2.3 Results And Discussion
2.3.3 MD simulations
MD simulations (100 ns) were carried out in water to gain insights about the type of turn mediated by the Asn-Gly, DPro-Gly, and Aib-DPro dipeptide motifs in the peptides. The trajectory files obtained following MD simulations were analyzed to examine the folding of the peptides. Distance between the α-carbons of terminal amino acid residues and that between the α-carbons of the residues preceding and succeeding the β-turn dipeptide motifs were calculated. The dihedral angles of the β-turn dipeptide motifs gave insights into the type of turn formed. Furthermore, the possibility of H-bonding between C=O of preceding residue and NH of the succeeding residue was investigated for certain representative peptides viz. AβFY-NG, AβFY-pG, AβYY-NG and AβYY-pG.
The data for AβFY-NG is shown in figure 2.3. AβFY-NG simulation was started with an extended peptide structure (φall=ψall=180°). The peptide folds to take a β- hairpin conformation after ~30 ns; the distance between K1Cα and E16Cα is ~0.6 nm and shows only small variations (black trace, panel A). The distance between the E7Cα and K10Cα is also ~0.6 nm, and little variation is observed through the simulation (red trace, panel A), wherein a hydrogen-bond is formed between Glu7 C=O and Lys10 NH (panels D and E), suggesting a β-turn [6, 9]. At 34 ns, the dihedral angles, φ and ψ for Asn are -56° and 91°, respectively. The angles show variations around φ and ψ values of ~-57° and ~105° for the largest cluster (panel B). Gly, on the other hand, adopts φ and ψ values of 56° and 51° after 34 ns. After 34 ns, Gly9 φ samples values largely between 40 - 120°, spread around +66° (panel C). The values of ψ range from -60 - +60° with ψ = 30° as the largest cluster. The dihedral angles of the largest cluster indicate a type II β-turn. At 34 ns, the distance between E7CO and K10NH is ~0.19 nm (panel D) with angle HNO of 19° (panel E), indicating an H-bond. Panels F-K show the snapshots at 20 ns interval supporting hairpin structures.
Figure 2.3 MD simulation of AβFY-NG. (A), distance between K1Cα and E16Cα (black) and that between E7Cα and K10Cα (red); (B), dihedral angles, φ (black) and ψ (red) of Asn residue; (C), dihedral angles, φ (black) and ψ (red) of Gly residue; (D), distance between amide oxygen of Glu7 and amide proton of Lys10; (E), angle HNO, wherein HN belongs to Lys10 amide whereas O belongs to the Glu7 amide; after ~32 ns, the distance and the angle are, mostly, within 0.35 nm and 60°, respectively; (F-K), snapshots from the MD trajectory at 20 ns interval.
AβFY-pG simulation was started with extended conformation; φ and ψ values for
DPro, however, were taken 70° and -45°, respectively. The peptide folds into a β- hairpin-like structure around 19 ns (Figure 2.4). The distance between K1Cα and E16Cα as well as that between E7Cα and K10Cα is ~0.5-0.6 nm and is invariant over time. After 19 ns, the largest cluster takes the φ and ψ values for DPro spread around +60° and ~ -110°, respectively (panel B). Gly9 takes φ and ψ values around -120° and 30°, respectively (panel C). The DPro and Gly dihedral angles conform to a type II′ turn. The hairpin appears to be stabilized by an i ‒ i+3 hydrogen-
bond in the βturn region as suggested by the distance between E7CO and K10NH (panel D) and angle HNO (panel E).
Figure 2.4 MD simulation of AβFY-pG. (A), distance between K1Cα and E16Cα (black) and that between E7Cα and K10Cα (red); (B), dihedral angles, φ (black) and ψ (red) of DPro residue; (C), dihedral angles, φ (black) and ψ (red) of Gly residue; (D), distance between amide oxygen of Glu7 and amide proton of Lys10; (E), angle HNO, wherein HN belongs to Lys10 amide whereas O belongs to the Glu7 amide; after ~11 ns, the distance and the angle are, mostly, within 0.35 nm and 60°, respectively; (F-K), snapshots from the MD trajectory at 20 ns interval.
MD simulation data for AβYY-NG are shown in figure 2.5. The data show a K1Cα and E16Cα distance of ~0.6-0.7 nm within 10 ns of simulation (Panel A). However, β-turn is not mediated by residues 7-10, but by residues 8-11. Instead of Asn8 and Gly9, Gly9 and Lys10 happen to be the i+1 and i+2 turn residues, respectively.
This results in a shift in the β-strand registry (Panels F-K). Large fluctuations observed in the distance between K1Cα and E16Cα could be attributed to the
flexible N-terminal residue. Around 40 ns, Gly9 takes up φ and ψ values spread around +65° and -105°, respectively (Panel B). Lys10 takes φ and ψ values around -90° and -30°, respectively (Panel C). The distance between N8Cα and L11Cα are also within 0.6 nm. The distance between N8CO and L11NH is largely within 0.3 nm (panel D) with angle HNO within 60° (panel E) indicating an H-bond. The presence of H-bond between ith and (i+3)th residues and the dihedral angles of the i+1 and i+2 residues indicate a type II′ β-turn.
Figure 2.5 MD simulation of AβYY-NG. (A), distance between K1Cα and E16Cα (black) and that between N8Cα and L11Cα (red); (B), dihedral angles, φ (black) and ψ (red) of Gly residue; (C), dihedral angles, φ (black) and ψ (red) of Lys10 residue; (D), distance between backbone amide oxygen of Asn8 and amide proton of Leu11; (E), angle HNO, wherein HN belongs to Leu11 amide whereas O belongs to the Asn8 backbone amide;
after 40 ns, the distance and the angle are, mostly, within 0.35 nm and 60°, respectively;
(F-K), snapshots from the MD trajectory at 20 ns interval.
AβYY-pG rapidly folds into a β-hairpin structure (Figure 2.6). Around 7 ns, the distance between K1Cα and E16Cα and that between E7Cα and K10Cα are within 0.6 ns and are largely invariant throughout the simulation (Panel A). DPro8 takes up φ and ψ values spread around +60° and -110°, respectively (Panel B). Gly9 takes up φ and ψ values spread around -90° and 0°, respectively (Panels C); ψ values largely reside around 30° and -30° suggesting two major clusters around 0°. The data suggest a classic type II′ turn mediated by DPro8 and Gly9.
Figure 2.6 MD simulation of AβYY-pG. (A), distance between K1Cα and E16Cα (black) and that between E7Cα and K10Cα (red); (B), dihedral angles, φ (black) and ψ (red) of DPro residue; (C), dihedral angles, φ (black) and ψ (red) of Gly residue; (D), distance between amide oxygen of Glu7 and amide proton of Lys10; (E), angle HNO, wherein HN belongs to Lys10 amide whereas O belongs to the Glu7 amide; after ~6 ns, the distance and the angle are, mostly, within 0.35 nm and 60°, respectively; (F-K), snapshots from the MD trajectory at 20 ns interval.
The hairpin appears to be stabilized by an i ‒ i+3 hydrogen-bond in the beta-turn region as suggested by the distance between E7CO and K10NH (panel D) and angle HNO (panel E). Panels F-K show the snapshots at 20 ns interval and confirm the β-hairpin structure.
Snapshots of the MD simulations of AβFF-NG, AβFF-pG, AβWW-NG, AβWW- pG, AβFF-Up, AβFY-Up, AβYY-Up, and AβWW-Up are shown from figures 2.7- 2.14, respectively. AβFF-NG folds into a β-hairpin structure. The turn, however, is a non-classical one (Figure 2.7). AβFF-pG takes up a β-hairpin structure mediated by a β-turn within 20 ns (Figure 2.8). DPro mostly takes up the dihedral angles characteristic of a type II′ turn, but Gly shows high conformational flexibility (Panels B and C, Figure 2.8). AβWW-NG coils but a β-hairpin-like conformation is obtained only around 96 ns (Figure 2.9, panel F). The hairpin, however, is not mediated by a β-turn. As suggested by the unstable dihedral angles near the end of the simulation, a 100 ns simulation appears to be insufficient to comment if the hairpin is indeed the kinetically-stable structure.
AβWW-pG takes up a β-hairpin structure, but the turn is neither of the classical β-turns (Figure 2.10). AβFF-Up takes type I′ β-turn (Figure 2.11). In AβFY-Up, the turn is not only mediated by Aib and DPro but Lys11 as well, suggesting a γ turn (Figure 2.12). AβYY-Up folds to take a hairpin-like structure (Figure 2.13). The Aib and DPro dihedral angles take up values those of classical type I′ turn in the 10- 25 ns region; the chains, however, do not take up a β-conformation. It appears that the bulky Tyr side-chains disrupt the β-sheet structure due to steric clashes;
the β-hairpin structure is finally achieved with a shift in the β-strand registry, possibly to ease out the steric clashes. AβWW-Up failed to form any β-hairpin- like structure, possibly due to the steric hindrance caused by bulky indole groups of Trp residues (Figure 2.14).
Figure 2.7 MD simulation of AβFF-NG. (A), distance between K1Cα and E16Cα (black) and that between E7Cα and K10Cα (red); (B), dihedral angles, φ (black) and ψ (red) of Asn residue; (C), dihedral angles, φ (black) and ψ (red) of Gly residue; (D-I), snapshots from the MD trajectory at 20 ns interval.
Figure 2.8 MD simulation of AβFF-pG. (A), distance between K1Cα and E16Cα (black) and that between E7Cα and K10Cα (red); (B), dihedral angles, φ (black) and ψ (red) of DPro residue; (C), dihedral angles, φ (black) and ψ (red) of Gly residue; (D-I), snapshots from the MD trajectory at 20 ns interval.
Figure 2.9 MD simulation of AβWW-NG. (A), distance between K1Cα and E16Cα (black) and that between E7Cα and K10Cα (red); (B), dihedral angles, φ (black) and ψ (red) of Asn residue; (C), dihedral angles, φ (black) and ψ (red) of Gly residue; (D-I), snapshots from the MD trajectory at 20 ns interval.
Figure 2.10 MD simulation of AβWW-pG. (A), distance between K1Cα and E16Cα (black) and that between E7Cα and K10Cα (red); (B), dihedral angles, φ (black) and ψ (red) of
DPro residue; (C), dihedral angles, φ (black) and ψ (red) of Gly residue; (D-I), snapshots from the MD trajectory at 20 ns interval.
Figure 2.11 MD simulation of AβFF-Up. (A), distance between K1Cα and E16Cα (black) and that between E7Cα and K10Cα (red); (B), dihedral angles, φ (black) and ψ (red) of Aib residue; (C), dihedral angles, φ (black) and ψ (red) of DPro residue; (D-I), snapshots from the MD trajectory at 20 ns interval.
Figure 2.12 MD simulation of AβFY-Up. (A), distance between K1Cα and E16Cα (black) and that between E7Cα and K10Cα (red); (B), dihedral angles, φ (black) and ψ (red) of Aib residue; (C), dihedral angles, φ (black) and ψ (red) of DPro residue; (D-I), snapshots from the MD trajectory at 20 ns interval.
Figure 2.13 MD simulation of AβYY-Up. (A), distance between K1Cα and E16Cα (black) and that between E7Cα and K10Cα (red); (B), dihedral angles, φ (black) and ψ (red) of Aib residue; (C), dihedral angles, φ (black) and ψ (red) of DPro residue; (D-I), snapshots from the MD trajectory at 20 ns interval.
Figure 2.14 MD simulation of AβWW-Up. (A), distance between K1Cα and E16Cα (black) and that between E7Cα and K10Cα (red); (B), dihedral angles, φ (black) and ψ (red) of Aib residue; (C), dihedral angles, φ (black) and ψ (red) of DPro residue; (D-I), snapshots from the MD trajectory at 20 ns interval.