Figure 3

The VV mutations in VAMP2 TMD reduce structural dynamics. Structural features of VAMP2 WT or VAMP2 VV full-length proteins were measured by infrared spectroscopy (PMIRRAS) in a Langmuir through at different lateral pressure. Note that increases in pressure increase local protein concentrations and peptide/lipid ratios. All experiments were performed at room temperature. Full-length proteins were embedded in DMPC monolayer at the protein/lipid ratio of 1/50 (Comparable results were obtained at peptide/lipid ratios of 1/20, data not shown). α-helical conformations and β-sheets conformations were detected at 1653 cm⁻¹ and 1630 cm⁻¹, respectively. (a) Structural behaviour of either VAMP2 WT or VV-TMDs during compression of the DMPC monolayer (ai and aii, respectively). (a i) Starting from a low lateral pressure (5 mN.m⁻¹, black curve), a shoulder at 1653 cm⁻¹ (corresponding to α helices) was detected. During compression to 44 mN.m⁻¹, this shoulder becomes less prominent (red curve, arrowhead). (a ii) Identical procedure performed with VV from 4 mN.m⁻¹ (black curve) to 55 mN.m⁻¹ (red curve). Note the persistence of the shoulder (arrowhead) at high pressure. (b) Structural behaviour of either VAMP2 WT or VV-TMDs during decompression of the DMPC monolayer (bi and bii, respectively). (b i) To facilitate comparison, the high pressure curve from A has been superimposed (red traces). Decompression of the monolayer from 44 to 4 mN.m⁻¹ restores the α helix shoulder (bi, blue curve, arrowhead), whereas decompression from 55 mN.m⁻¹ to 4 mN.m⁻¹ on membrane containing VV does not change the absorbance curve (b ii, blue curve and arrowhead).