Interaction of a peptide corresponding to the loop domain of the S2 SARS-CoV virus protein with model membranes

Figures & data

Figure 1.  Hydrophobic moment, hydrophobicity, interfaciality distribution, and relative position of the SARS_L peptide used in this study along the SARS-CoV spike S2 domain, assuming it forms an α-helical wheel [16]. Only positive bilayer-to-water transfer free-energy values are depicted (the darker, the higher). The scheme of the structure of SARS-CoV spike glycoprotein S2 (amino acid residues 758–761 to 1255 for the full-length) according to literature consensus is also shown. The relevant functional regions are highlighted: the putative fusion peptide (FP), the internal fusion peptide (IFP), the predicted heptad repeat regions pertaining HR1, HR2, and the pre-transmembrane (PTM), transmembrane (TM) domains [14], [16], [19], [20], [34]. The sequence of the peptide used in this work is also shown.

Figure 2.  (A) Change on the Trp fluorescence intensity of the SARS_L peptide in the presence of increasing lipid concentration, (B) Stern-Volmer plots of the quenching of the Trp fluorescence emission of SARS_L by acrylamide in aqueous buffer (∇), and in the presence of LUVs, (C) Doxyl NS quenching (5NS, open symbols, and 16NS, black symbols) of the Trp fluorescence of the SARS_L peptide in the presence of LUVs at different lipid compositions. The lipid compositions used were EPC (▪), BPS (▴), EPG (•), EPA (♦). The spin label concentration is the effective quencher concentration calculated as stated in the Material and Methods section.

Table I.  Partition coefficient (Kp), spectral shift (Δλ), anisotropy value, acrylamide Stern-Volmer (K_SV) quenching constant obtained from the fluorescence of the Trp residue of the SARS_L peptide in buffer, in the presence of different model membranes.

System	Kp	▵λ (nm) *	<r> *	KSV
Buffe	–	–	0.065	16
EPC	6.8·10^5	1	0.073	5.87
EPG	3.9·10^6	5	0.091	4.81
BPS	4.8·10^6	4	0.094	4.54
EPA	8.5·10^6	6	0.104	4.78

* Δλ, <r> correspond to a peptide/lipid ratio of 1:30.

Table II.  Fluorescence lifetime components τ_i, normalized amplitudes α_ι, and fluorescence mean lifetime (amplitude weighted, , and intensity weighted, <τ > ) of the Trp residue of the SARS_L peptide in buffer, in the presence of phospholipid model membranes at 25°C. The reduced χ² values of the fits to the experimental fluorescence decays are also given. The peptide/lipid ratio was 1:30.

System	τ1 (ns)	τ2 (ns)	τ3 (ns)	α1	α2	α3	(ns)	<τ> (ns)	χ^2
Buffer 15°C	0.305	1.343	4.302	0.403	0.387	0.210	1.55	2.99	1.16
Buffer 25°C	0.212	0.986	3.531	0.513	0.365	0.121	0.897	2.11	1.19
Buffer 50°C	0.243	1.039	2.72	0.362	0.475	0.162	1.02	1.70	1.18
EPC	0.25	0.934	3.186	0.486	0.373	0.141	0.918	1.94	1.20
EPG	0.259	1.14	4.107	0.440	0.389	0.171	1.26	2.72	1.28
BPS	0.308	1.229	3.939	0.507	0.345	0.147	1.16	2.46	1.10
EPA	0.268	1.09	3.485	0.463	0.361	0.176	1.13	2.30	1.18

Table III.  Long rotational correlation time φ from the anisotropy decay of the peptide SARS_L in buffer solution at different temperatures. The global chi-square is also given. For comparison, the rotational correlation time for different aggregates of the SARS_L peptide at those temperatures is also indicated. The inclusion of a very short rotational correlation time (<100 ps) was necessary to account for the initial depolarization, but its value could not be accurately determined.

System	φexperimental (ns)	φmonomer (ns)*	φdimer (ns)*	φtrimer (ns)*	χG^2
Buffer 15°C	2.43	0.97–1.4	2.0–2.9	2.9–4.3	1.44
Buffer 25°C	1.80	0.74–1.1	1.5–2.2	2.2–3.3	1.09

*From the Stokes-Einstein equation. The lower, upper bounds are calculated for non-hydrated, maximally hydrated peptide respectively.

Table IV.  Fitting parameters for the anisotropy decay of the peptide SARS_L (rotational correlation time φ, associated depolarization β, and limiting anisotropy r_∞) in lipid membranes of several compositions at 25°C. The global chi-square is also given.

System	β^a	φexperimental (ns)	r∞	χG^2
EPC	0.075	2.44	0.027	1.16
EPG	0.061	3.58	0.053	1.28
BPS	0.032	2.34	0.078	1.17
EPA	0.049	5.94	0.070	1.12

^aThe inclusion of a very short rotational correlation time (<100 ps) was necessary to account for the initial depolarization, but its value could not be accurately determined.

Figure 3.  (A) Effect of the SARS_L peptide on membrane rupture of LUVs at different lipid compositions, at different lipid-to-peptide molar ratios. The lipid compositions used were EPC (▪), EPC/BPS/CHOL at a molar ratio of 5:3:1 (▴), EPG (•), EPC/EPG/CHOL at a molar ratio of 5:3:1 (▾), EPC/EPA/Chol at a molar ratio of 5:3:1 (♦). (B) Insertion of the SARS_L peptide into lipid monolayers. Increments in surface pressure (Δπ) of lipid monolayers due to the addition of SARS_L peptide into the subphase is illustrated as a function of the initial pressure (π₀). The lipid compositions used were POPC (•), POPS (▴), POPG (▪).

Figure 4.  Infrared spectra in the Amide I’ region of the SARS_L peptide in solution (A), and in the presence of liposomes composed of (B) EPC, (C) EPG at a phospholipid/peptide molar ratio of 15:1 in D₂O buffer at 25°C.

Table V.  Fluorescence lifetime components τ_ι, normalized amplitudes α_ι, and fluorescence mean lifetimes (amplitude weighted, , and intensity weighted, <τ > ) of DPH in phospholipid model membranes in the absence, in the presence of the SARS_L peptide below, above the phospholipid T_m. ^* From ref. [34].

T (°C)	System	τ1 (ns)	τ2 (ns)	τ3 (ns)	α1	α2	α3	(ns)	<τ> (ns)	χ^2
15°C	DMPG + SARSL	1.490	6.200	11.54	0.072	0.314	0.613	5.86	6.110	1.13
	DMPG^*	0.876	6.170	12.17	0.136	0.085	0.778	10.12	11.72	1.24
	POPE + SARSL	0.810	5.397	9.310	0.168	0.163	0.669	6.060	6.410	1.10
	POPE	1.030	––	8.840	0.454	0	0.546	5.300	8.150	1.20
35°C	DMPG + SARSL	1.130	5.470	10.94	0.145	0.438	0.417	4.840	5.330	1.04
	DMPG^*	1.500	––	9.190	0.182	––	0.818	7.790	8.920	1.06
	POPE + SARSL	0.649	3.220	8.060	0.124	0.108	0.768	2.900	3.150	1.09
	POPE	0.986	––	8.160	0.249	––	0.751	6.370	7.880	1.17

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