Origin of β-Hairpin Stability in Solution:  Structural and Thermodynamic Analysis of the Folding of a Model Peptide Supports Hydrophobic Stabilization in Water

Abstract

The origin of the stability of isolated β-hairpins in aqueous solution is unclear with contrasting opinions as to the relative importance of interstrand hydrogen bonding, hydrophobic interactions, and conformational preferences, the latter being associated largely with the turn sequence. We have designed an unconstrained 16-residue peptide that we show folds autonomously in water to form a β-hairpin that mimics the two-stranded anti-parallel β-sheet DNA binding motif of the met repressor dimer. The designed peptide, with a type I‘ turn (INGK), is shown by CD and a range of NMR parameters to be appreciably folded (≈50% at 303 K) in aqueous solution with the predicted alignment of the peptide backbone. We show that the folding transition approximates to a two-state model. The hairpin has a marked temperature-dependent stability, reaching a maximum value at 303 K in water with both lower and higher temperatures destabilizing the folded structure. Van't Hoff analysis of Hα chemical shifts, reveals that folding is endothermic and entropy-driven in aqueous solution with a large negative ΔC p, all of which are reminiscent of proteins with hydrophobic cores, pointing to the hydrophobic effect as the dominant stabilizing interaction in water. We have examined the conformational properties of the C-terminal β-strand (residues 9−16) in isolation and have shown that 3 J α N values and backbone intra- and inter-residue Hα-NH NOE intensities deviate from those predicted for a random coil, indicating that the β-strand has a natural predisposition to adopt an extended conformation in the absence of secondary structure interactions. A family of β-hairpin structures calculated from 200 (distance and torsion angle) restraints using molecular dynamics shows that the conformation of the hairpin mimics closely the DNA binding face of the met repressor dimer (backbone RMSD between corresponding β-strands of 1.0 ± 0.2 Å).

A fully atomistic computer simulation study of cold denaturation of a β-hairpin

“…From the simulated melting curves, thermodynamic parameters were obtained by best fit to a two-state folding model 12 ( Table 1). Consistent with the NMR-based result, the predicted DH 298 K and DS 298 K on unfolding are both negative.…”

“…The coordinates of the NMR native structure of the bhairpin peptide (MrH1) are not directly available from the literatures. Hence, the native structure of this peptide was computer-modeled by taking advantage of 30 NMR f/c angle restraint data 12 . This peptide is then solvated by a total of 2,577 water molecules ions in a dodecahedron box of a size 48.4 Å.…”

“…This model peptide contains a total of 16 residues, of which 6 are hydrophobic (TYR3, VAL5, ILE7, ILE12, VAL14 and ILE16). Furthermore, a single hydrophobic cluster formed by these non-polar residues provides the major stabilizing factor of the b-hairpin structure in water 12 . Under ambient conditions, MrH1 is marginally stable in water (a folding fraction of about 50%), but when the temperature is decreased from the temperature of maximum folding fraction (T max ¼ 303 K), this peptide displays a gradual loss of the native structure, as shown by its NMR-based melting profile (folding fraction versus temperature) 12 .…”

“…Furthermore, a single hydrophobic cluster formed by these non-polar residues provides the major stabilizing factor of the b-hairpin structure in water 12 . Under ambient conditions, MrH1 is marginally stable in water (a folding fraction of about 50%), but when the temperature is decreased from the temperature of maximum folding fraction (T max ¼ 303 K), this peptide displays a gradual loss of the native structure, as shown by its NMR-based melting profile (folding fraction versus temperature) 12 . On the basis of the thermodynamic ground of protein stability, characteristic features of cold denaturation can be investigated by producing these melting curves.…”

Cross-Strand Coupling and Site-Specific Unfolding Thermodynamics of a Trpzip β-Hairpin Peptide Using 13C Isotopic Labeling and IR Spectroscopy

“…Studies of isolated, water-soluble -hairpins can provide valuable insight into properties of -sheet structure and folding since -hairpins are considered to act as possible nucleation sites for protein folding. [1][2][3][4][5][6][7][8][9][10][11][12][13][14] Additionally, their detailed structural interactions can be relevant in developing an understanding of the mechanism for forming various -sheet structures such as found in many (amyloid-like) neurodegenerative diseases in which protein aggregation is an important pathology. [15][16][17][18] During the past decade, there have been many reports discussing de novo designed, water-soluble -hairpin peptide systems, as well documented in several reviews.…”