Biophysical Society Thematic Meeting | Ascona, Switzerland

Liposomes, Exosomes, and Virosomes: From Modeling Complex Membrane Processes to Medical Diagnostics and Drug Delivery

Poster Abstracts

37-POS Board 19 Single-Molecule FRET Reveals Structural Basis for Conformational Misfolding of a Cystic Fibrosis Mutation in CFTR Georg Krainer 1 , Antoine Treff 1 , Andreas Hartmann 1 , Henry Chang 2,3 , Tracy Stone 2,3 , Arianna Rath 2 , Charles M. Deber 2,3 , Michael Schlierf 1 . 3 University of Toronto, Toronto, ON, Canada. 1 TU Dresden, Dresden, Germany, 2 Hospital for Sick Children, Toronto, ON, Canada, Cystic fibrosis (CF) is the most common lethal genetic disease among the Western population caused by mutations in the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR). The development of effective therapeutic agents against CF requires a molecular understanding of how mutation-related structural alterations lead to impaired functioning of CFTR. The main cause for CF are processing mutations frequently found within CFTR-transmembrane (TM) segments which affect co-translational folding and insertion. However, the underlying molecular mechanisms for misfolding remain obscure, mainly because of the unavailability of high- resolution structures and a lack of methods suitable for studying membrane protein structure- function relationships. Devising a divide-and-conquer approach in combination with single-molecule FRET (smFRET), we present here a strategy to gain novel insights into the structural basis for conformational misfolding in CFTR. Using this approach, we study the CF-phenotypic polar mutation V232D located within the fourth TM helix (TM4) of CFTR. In vivo folding and insertion of TM4 is functionally coupled with TM3 where both TM segments are co-translationally inserted into the membrane as a TM helix-loop-helix hairpin motif (TM3/4). Utilizing this minimal folding unit, we employ smFRET as a molecular ruler to readout structural alterations imposed on the helical packing of the TM3/4 hairpin upon mutation. In contrast to earlier findings that suggested a TM lock between TM3 and TM4 by a non-native H-bond, our results reveal that V232D TM3/4 associates with membranes in an open conformation. This implies that the charged residue imposes an energy penalty on membrane insertion of the hairpin. We propose a model for CF pathogenesis where partitioning of V232D TM3/4 into the interfacial region leads to misfolding of CFTR during co-translational membrane insertion and inhibits maturation by trapping the protein as a partially folded intermediate.

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