewise, overexpression of certain ABC transporters, such as P-glycoprotein and members of the multidrug resistance associated protein (MRP) family, is associated with multidrug resistance in various cells and organisms. Understanding the structure and molecular mechanisms of transport of the ABC transporters in normal tissues and their possibly altered function in human diseases requires large amounts of purified and active proteins. For this, efficient expression systems are needed. The methylotrophic yeast Pichia pastoris has proven to be an efficient and inexpensive experimental model for high-level expression of many proteins, including ABC transporters. In the present review, we will summarize recent advances on the use of this system for the expression, purification, and functional characterization of P-glycoprotein and two members of the MRP subfamily.
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No. Sentence Comment
188 Studies with mutants at homologous positions in the Walker A (K429R/K1072R) and B (D551N/ D1196N) sequence motifs of NBD1 and NBD2 showed that alterations at either position completely inactivate the ATPase activity of P-gp.
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ABCA4 p.Lys429Arg 12586381:188:62
status: NEW211 Under such conditions the trypsin sensitivity profiles of double P-gp mutants K429R/K1072R and D551N/D1196N and of single mutants K429R, K1072R, and D1196N were very similar and clearly distinct from the wild-type protein.
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ABCA4 p.Lys429Arg 12586381:211:78
status: NEWX
ABCA4 p.Lys429Arg 12586381:211:130
status: NEW[hide] Analysis of catalytic carboxylate mutants E552Q an... Biochemistry. 2003 Nov 11;42(44):12875-85. Carrier I, Julien M, Gros P
Analysis of catalytic carboxylate mutants E552Q and E1197Q suggests asymmetric ATP hydrolysis by the two nucleotide-binding domains of P-glycoprotein.
Biochemistry. 2003 Nov 11;42(44):12875-85., 2003-11-11 [PMID:14596601]
Abstract [show]
In the nucleotide-binding domains (NBDs) of ABC transporters, such as mouse Mdr3 P-glycoprotein (P-gp), an invariant carboxylate residue (E552 in NBD1; E1197 in NBD2) immediately follows the Walker B motif (hyd(4)DE/D). Removal of the negative charge in mutants E552Q and E1197Q abolishes drug-stimulated ATPase activity measured by P(i) release. Surprisingly, drug-stimulated trapping of 8-azido-[alpha-(32)P]ATP is still observed in the mutants in both the presence and absence of the transition-state analogue vanadate (V(i)), and ADP can be recovered from the trapped enzymes. The E552Q and E1197Q mutants show characteristics similar to those of the wild-type (WT) enzyme with respect to 8-azido-[alpha-(32)P]ATP binding and 8-azido-[alpha-(32)P]nucleotide trapping, with the latter being both Mg(2+) and temperature dependent. Importantly, drug-stimulated nucleotide trapping in E552Q is stimulated by V(i) and resembles the WT enzyme, while it is almost completely V(i) insensitive in E1197Q. Similar nucleotide trapping properties are observed when aluminum fluoride or beryllium fluoride is used as an alternate transition-state analogue. Partial proteolytic cleavage of photolabeled enzymes indicates that, in the absence of V(i), nucleotide trapping occurs exclusively at the mutant NBD, whereas in the presence of V(i), nucleotide trapping occurs at both NBDs. Together, these results suggest that there is single-site turnover occurring in the E552Q and E1197Q mutants and that ADP release from the mutant site, or another catalytic step, is impaired in these mutants. Furthermore, our results support a model in which the two NBDs of P-gp are not functionally equivalent.
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No. Sentence Comment
124 This is in agreement with previous studies of catalytic residue mutants of the Walker A and B signature motifs (K429R, K1072R, D551N, and D1196N) which severely affect the catalytic activity of mouse Mdr3 but have little effect on the nucleotide-binding affinity of the protein (49).
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ABCA4 p.Lys429Arg 14596601:124:112
status: NEW