Structural basis for misfolding at a disease phenotypic position in CFTR: comparison of TM3/4 helix-loop-helix constructs with TM4 peptides.

Understanding the residue-dependent effects of disease-phenotypic mutations in multi-spanning membrane proteins is an essential step toward the development of corrective therapies. As a systematic approach to further elucidate mutant-dependent mis-folding consequences, we prepared two libraries: one consisting of 20 helix-loop-helix ("hairpin") constructs derived from helices 3 and 4 of the human cystic fibrosis transmembrane conductance regulator (CFTR) (residues 194-241) in which the CF-phenotypic position Val-232 was substituted individually to each of the 20 commonly-occurring amino acids; and a second library consisting of 20 single-stranded TM4 peptides (CFTR residues 221-241) similarly substituted at position 232. Both libraries were analyzed to measure mutant-dependent variations in mobility on SDS-PAGE; size and shape on size exclusion chromatography; retention times on reverse phase HPLC; and helical content by circular dichroism spectroscopy. Analysis of a scatter plot between TM3/4 hairpin and TM4 peptide retention times showed a strong correlation (r=0.94, p<0.05), with retention times largely a function of residue hydrophobicity. In contrast, while the hairpin library migrated over a significant range on SDS-PAGE, migration rates for TM4 hydrophobic residues at position 232 converged at a single value, suggesting that residue-dependent re-orientations of hairpin van der Waals interfaces may expose varying faces of the TM3 and/or TM4 helices to the SDS detergent. The overall results suggest that mutant-mediated variations are a principal determinant of tertiary interhelical folding interactions in membranes.