Membrane interactions of peptides representing the polybasic regions of three Rho GTPases are sensitive to the distribution of arginine and lysine residues

Figures & data

Figure 1.  Aligned C-terminal sequences of the three human Rho protein family members Rac1, TCL and Cdc42, indicating (in upper case) the lysine- and arginine- rich polybasic region.

Figure 2.  ³¹P MAS NMR spectra of MLVs of DOPC/DOPG (2:1 molar ratio) at 4°C. Spectra are shown for the lipid sample alone (a) and after the addition of the 5 basic hexapeptides to a lipid/peptide molar ratio of 20:1 (b–f).

Figure 3.  ³¹P MAS NMR spectra of MLVs of DMPC/DOPG (2:1 molar ratio) at 4°C. Spectra are shown for the lipid sample alone (a) and after the addition of the 5 basic hexapeptides to a lipid/peptide molar ratio of 20:1 (b–f).

Table I.  Summary of ³¹P NMR isotropic chemical shifts (σ_i) and line widths at half height (Δν_½) at 4°C for MLVs of DOPC/DOPG alone and in the presence of the 5 basic hexapeptides at a lipid/peptide molar ratio of 20:1.

Sample	σi (ppm)		Δν½ (ppm)
	DOPG	DOPC	DOPG	DOPC
Lipid only	0.28	−0.82	0.24	0.27
+ K6	0.23	−0.87	0.42	0.46
+ KKRKRK	0.18	−0.89	0.34	0.37
+ KKKKKR	0.21	−0.89	0.29	0.31
+ PKKSRR	0.26	−0.85	0.32	0.39
+ R6	0.23	−0.87	0.39	0.41

Table II.  Summary of ³¹P NMR isotropic chemical shifts (σ_i) and line widths at half height (Δν_½) at 4°C and apparent chemical shift anisotropy (Δσ) at 30°C for MLVs of DMPC/DOPG alone and in the presence of the 5 basic hexapeptides at a lipid/peptide molar ratio of 20:1.

Sample	σi (ppm)		Δν½ (ppm)		Apparent Δσ (ppm)
	DOPG	DMPC	DOPG	DMPC
Lipid only	0.31	−0.85	0.31	0.63	42.2
+ K6	−0.05	−0.98	0.28	0.67	42.7
+ KKRKRK	0.38	−0.74	0.29	0.57	47.2
+ KKKKKR	0.42	−0.59	0.23	0.58	44.6
+ PKKSRR	0.50	−0.50	0.25	0.94	45.4
+ R6	0.42/0.12	−0.71	split	1.03	47.2

Figure 4.  An illustration of the effect of lipid phase separation on the ³¹P MAS NMR spectra of DMPC/DOPG membranes. The diagram at the top illustrates the composition of the homogeneously mixed vesicles prepared from DMPC/DOPG in a 2:1 molar ratio (left) and from a mixture of preformed DMPC and preformed DOPG vesicles in the same ratio (right). Spectra corresponding to the two samples are shown underneath each diagram.

Figure 5.  Wide-line ³¹P NMR spectra of MLVs of DMPC-d₄/DOPG (2:1 molar ratio) at 30°C. Spectra are shown for the lipid sample alone (a) and after the addition of the 5 basic hexapeptides to a lipid/peptide molar ratio of 20:1 (b–f).

Figure 6.  Wide-line ²H NMR spectra of MLVs of DMPC-d₄/DOPG (2:1 molar ratio) at 30°C. Spectra are shown for the lipid sample alone (a) and after the addition of the 5 basic hexapeptides to a lipid/peptide molar ratio of 20:1 (b–f). The quadrupole splittings for deuterons at the α- and β-choline positions of DMPC-d₄ are denoted by the separation between the dotted lines.

Figure 7.  A plot of the ²H quadrupolar splittings (Δν_Q) at 30°C for the α- and β- deuterons of DMPC-d₄ in the mixed DMPC-d₄/DOPG membranes alone (open symbols) and after the addition of the basic hexapeptides to a lipid/peptide molar ratio of 50:1 (half-filled symbols) and 20:1 (full symbols).