ABCMdb

PMID: 11938353

Wigley WC, Corboy MJ, Cutler TD, Thibodeau PH, Oldan J, Lee MG, Rizo J, Hunt JF, Thomas PJ
A protein sequence that can encode native structure by disfavoring alternate conformations.
Nat Struct Biol. 2002 May;9(5):381-8., [PubMed]

Sentences

No. Mutations Sentence Comment

21 ABCC7 p.Pro205Ser ABCC7 p.Pro205Ser ABCC7 p.Pro205Ser Correspondence should be addressed to P.J.T. email: Philip.Thomas@UTSouthwestern.edu nature structural biology • volume 9 number 5 • may 2002 381 WT P205S P205S + lac P205S∆FWT B C a b Fig. 1 P205S mutant CFTR misfolds and aggregates in vivo. Login to comment 22 ABCC7 p.Pro205Ser a, The subcellular disposition of wild type and P205S CFTR transiently expressed in HEK293 cells was evaluated by indirect immunofluorescence. Login to comment 23 ABCC7 p.Pro205Ser Staining with polyclonal antiserum R3194 (directed against the CFTR C-terminus; (red) demonstrates surface expression of mature wild type protein (left) and ER-retention of P205S (center)). Login to comment 25 ABCC7 p.Pro205Ser Treatment of mutant expressing cells with the proteasome inhibitor lactacystin (lac, 10 µM for 12 h) results in the accumulation of P205S CFTR in a single perinuclear aggregate or inclusion (right). Login to comment 26 ABCC7 p.Pro205Ser b, Maturation of wild type (WT) and mutant (both ∆F508 and P205S) CFTR expressed in HEK293 cells was evaluated by western blotting using monoclonal anti-CFTR M3A7. Login to comment 32 ABCC7 p.Pro205Ser However, the CF mutation P205S, in the center of the third TM α-helix (m3), results in maturation-deficient molecules that fail to correctly fold and traffic to the plasma membrane (Fig. 1a). Login to comment 40 ABCC7 p.Pro205Ser Under these conditions, the wild type peptide is predominantly α-helical, whereas the P205S mutant forms a non-native structure, with a significantly decreased α-helical component and increased non-native structure (Fig. 2a). Login to comment 41 ABCC7 p.Pro205Ala ABCC7 p.Pro205Leu ABCC7 p.Pro205Gly This alternate conformation is apparently not induced specifically by the Ser residue, because the control peptides (P205G, P205A and P205L) each assume a similar non-native structure under these conditions. Login to comment 42 ABCC7 p.Pro205Leu Consistent with this conclusion, full-length P205L mutant CFTR expressed in cultured cells also misfolds, fails to achieve mature glycosylation and is retained in the ER (W.C.W., M.J.C. and P.J.T., unpublished observations). Login to comment 49 ABCC7 p.Pro205Ser ABCC7 p.Pro205Ala ABCC7 p.Pro205Leu ABCC7 p.Pro205Gly CD spec- troscopy30 evaluated the seconday structure of peptides representing wild type m3, the CF-causing mutant P205S, and control peptides P205G, P205A and P205L solubilized in either micellar SDS (0.5% (w/v) SDS and 5mM phosphate buffer, pH 7.2) or polyfluorinated organic solvents (10% HFIP, 40% TFE and 50% (v/v) H2O). Login to comment 50 ABCC7 p.Pro205Ser ABCC7 p.Pro205Ala ABCC7 p.Pro205Leu ABCC7 p.Pro205Gly The lines used to represent each peptide are wild type m3, dashed red; P205S, dashed blue; P205G, solid green; P205A, solid light purple; and P205L, solid black. Login to comment 63 ABCC7 p.Pro205Ser Wild type peptides integrated well (>18%) in a predominately α-helical conformation, as opposed to P205S, which did not measurably integrate (data not shown). Login to comment 72 ABCC7 p.Pro205Ser ABCC7 p.Pro205Leu Constructs are denoted as follows: P, TfR-m3 wt; S, TfR-m3 P205S; and L, TfR-m3 P205L. Login to comment 81 ABCC7 p.Pro205Ser b, The secondary structure of misfolded peptide P205S aggregates was assessed by ATR-FTIR spectroscopy (see Methods). Login to comment 92 ABCC7 p.Pro205Ser ABCC7 p.Pro205Leu Consistent with the peptide results, the wild type chimera properly integrates better than either the P205S or P205L mutants (Fig. 3b). Login to comment 100 ABCC7 p.Pro205Ser Consistent with this observation, the misfolded, aggregated P205S peptide binds thioflavin T (ThT), a fluorescent stain used to identify β-amyloid33 (data not shown). Login to comment 104 ABCC7 p.Pro205Ala In each of four mutant peptides, Pro 205 was replaced by an Ala, and extant Ala residues at positions 196 (red), 198 (blue), 204 (green) or 209 (light purple) were individually replaced with Pro. Login to comment 133 ABCC7 p.Pro205Leu A representative IR spectrum (solid line) of the amide I region is presented for the mutant peptide (P205L). Login to comment 136 ABCC7 p.Pro205Leu b, Wild type and mutant (P205L) peptides were first solubilized in neat TFE at 5 mg ml-1 and then unfolded by rapid dilution (1/10) in the strong denaturant 6 M guanidinium-SCN. Login to comment 142 ABCC7 p.Pro205Leu The symbols used are wild type (filled circle) and P205L (open circle). Login to comment 143 ABCC7 p.Pro205Ser An effect on apparent stability of similar magnitude is also observed for the mutant P205S (not shown). Login to comment 187 ABCC7 p.Pro205Ser TfR-m3 chimeras were then generated by replacing the single TfR TM span with that of wild type CFTR m3 (residues 190-220), followed by site-directed mutagenesis (QuickChange, Stratagene) to introduce the P205S and L missense mutations. Login to comment