Data from: Free-energy changes of bacteriorhodopsin point mutants measured by single-molecule force spectroscopy

Single-amino-acid mutations provide quantitative insight into the
energetics that underlie the dynamics and folding of membrane proteins.
Chemical denaturation is the most widely used assay and yields the change
in unfolding free energy (ΔΔG). It has been applied to >80
different residues of bacteriorhodopsin (bR), a model membrane protein.
However, such experiments have several key limitations: (i) a non-native
lipid environment, (ii) a denatured state with significant secondary
structure, (iii) error introduced by extrapolation to zero denaturant, and
(iv) the requirement of globally reversible refolding. We overcame these
limitations by reversibly unfolding local regions of an individual protein
with mechanical force using an atomic-force-microscope assay optimized for
2-ms time resolution and 1-pN force stability. In this assay, bR was
unfolded from its native bilayer into a well-defined, stretched state. To
measure ΔΔG, we introduced two alanine point mutations into an
8-amino-acid region at the C-terminal end of bR’s G helix. For each, we
reversibly unfolded and refolded this region hundreds of times while the
rest of the protein remained folded. Our single-molecule-derived ΔΔG for
mutant L223A (−2.3 ± 0.6 kcal/mol) quantitatively agreed with past
chemical-denaturation results while our ΔΔG for mutant V217A was 2.2-fold
larger (−2.4 ± 0.6 kcal/mol). We attribute the latter result, in part, to
contact between Val217 and a natively bound squalene lipid, highlighting
the contribution of membrane-protein–lipid contacts not present in
chemical-denaturation assays. More generally, we established a platform
for determining ΔΔG for a fully folded membrane protein embedded in its
native bilayer.