Structural insights for designed alanine-rich helices: comparing NMR helicity measures and conformational ensembles from molecular dynamics simulation

Abstract

The temperature dependence of helical propensities for the peptides Ac-ZGG-(KAAAA)(3)X-NH(2) (Z = Y or G, X = A, K, and D-Arg) were studied both experimentally and by MD simulations. Good agreement is observed in both the absolute helical propensities as well as relative helical content along the sequence; the global minimum on the calculated free energy landscape corresponds to a single alpha-helical conformation running from K4 to A18 with some terminal fraying, particularly at the C-terminus. Energy component analysis shows that the single helix state has favorable intramolecular electrostatic energy due to hydrogen bonds, and that less-favorable two-helix globular states have favorable solvation energy. The central lysine residues do not appear to increase helicity; however, both experimental and simulation studies show increasing helicity in the series X = Ala --> Lys --> D-Arg. This C-capping preference was also experimentally confirmed in Ac-(KAAAA)(3)X-GY-NH(2) and (KAAAA)(3)X-GY-NH(2) sequences. The roles of the C-capping groups, and of lysines throughout the sequence, in the MD-derived ensembles are analyzed in detail.

FIGURE 1

Structuring shift melts for the non-acetylated (KAAAA)₃X peptides with X = Lys (a) and d-Arg (b). Random coil shifts were derived from AcGKAAAKG-NH₂ and thus assume no differences in sequence effect for Lys and D-Arg. The more remote probes, A3 and A8, are, presumably, not influenced by the C-terminal substitution; these indicate that the D-Arg species is slightly more helical and resistant to melting.

FIGURE 2

The CSDs observed for Ac-(KAAAA)₃XGY-NH₂ (X = A, K, d-Arg) at the probed helix positions (initial K = #1) at 280K are shown as filled symbols. The corresponding points for KAAAA(KAAAA)₂-X-GYNH₂ (X = K, d-Arg) are shown as open symbols. The lines are trendlines drawn through the data to guide the eye and have no theoretical significance. Throughout C-caps are color-coded: X = d-Arg points and lines are in blue, Lys in black, and Ala in red. The same color-coding is used in Figure 3.

FIGURE 3

Fractional helicities at each residue of peptides A19, K19 and dR19. The continuous lines connect perresidue values obtained from REMD ensembles at 275 K. The experimental values (points) are from ¹³C-NMR for the corresponding Ac-YGG-capped peptides; the A19 data was for the peptide lacking the N-terminal acetyl. Since the NMR data reports on the amide linkage between residue i and i+1, we place the experimental point based on the ¹³C=O CSD of residue i halfway between i and i+1 on this plot.

FIGURE 4

Average α-helical propensities of representative residues at different temperatures. Different symbols are used for each residue examined. Helical propensity was calculated using local backbone conformation of the residue (left) and DSSP (right). The inset in A shows the NMR shift melts.

FIGURE 5

Lifson-Roig based melting curves for the helical segment (K4 through X19, normalized for this region of the three peptides.

FIGURE 6

Temperature-dependent populations of structures containing different number of continuous helical segments (shown using different line styles) sampled in the REMD simulation ensembles. The numbers in the legend refer to the number of continuous helical segments. Different peptide sequences are indicated using different colors.

FIGURE 7

The free energy landscape for peptide K19 at 275 K, along with representative structures obtained from cluster analysis of the ensemble. X and Y axes represent the first 2 principle components. Relative free energy values (in kcal/mol) are represented by color as indicated by the legend. Representative structures for each basin are shown. The colors of the structures reflect secondary structure type: alpha helix – purple, extended beta – yellow, turn – cyan, coil – orange. Only lysine side chains are shown. The populations of the clusters are shown in parentheses.

FIGURE 8

The normalized average helical propensity as a function of sequence for each cluster shown in Figure 7. Similar to the overall ensemble shown in Figure 3, helical content is reduced at the termini of most clusters. Most clusters also have nearly flat profiles along the middle of the sequence, with the exception of clusters 2 and 3 which correspond to helix-turn-helix motifs. Cluster 6 shows no helical content.

FIGURE 9

The distributions of the radius of gyration for peptide K19 at different temperatures.