PMID: 10820025

Booth CL, Pulaski L, Gottesman MM, Pastan I
Analysis of the properties of the N-terminal nucleotide-binding domain of human P-glycoprotein.
Biochemistry. 2000 May 9;39(18):5518-26., 2000-05-09 [PubMed]
Sentences
No. Mutations Sentence Comment
8 ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:8:8
status: NEW
view ABCB1 p.Asp555Asn details
Using a D555N mutated protein, we found that the conserved aspartate residue in the Walker B motif plays a role in magnesium-enhanced ATP-binding. Login to comment
45 ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:45:24
status: NEW
view ABCB1 p.Asp555Asn details
pTM1-MDR1 and pTM1-MDR1-D555N were supplied by Dr. Christine Hrycyna (25, 29). Login to comment
55 ABCB1 p.Cys431Ala
X
ABCB1 p.Cys431Ala 10820025:55:100
status: NEW
view ABCB1 p.Cys431Ala details
The resulting expression vector was used as a template for site-directed mutagenesis to introduce a C431A mutation. Login to comment
58 ABCB1 p.Cys431Ala
X
ABCB1 p.Cys431Ala 10820025:58:28
status: NEW
view ABCB1 p.Cys431Ala details
The coding sequence for the C431A mutant primer was 5'-GTTGGAAACAGTG- GCGCTGGGAAGAGCACA-3'. Login to comment
61 ABCB1 p.Cys431Ala
X
ABCB1 p.Cys431Ala 10820025:61:83
status: NEW
view ABCB1 p.Cys431Ala details
ABCB1 p.Cys431Ala
X
ABCB1 p.Cys431Ala 10820025:61:128
status: NEW
view ABCB1 p.Cys431Ala details
The plasmid obtained contained a sequence encoding MDR1 amino acids 358-707 with a C431A mutation and was labeled pET- NBD1MDR1-C431A (358-707). Login to comment
62 ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:62:301
status: NEW
view ABCB1 p.Asp555Asn details
ABCB1 p.Cys431Ala
X
ABCB1 p.Cys431Ala 10820025:62:157
status: NEW
view ABCB1 p.Cys431Ala details
ABCB1 p.Cys431Ala
X
ABCB1 p.Cys431Ala 10820025:62:295
status: NEW
view ABCB1 p.Cys431Ala details
ABCB1 p.Cys431Ala
X
ABCB1 p.Cys431Ala 10820025:62:439
status: NEW
view ABCB1 p.Cys431Ala details
ABCB1 p.Cys431Ala
X
ABCB1 p.Cys431Ala 10820025:62:578
status: NEW
view ABCB1 p.Cys431Ala details
ABCB1 p.Cys431Ala
X
ABCB1 p.Cys431Ala 10820025:62:716
status: NEW
view ABCB1 p.Cys431Ala details
ABCB1 p.Cys431Ala
X
ABCB1 p.Cys431Ala 10820025:62:836
status: NEW
view ABCB1 p.Cys431Ala details
ABCB1 p.Cys431Ala
X
ABCB1 p.Cys431Ala 10820025:62:961
status: NEW
view ABCB1 p.Cys431Ala details
ABCB1 p.Cys431Ala
X
ABCB1 p.Cys431Ala 10820025:62:1086
status: NEW
view ABCB1 p.Cys431Ala details
ABCB1 p.Cys431Ala
X
ABCB1 p.Cys431Ala 10820025:62:1211
status: NEW
view ABCB1 p.Cys431Ala details
A plasmid, containing a Table 1: Cloning Summary for Expression Vectors plasmid name cloning vector PCR primers (5' f 3')a restriction enzymesb pET-NBD1MDR1-C431A (358-707) pET3a A: GCAATACATATGGCAAGAGGAGCAGCTTATGAAATCTTC A: NdeI B: AAAGGATCCTCATTCAGTTAAATTTAGCTTCATAATCCT B: BamHI pET-NBD1MDR1-C431A/D555N (358-707) pET3a A: GCAATACATATGGCAAGAGGAGCAGCTTATGAAATCTTC A: NdeI B: AAAGGATCCTCATTCAGTTAAATTTAGCTTCATAATCCT B: BamHI pET-NBD1MDR1-C431A (412-574) pET23a A: GTTAAGATGCATATGGGCCTGAACCTGAAGGTGCAGAGT A: NdeI B: ACCTTTGAATTCTCAATCCAGAGCCACCTGAACCACTGC B: EcoRI pET-NBD1MDR1-C431A (358-590) pET3a A: GCAATACATATGGCAAGAGGAGCAGCTTATGAAATCTTC A: NdeI B: ATTGGATCCTCAAGACAAACGATGAGCTATCACAATGGT B: BamHI pET-NBD1MDR1-C431A (358-635) pET23a A: GAGATATACATATGGCAAGAGGAGCA A: NdeI B: TCTCCTCGAGCTAAACTTCATTTCCTGCTGTCTG B: XhoI pET-NBD1MDR1-C431A (409-635) pET23a A: GAGACATATGATCTTGAAGGGCCTGAACCTG A: NdeI B: TCTCCTCGAGCTAAACTTCATTTCCTGCTGTCTG B: XhoI pET-NBD1MDR1-C431A (375-707) pET23a A: GAGACATATGATTGACAGCTATTCGAAGAGT A: NdeI B: TCTCCTCGAGCTATTCAGTTAAATTTAGCTTCAT B: XhoI pET-NBD1MDR1-C431A (392-707) pET23a A: GAGACATATGTTGGAATTCAGAAATGTTCAC A: NdeI B: TCTCCTCGAGCTATTCAGTTAAATTTAGCTTCAT B: XhoI pET-NBD1MDR1-C431A (348-635) pET23a A: GAGACATATGGCATCTCCAAGCATTGAAGCATTTGCAAAT- GCAAGAGGAGCAGCT A: NdeI B: TCTCCTCGAGCTAAACTTCATTTCCTGCTGTCTG B: XhoI a A, forward primer; B, reverse primer. Login to comment
64 ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:64:63
status: NEW
view ABCB1 p.Asp555Asn details
ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:64:132
status: NEW
view ABCB1 p.Asp555Asn details
ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:64:185
status: NEW
view ABCB1 p.Asp555Asn details
ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:64:261
status: NEW
view ABCB1 p.Asp555Asn details
ABCB1 p.Cys431Ala
X
ABCB1 p.Cys431Ala 10820025:64:53
status: NEW
view ABCB1 p.Cys431Ala details
ABCB1 p.Cys431Ala
X
ABCB1 p.Cys431Ala 10820025:64:254
status: NEW
view ABCB1 p.Cys431Ala details
sequence encoding MDR1 amino acids 358-707 with both C431A and D555N mutations, was prepared as described above, with the pTM1-MDR1-D555N vector bearing the MDR1 coding sequence with a D555N mutation as the primary template, and was labeled pET-NBD1MDR1-C431A/ D555N (358-707). Login to comment
65 ABCB1 p.Cys431Ala
X
ABCB1 p.Cys431Ala 10820025:65:13
status: NEW
view ABCB1 p.Cys431Ala details
pET-NBD1MDR1-C431A was used as the template for construction of subsequent expression vectors as indicated in Table 1. Login to comment
92 ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:92:43
status: NEW
view ABCB1 p.Asp555Asn details
Sample consisted of NBD1 (358-707) or NBD1 D555N (358-707) proteins (~250 µg/mL) in 50 mM potassium phosphate/50 mM NaCl (pH 7.0). Login to comment
104 ABCB1 p.Cys431Ala
X
ABCB1 p.Cys431Ala 10820025:104:113
status: NEW
view ABCB1 p.Cys431Ala details
ABCB1 p.Cys431Ala
X
ABCB1 p.Cys431Ala 10820025:104:153
status: NEW
view ABCB1 p.Cys431Ala details
Recombinant proteins, encompassing amino acids 412-574, 358-590, and 358-707 of human P-glycoprotein each with a C431A mutation, encoded by pET-NBD1MDR1-C431A-based plasmids (Table 1) were expressed and purified from E. coli, purified from inclusion bodies under denaturing conditions, and renatured by rapid dilution. Login to comment
105 ABCB1 p.Cys431Ala
X
ABCB1 p.Cys431Ala 10820025:105:4
status: NEW
view ABCB1 p.Cys431Ala details
The C431A mutant was used to prevent disulfide interactions. Login to comment
129 ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:129:174
status: NEW
view ABCB1 p.Asp555Asn details
(A) Expression of NBD1 proteins before (lanes 1, 3, 5, and 7) and after (lanes 2, 4, 6, and 8) IPTG induction. NBD1 proteins were as follows: NBD1 (358-707), lanes 1-2; NBD1 D555N (358-707), lanes 3 and 4; NBD1 (412-574), lanes 5 and 6; and NBD1 (358-590), lanes 7 and 8. Login to comment
130 ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:130:111
status: NEW
view ABCB1 p.Asp555Asn details
(B) Purified NBD1 proteins were identified by colloidal blue staining as follows: NBD1 (358-707), lane 1; NBD1 D555N (358-707), lane 2; NBD1 (412-574), lane 3; and NBD1 (358-590), lane 4. Login to comment
133 ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:133:104
status: NEW
view ABCB1 p.Asp555Asn details
NBD1 proteins were as follows: (A) NBD1 (358-707), (B) NBD1 (358-590), (C) NBD1 (412-574), and (D) NBD1 D555N (358-707). Login to comment
136 ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:136:0
status: NEW
view ABCB1 p.Asp555Asn details
D555N Amino Acid Substitution in the Walker B Consensus Sequence Results in an Alteration in Mg2+ -Enhanced ATP Binding. Login to comment
137 ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:137:116
status: NEW
view ABCB1 p.Asp555Asn details
ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:137:117
status: NEW
view ABCB1 p.Asp555Asn details
To evaluate the effect of Mg2+ on [R-32 P]-8-azido-ATP labeling, the NBD1 (358-707) protein was mutated at position 555 from aspartate to asparagine in the Walker B consensus motif (36). Login to comment
139 ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:139:15
status: NEW
view ABCB1 p.Asp555Asn details
Thus, the NBD1 D555N (358-707) protein serves as a specificity control in the present work. Login to comment
140 ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:140:36
status: NEW
view ABCB1 p.Asp555Asn details
Expression and purification of NBD1 D555N (358-707) protein is shown in Figure 1. Login to comment
141 ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:141:22
status: NEW
view ABCB1 p.Asp555Asn details
As demonstrated, NBD1 D555N (358-707) protein was localized in the inclusion-body protein and was purified by the methods described previously. Login to comment
142 ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:142:94
status: NEW
view ABCB1 p.Asp555Asn details
Unlike the wild-type NBD1 (358-707) protein, [R-32 P]-8-azido-ATP labeling of the mutant NBD1 D555N (358-707) protein was greater in the absence of MgSO4 than in the presence of MgSO4 (Figure 2D). Login to comment
143 ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:143:84
status: NEW
view ABCB1 p.Asp555Asn details
In fact, in the presence of MgSO4 the ability of [R-32 P]-8-azido-ATP to label NBD1 D555N (358-707) protein was impaired severely (Figure 2D). Login to comment
144 ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:144:70
status: NEW
view ABCB1 p.Asp555Asn details
Incubation of [R-32P]-8-azido-ATP with NBD1 (358-707) protein or NBD1 D555N (358-707) protein in the presence of increasing concentrations of MgSO4 resulted in an increase in labeling with wild-type protein and a decrease in labeling with mutant protein (data not shown). Login to comment
145 ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:145:36
status: NEW
view ABCB1 p.Asp555Asn details
The specificity of labeling of NBD1 D555N (358-707) protein was explored by incubating the protein with [R-32 P]-8-azido-ATP in the presence of increasing concentrations of ATP in the absence of MgSO4. Login to comment
148 ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:148:36
status: NEW
view ABCB1 p.Asp555Asn details
Analysis of NBD1 (358-707) and NBD1 D555N (358-707) proteins by CD was performed in order to assess the secondary structural components and to evaluate the effect of Mg2+. Login to comment
149 ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:149:157
status: NEW
view ABCB1 p.Asp555Asn details
The predicted secondary structural components of these proteins were 25% R-helix, 23% -strand, and 19% -turn; no differences between NBD1 (358-707) and NBD1 D555N (358-707) proteins were demonstrated (data not shown). Login to comment
150 ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:150:82
status: NEW
view ABCB1 p.Asp555Asn details
Similarly, the predicted secondary structural elements of NBD1 (358-707) and NBD1 D555N (358-707) proteins in the presence of 2 mM MgSO4 were not different than in the absence of MgSO4 (data not shown). Login to comment
151 ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:151:95
status: NEW
view ABCB1 p.Asp555Asn details
A temperature melt was performed to determine the thermal stability of NBD1 (358-707) and NBD1 D555N (358-707) proteins. Login to comment
206 ABCB1 p.Cys431Ala
X
ABCB1 p.Cys431Ala 10820025:206:55
status: NEW
view ABCB1 p.Cys431Ala details
In the present work, human P-glycoprotein cDNAs with a C431A mutation were utilized to express a series of recombinant proteins containing NBD1 in E. coli. Login to comment
220 ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:220:57
status: NEW
view ABCB1 p.Asp555Asn details
Secondary structural analysis of NBD1 (358-707) and NBD1 D555N (358-707) proteins by circular dichroism showed 25% R-helix, 23% -strand, and 19% -turn with no significant difference in secondary structure between the two proteins. Login to comment
224 ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:224:88
status: NEW
view ABCB1 p.Asp555Asn details
More interesting was the lack of secondary structural change of NBD1 (358-707) and NBD1 D555N (358-707) proteins in the presence of Mg2+ . Login to comment
249 ABCB1 p.Asp555Asn
X
ABCB1 p.Asp555Asn 10820025:249:14
status: NEW
view ABCB1 p.Asp555Asn details
ABCB1 p.Cys431Ala
X
ABCB1 p.Cys431Ala 10820025:249:4
status: NEW
view ABCB1 p.Cys431Ala details
The C431A and D555N mutations within the Walker A and Walker B motifs, respectively, are identified by vertical bars. Login to comment