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Cation–π interactions are common in proteins, but their contribution to the stability and specificity of protein structure has not been well established. In this study, we examined the impact of cation–π interactions in a diagonal position of a β-hairpin peptide through comparison of the interaction of Phe or Trp with Lys or Arg. The diagonal interactions ranged from −0.20 to −0.48 kcal/mole. Our experimental values for the diagonal cation–π interactions are similar to those found in α-helical studies. Upfield shifting of the Lys and Arg side chains indicates that the geometries of cation–π interactions adopted in the 12-residue β-hairpin are comparable to those found in protein structures. The Lys was found to interact through the polarized Cε, and the Arg is stacked against the aromatic ring of Phe or Trp. Folding of these peptides was found to be enthalpically favorable (ΔH° ∼ −3 kcal/mole) and entropically unfavorable (ΔS°∼ −8 cal mole−1 K−1).
Protein folding to the single correct structure requires contributions from many noncovalent interactions. A typical protein is stable only by ∼5 to 10 kcal/mole. Hence, small differences in energy between a myriad of possible noncovalent interactions are summed up to provide one unique folded structure. The relevance of cation–π interactions in biological structure have only been recognized in recent years. Cation–π interactions have been observed in a number of systems, including protein structure (Dougherty 1996; Gallivan and Dougherty 1999b), particularly the nicotinic-acetylcholine receptor (Dougherty and Stauffer 1990; Nowak et al. 1995), protein–DNA binding (Wintjens et al. 2000), ion channels (Kumpf and Dougherty 1993), catalysis (Gruber et al. 1999), and molecular recognition (Kearney et al. 1993; Forman et al. 1995). Understanding of the cation–π interaction comes principally from small molecule cyclophane receptors, protein structures, and theoretical studies. Computational methods have implicated charge-quadrupole, charge-dipole, charge-induced dipole, charge transfer, dispersion forces, and hydrophobic packing as components of cation–π interactions (Duffy et al. 1993; Caldwell and Kollman 1995; Mecozzi et al. 1996; Cubero et al. 1998). This interaction has also been shown to stabilize α-helices (Andrew et al. 2002; Shi et al. 2002; Tsou et al. 2002). Studies in proteins and cyclophanes have defined geometries, distances, and strengths of interactions. Although widespread, the contribution of the cation–π interaction to protein stability and specificity has not been well established. Here, we describe the use of a β-hairpin peptide as a model system to probe the stability and geometry of cation–π interactions in a biologically relevant environment in the absence of tertiary interactions.
Many components of β-hairpin structure have been shown to impact stability, including cross-strand interactions (Smith and Regan 1995; Russell and Cochran 2000; Syud et al. 2001), turn propensity (Chung et al. 1998; Stanger and Gellman 1998; Fisk et al. 2000), β-sheet propensity (Distefano et al. 2002), and strand length (Stanger et al. 2001). Recently, diagonal interactions have been implicated in β-hairpin stability (Syud et al. 2001). This initial study indicates that diagonal interactions may be as stabilizing as cross-strand interactions. Geometric descriptions of β-sheet structure derived from the Protein Data Bank (PDB) indicate that diagonal side chains are in close proximity. Each residue side chain is defined as a shell from the geometric mean of side-chain atoms from coordinates in the PDB. Similar side-chain separation is observed for diagonal residues in the i and j − 2 positions (3.0 Å) as for cross-strand residues (2.8 Å; Cootes et al. 1998), supporting the observation of diagonal interactions. In contrast, the side-chain separation for the i and j + 2 side chains is 4.5 Å, which is too great a distance for any natural side chain to span.
Our initial interest into diagonal cation–π interactions arose from an unexpected finding in a related system. The two β-hairpin peptides Ac-RFVEVNGOKI(Cha)Q-NH2 and Ac-R(Cha)VEVNGOKIFQ-NH2, where Cha is cyclohexylalanine, were investigated (C.D. Takto and M.L. Waters, unpubl.). Each peptide has the same 12 residues, although in different orientations, but the degree that they are folded differs, at 49% and 38%, respectively. As these peptides have the same 12 residues, the total β-sheet propensity of each peptide is identical, and all lateral interactions are preserved. Seemingly, the only difference is that one peptide has the potential for a diagonal cation–π interaction, whereas the other does not. The determination of the difference in stability of 11% or 0.3 kcal/mole led us to the current investigation.
The present study investigates the influence of cation–π interactions between two diagonal residues on β-hairpin stability. We have used double-mutant cycles to determine the interaction of residues in diagonal positions. We have also obtained information on the geometry of the interaction through probing side-chain chemical shifts. Our results indicate that cation–π interactions provide stabilization of the hairpin through a diagonal interaction. Moreover, the geometries observed for cation–π interactions in this minimalist system correspond to those observed in proteins. Thermal denaturation studies of these peptides indicate that folding is enthalpically favorable and entropically unfavorable, indicating a driving force other than the classical hydrophobic effect. Taken together, these results provide insight into the driving force and significance of cation–π interactions to protein structure and stability.
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- Materials and methods
- Electronic supplemental material
- Supporting Information
We have exploited the NHB cluster on the face of a β-hairpin to investigate the nature of a diagonal interaction between an aromatic and cationic residue. The strength of noncovalent interactions involving Phe or Trp with Lys or Arg were assessed through double-mutant cycles. The diagonal interactions observed ranged from −0.20 to −0.48 kcal/mole, which are similar in magnitude to cross-strand interactions (Smith and Regan 1995; Merkel et al. 1999) and the stability gained from β-sheet propensities (Minor and Kim 1994; Smith et al. 1994).
The magnitude of the cation–π interaction in this system is significantly lower than that predicted by theory (Gallivan and Dougherty 2000), but the observed order of stabilities correlates well with that determined for proteins (Gallivan and Dougherty 1999a, 2000). In this system, we have found that Trp is the preferred aromatic group and Arg is the preferred cation for formation of a cation–π interaction, in agreement to the preferences found in proteins. The magnitudes of the interactions reported here are similar to those measured in α-helical model systems (Andrew et al. 2001; Olson et al. 2001; Shi et al. 2002; Tsou et al. 2002). These investigations found that both Orn–Phe and Arg–Trp interactions in the i, i + 4 positions of an α-helix yielded a stabilizing interaction (Andrew et al. 2002; Shi et al. 2002; Tsou et al. 2002). The magnitude of the cation–π interactions in α-helices varied but did not exceed −0.4 kcal/mole, compared with a range of −0.2 and −0.5 kcal/mole observed here.
Based on upfield shifting of the cationic side chain and NOE resonances, the favored geometries for FK, WK, and cyclic WK were found to direct the ε-methylene, and to a lesser extent the δ-methylene, of Lys at the aromatic ring. This orientation allows the protonated amine to remain solvated by water while packing the ε-methylene against the aromatic ring. Analysis of the PDB has shown that cation–π interactions with Lys typically involve the ε-methylene (Cε). As many as 60% of all Lys residues in proteins interact through Cε, as we have seen in our model system (Gallivan and Dougherty 1999b). The interaction of Lys via Cε likely reflects some combination of hydrophobic and electrostatic forces, because the methylene is highly polarized by the neighboring ammonium group yet poorly solvated by water (Reetz 1988). It is notable that NMR chemical shifts in the α-helical studies indicate an interaction with the Orn δ-methylene and Lys ε-methylene, indicating a similar type of geometry as observed here (Tsou et al. 2002).
In cation–π interactions with Arg, we have found that the guanidinium group orients itself parallel to the Trp ring, as evidenced by the side-chain chemical shifts. The FR peptide is less definitive, as the Cδ is more shielded than are the amines, but the shifting of the amines implicates at least some parallel geometry. The stacking of Arg with Trp is also in accord with findings in protein structures. In the stacked geometry, the delocalized π system in the guanidinium group allows for favorable van der Waals contacts with the π system of the aromatic side chain as well as electrostatic interactions. In addition, Arg is less well solvated than is Lys so the desolvation penalty for interaction with the aromatic ring is reduced. Although the cation–π interaction in “T” geometry has been proposed to be stronger than in the parallel geometry, the nonelectrostatic contacts seem to favor the parallel orientation, as has been observed in proteins (Gallivan and Dougherty 1999b).
With regard to the aromatic residues, we have found that Trp interacts with a cation more favorably than does Phe, although the observed differences in this system are modest. This trend is also seen in proteins, with a remarkable 24% of all Trp residues interacting with cationic residues in proteins, according to a study of the PDB (Gallivan and Dougherty 1999a).
Thermal denaturation studies have shown that folding of these peptides is enthalpically favorable and entropically unfavorable. Although there are still only a few β-hairpins for which thermodynamic parameters have been reported, most that are well folded also demonstrate enthalpically driven folding. Typically, this is associated with an aromatic (Espinosa and Gellman 2000; Cochran et al. 2001; Tatko and Waters 2002) or hydrophobic (Tatko and Waters 2002) cluster on the NHB face of the β-hairpin. Interestingly, those peptides with hydrophilic residues on the NHB face of the hairpin are the ones that demonstrate cold denaturation and a classical hydrophobic driving force (Ciani et al. 2003; Kiehna and Waters 2003). This may be a result of hydrophobic interactions on the HB face of these hairpins. Espinosa and Gellman (2000) has proposed that the enthalpic driving force in a peptide with an aromatic cluster may be the result of a “tight” interaction, as defined by Diederich et al. (Smithrud and Diederich 1990; Smithrud et al. 1991). In the system described here, a “tight” interaction may result from the contribution of an electrostatic component between the aromatic residue and the cationic residue, which may provide specificity to the interaction. Further studies are underway to explore this possibility.
In our study of cation–π interactions in a minimalist β-hairpin system, we have found the same geometric preferences for these interactions as have been observed in protein structures. Specifically, Lys prefers to interact via the ε-CH2 group, and Arg prefers to stack with the aromatic ring. The energetic trends observed here agree with the predicted trends, although the magnitude of the interactions was significantly lower than predicted. Nonetheless, the interaction energies are in good agreement with those found in model α-helical systems. Trp was found to be the preferred aromatic residue, and Arg was the preferred cationic residue. Thermal denaturation studies indicate that folding of these peptides is enthalpically driven, similar to other β-hairpins containing aromatic or hydrophobic clusters.