ABCMdb

PMID: 21594798

Kanelis V, Chong PA, Forman-Kay JD
NMR spectroscopy to study the dynamics and interactions of CFTR.
Methods Mol Biol. 2011;741:377-403., [PubMed]

Sentences

No. Mutations Sentence Comment

78 ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys (b) HSQC spectrum of the G550E/R553M/R555K mutant NBD1-RE (398-673). Login to comment 102 ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys The interacting peptide is in red and the NBD1-RE structure is colored blue for residues for which we have resonance assignments, light grey for those not assigned, and dark grey for those assigned in the G550E/R553M/R555K mutant but not transferable to WT NBD1-RE (19). Login to comment 127 ABCC7 p.Phe833Leu As a disordered protein having a high fraction of charged residues, the R region was more easily amenable to solution NMR studies, once a better behaved construct containing the polymorphism F833L was identified (20). Login to comment 134 ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys The solubility of mNBD1 is greatly improved by the inclusion of the RE (14), which transiently populates helical structures that interact with NBD1 (19, 20), and incorporation of the revertant mutations, G550E, R553M, and R555K (43-45), yielding an NBD1-RE construct that is sufficiently soluble for NMR assignment experiments. Login to comment 136 ABCC7 p.Phe429Ser ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg Alternatively, the protein can be modified to increase solubility by specific point mutations (F494N and to a lesser degree F429S and Q637R) without deleting the RI (46). Login to comment 140 ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg555Lys ABCC7 p.Phe429Ser ABCC7 p.Phe494Asn ABCC7 p.Phe494Asn ABCC7 p.Gln637Arg Higher concentrations of glycerol and lower temperatures further stabilize the protein, but increase the viscosity of the solution, leading to Table 25.1 List of preferred CFTR constructs for NMR studies Construct Boundaries "Solubilizing" mutations mNBD1-RE 389-673 G550E, R553M, R555K hNBD1a 387-404, 437-646 None hNBD1-REa 387-404, 437-678 None hNBD1-RE 389-678 F494N hNBD1-RE 389-678 F429S, F494N, Q637R aThe RI (residues 405-436) have been deleted in these constructs. Login to comment 187 ABCC7 p.Gly550Glu ABCC7 p.Gly550Glu ABCC7 p.Arg553Met ABCC7 p.Arg553Met ABCC7 p.Arg555Lys ABCC7 p.Arg555Lys HSQC spectra recorded on samples specifically 15N labeled on Leu residues, aromatic residues (Phe, Tyr, and Trp), or the combination of Gly, Ser, Asp, and Asn residues were used to assist in identification of residue type in order to achieve 70% assignment of the G550E, R553M, R555K mutant NBD1-RE, which were then transferred to the WT protein (19), as the level of uniformity of lineshapes was greater for the G550E, R553M, R555K mutant than either WT or F508del (compare Fig. 25.2b, c). Login to comment