Water: foldase activity in catalyzing polypeptide conformational rearrangements

Abstract

Polypeptide conformer interconversion in a low dielectric environment is shown to be highly dependent on water concentration. Water increases this rate by 10(3), apparently by catalyzing hydrogen bond exchange, and thereby presenting functional properties analogous to that of a foldase. This catalytic effect is demonstrated on the interconversion of a parallel gramicidin dimer into an antiparallel dimer. A Hill coefficient of 6.5 is observed, illustrating the highly cooperative nature of the process. Protein folding in nonpolar environs, such as the hydrophobic core of a protein or the hydrophobic domain of a lipid bilayer, may be contingent on and rate-limited by the scarcity of water.

Figure 1

The GCOSY fingerprint regions for gA (12 mM) solutions during structural rearrangement in the presence (A–C) and “absence” (D–F) of water. The red cross peaks in the spectra represent the backbone NH-C^αH cross peaks of an antiparallel left-handed helical dimer (species 3), whereas the black cross peaks are from a mixture of parallel conformers. The horizontal axis is the actual time scale within which the NMR spectra were acquired and the vertical axis corresponds to the increase of the species 3 population over time. The initial and final peptide structures are shown as dimers with red/white double strands for the species 3 structure and black/white strands for the parallel structures. Trp₁₅ at the peptide C terminus is displayed to emphasize the relative orientation of the two peptide strands.

Figure 2

(A) The initial conformational interconversion rates as a function of water concentration. The rate constants were measured from buildup curves of the fully assigned resonances for the antiparallel conformer, and they range from 1.4 ± 0.21 × 10⁻⁵ to 1.0 ± 0.2 × 10⁻² min⁻¹. The much increased error bars for the faster rates reflect the time required for acquiring the initial GCOSY data set for each sample (≈ 18 min). The kinetic results were fit by using Eq. 1. (B) The Hill plot with a slope of 6.5 indicates a very substantial cooperativity for the catalytic activity of water, suggesting that the breaking of one pair of peptide hydrogen bonds rapidly facilitates the subsequent disrupting of neighboring and next nearest neighboring hydrogen-bond pairs. The data corresponding to 3–95% kinetic rate saturation are used for calculating the maximal slope for the Hill coefficient.

Figure 3

A proposed free energy profile for water-catalyzed gramicidin conformational interconversion. (Inset) A parallel dimer model showing from this view six inter-strand hydrogen-bond pairs, each associated with an interacting water molecule. It is proposed here that these water molecules compete with and break the interpeptide hydrogen bonds, hence destabilizing the structure. In addition, water can substantially reduce the transition-state energy barrier by forming hydrogen bonds with those exposed polar backbone amide and carbonyl groups in nonpolar environments during the structural rearrangement. The free energies corresponding to the initial and transition states are labeled G_↑↑ and G^†_↑↑, respectively. Note that the structure for the intermediate is drawn so as to suggest that one monomer has moved with respect to the other by a dipeptide. The subscripts +H₂O/-H₂O and black/purple lines denote the peptide system in the presence and absence of water, respectively. The interconversion rate enhancement is determined by ΔΔG = [(G^†_↑↑(+H2O) − G^†_{↑↑(−H2O)}) − (G_↑↑(+H2O) − G_{↑↑(−H2O)})], which reflects the additive effects of transition state stabilization and initial/final state destabilization.