Figure 4.

Structure of the F1 loop and its function in membrane binding. (A) Superposition of the 20 lowest energy structures calculated for the F1-loop peptide in 35% TFE. Superposition was performed separately on the C-terminal helix (residues 154–167, left) and the N-terminal helix (residues 144–152, right). The middle part of the helix is not well defined in the ensemble of the calculated structure because of its lower stability, introducing a variable kink in the peptide structure. (B) Ribbon diagram of a low energy structure of the F1-loop peptide in 35% TFE corresponding to a continuous helix. Residues experiencing resonance broadening on the addition of 1:4 POPS:POPC SUVs are highlighted in red. (C) Surface charge distribution for the structure in (B). The views differ by a 180° rotation and are optimized to show the positively charged face. (D) HN/Hα region of the 2D DQF-COSY spectra of the loop peptide alone (Free) and in the presence of 2 mM SUVs composed of POPC (PC), 1:4 POPS:POPC (PC:PS) and 1:19 PIP2:POPC (PIP2) SUVs illustrating resonance broadening on addition of negatively charged lipids. (E) Secondary structure analysis of the F1-loop (145–168) peptide by circular dichroism (CD). Left—free peptide; right—superposition of the CD spectra of the free peptide (red) and the peptide in the presence of 1:4 POPS:POPC SUVs (blue) in the 215–250 nm region. Strong contribution from the SUVs to the spectrum below 215 nm prevents the detection of the peptide signal. (F) Superposition of ¹H projections of F1 HSQC spectra illustrating the reduction in resonance intensities in the presence of POPS (red) and PIP2 (black), but not pure POPC (blue).