Biophysical Society Thematic Meeting

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

, Antoine Treff

, Andreas Hartmann

, Henry Chang

2,3

, Tracy Stone

2,3

, Arianna

Rath

, Charles M. Deber

2,3

, Michael Schlierf

University of Toronto, Toronto, ON, Canada.

TU Dresden, Dresden, Germany,

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.