Fig. 2: The M105I mutation of α-SNAP does not affect the secondary structure of the protein but reduces its membrane interaction properties.

a Secondary structure throughout the 5 µs of molecular dynamics simulation. No significant differences were observed in the percentage of coils and helices between α-SNAP WT and M105I mutant protein. b Energy profiles obtained with the eABF method of the α-SNAP WT (gray) and M105I (blue) system. Mean ± SEM are shown. c Representation of the initial (top), and final position (bottom) of the WT (left) and M105I (right) proteins on the studied reaction coordinate. The small red cylinder represents the 1 Å radius harmonic constraint applied to the center of mass of the carbon atoms of the entire protein. The yellow cylinder represents the 6 Å radius harmonic constraint in which the center of mass of residues F27 and F28 was retained. d, e Left: The distances of the WT (d) or the M105I (e) systems and the membrane during the ABF calculations are shown. The protein center is defined as the geometrical center of the alpha carbons of the protein for each system. The up-bilayer corresponds to the z-component of the PO₄ atoms of the upper-leaflet of the lipid bilayer. Residues F27–F28 or L75–Q76 correspond to the geometrical center of both residues. The bilayer-center is defined as the geometrical center between the PO₄ atoms of both leaflets (upper and lower). Right: Representation of protein positions in different time points (indicated as circles 1 and 2 in the graph at the left). The residues F27, F28, and L75 are represented within the blue color palette, while residue Q76 is presented in green. Amino acid 105 is depicted in orange. Insertion frequency (%) and insertion depth (Å) of WT (f) and M105I protein (g) are shown for each residue. These metrics are measured as the percentage of frames in which the side chain geometric center was below the phosphate (PO₄) beads of the upper membrane leaflet. The corresponding distance below this plane is also reported. Negative values indicate deeper penetration. Data was aggregated across all five eABF simulation replicas. For (a) figure data is presented as mean ± SEM, with sample sizes of n = 3. Group differences were evaluated using two-tailed unpaired Student’s t tests. No significant differences were detected for (a) coil (t = 1.17, df = 4, P = 0.3064, η² = 0.26) or (a) helix (t = 1.15, df = 4, P = 0.3144, η² = 0.25). For panels (f) and (g) data is shown for descriptive purposes only; no statistical analysis was performed.