ABCMdb

PMID: 26384709

Aleksandrov LA, Jensen TJ, Cui L, Kousouros JN, He L, Aleksandrov AA, Riordan JR
Thermal stability of purified and reconstituted CFTR in a locked open channel conformation.
Protein Expr Purif. 2015 Dec;116:159-66. doi: 10.1016/j.pep.2015.09.018. Epub 2015 Sep 15., [PubMed]

Sentences

No. Mutations Sentence Comment

20 ABCC7 p.Ile539Thr ABCC7 p.Ala534Pro ABCC7 p.Ser492Pro Abbreviations used: CFTR, cystic fibrosis transmembrane conductance regulator; ABC, ATP-binding cassette; NBD1, N-terminal nucleotide-binding domain; CF, cystic fibrosis; CHO, Chinese hamster ovary; HEK, human embryonic kidney; BHK, baby hamster kidney; Tm, melting temperature; Ti, inactivation temperature; RI, Regulatory Insertion (residues 404-435); 2PT, variant with NBD1 mutations S492P, A534P and I539T; Q loop, residues contacting the gamma-phosphate of ATP; SDR, structurally divers region; DMNG, Decyl Maltose Neopentyl Glycol; MALS, multi-angle light scattering analysis; DOPE, 1,2-dioleoyl-sn-glycero-3-phosphoethanola mine; DOPC, 1,2-dioleoyl-sn-glycero-3-phosphocholine; DOPS, 1,2-dioleoyl-sn- glycero-3-phospho-L-serine; SUV, small unilamellar vesicles; LMV, large multilamellar vesicles; LULV, large unilamellar vesicles; PKA, protein kinase A; RIPA, radioimmunoprecipitation assay; ER, endoplasmic reticulum; RAMP, gradual increase with constant slope. Login to comment 107 ABCC7 p.Ile539Thr ABCC7 p.Ala534Pro ABCC7 p.Ser492Pro As seen in Fig. 2a the ''2PT" variant with NBD1 mutations S492P, A534P and I539T and the DRI variant, from which the Regulatory Insertion (residues 404-435) was deleted both increased expression levels substantially compared to the wild type. Login to comment 109 ABCC7 p.Ala534Pro ABCC7 p.Ser492Pro Channel open probability was substantially reduced in 2PT due to the introduction of the two prolines into the mobile Q loop (S492P) and SDR (A534P) regions of NBD1 (third tracing). Login to comment 112 ABCC7 p.His1402Ser When the H1402S mutations was added to the combined NBD1 modifications, the amount of CFTR expressed by the cells (and present in isolated membranes) was increased 3 to 5-fold compared to the wild-type and the mature/immature band intensity ratio as an index of maturation also was elevated (not shown). Login to comment 114 ABCC7 p.His1402Ser The thermal stability of the DRI/2PT/H1402S channel in isolated microsomal membranes was evaluated and it was found to retain full activity at 45 &#b0;C for at least 1 h (Fig. 3, top tracing) and remain active during a temperature ramp from 45 &#b0;C to 50 &#b0;C albeit with open state subconductances most evident in the extended lower tracing at 50 &#b0;C. Thus, the increased amounts of protein expressed and greater thermal stability encouraged us to proceed with purification and characterization of this variant. Login to comment 118 ABCC7 p.His1402Ser This number of cells yielded 1 g of microsomal membranes from which 2 mg of wild-type and 10 mg of the DRI/2PT/H1402S variant could be purified. Login to comment 120 ABCC7 p.His1402Ser The purified DRI/2PT/ H1402S protein was highly homogeneous as judged by heavily loaded SDS-PAGE gels (Fig. 4a) and appeared monodisperse in the low concentration of the DMNG (at 3 CMC) detergent in which it was purified (Fig. 4b). Login to comment 123 ABCC7 p.His1402Ser The gel filtration profile of the purified DRI/2PT/H1402S protein was unaltered after storage at 4 &#b0;C for at least two weeks and little changed when kept at 16 &#b0;C for this period of time (data not shown). Login to comment 128 ABCC7 p.His1402Ser In marked contrast to the minimal CFTR channel activity observed with the purified wild-type protein, the purified DRI/2PT/H1402S protein was fully active at temperatures of 25, 30 and 37 &#b0;C with very high open probability in all cases (Fig. 6, left 3 tracings). Login to comment 163 ABCC7 p.His1402Ser When this set of NBD1 stabilizing mutations were combined with the H1402S substitution in NBD2 the level of mature protein expression in BHK cells increased several fold, enabling purification of milligram quantities of homogeneous protein that remained monodisperse at concentrations >3 mg/ml in a low concentration of DMNG (3 CMC). Login to comment 171 ABCC7 p.His1402Ser Thermal sensitivity of NBD1 and NDB2 modified variant, DRI/2PT/H1402S. Login to comment 176 ABCC7 p.His1402Ser Purified DRI/2PT/H1402S CFTR protein before and after reconstitution. Login to comment 187 ABCC7 p.His1402Ser Thermal stability of purified and reconstituted DRI/2PT/H1402S CFTR. Login to comment