On the role of nuclear quantum effects on the stability of peptides

TL;DR

This study addresses how nuclear quantum effects influence peptide stability and isotope substitution by applying path-integral molecular dynamics with a high-fidelity, ab initio-trained interatomic potential. It demonstrates that NQEs destabilize folded peptide structures across diverse motifs, with substantial contributions arising from non-HB C–H vibrations rather than HB strengthening, and shows that active-hydrogen deuteration stabilizes folds with an intrinsic isotope effect comparable to solvent isotope effects. The work provides mechanistic insight that challenges HB-centric views of NQEs in biomolecules and reveals the importance of intrinsic isotope effects in determining peptide stability under deuteration. Together, these findings enable more accurate predictions of isotope effects in biology and offer design principles for isotopic substitution strategies in biotech and pharmacology.

Abstract

Nuclear quantum effects (NQEs) arising from the light mass of hydrogen can influence the structure and stability of hydrogen-bonded biomolecules, yet their role in determining peptide and protein folding remains unclear. Experiments show that substituting H$_2$O with D$_2$O often stabilizes folded states, but the microscopic mechanism associated with this phenomena remains unresolved. Through ab initio-level path-integral molecular dynamics simulations enabled by machine-learning interatomic potentials, we address the fundamental question of the role of NQEs in peptides by investigating both their overall impact and isotope substitution effects. Overall, NQEs systematically destabilize compact three-dimensional structures across peptide systems, independent of secondary structure type or side-chain interactions. Contrary to the conventional picture that places central importance on hydrogen bonds, we find that the dominant destabilization instead arises from the quantum C-H vibrations. In addition, we reveal microscopic insights into the stabilization of folded peptides upon H$_2$O to D$_2$O substitution, showing that the H/D isotope substitution of active peptide hydrogens, previously considered unimportant, produces free-energy changes within the range of experimentally observed shifts. These findings provide a new interpretation of isotope effects in biological systems, indicating that seemingly small H$\to$D substitutions within peptides can be as important as, or even outweigh, solvent contributions.

Figures (5)

• Figure 1: Optimized structures of the peptide fragments investigated in this work. (a) ALA-10 in -helical conformation and (b) -hairpin conformation. Protein fragment structures from the Protein Data Bank: (c) frag-1 (from 5QI6 5qib_pdb), (d) frag-2 (from 3BWH 3bwh_pdb), (e) frag-3 (from 6F54 6f54_pdb), and (f) frag-4 (from 6HMD 6hmd_pdb). All structures are shown in stick representation (carbon in light cyan, oxygen in red, nitrogen in blue, sulfur in yellow, hydrogen in white), with secondary structure elements highlighted as ribbon representation. Hydrogen bonds that stabilize the secondary structures are shown as yellow dashed lines.
• Figure 2: Thermodynamic cycles illustrating the free energy relationships in peptide state transitions. (a) The gas-phase thermodynamic cycle showing relationships between peptide conformational states with classical and quantum nuclei. The vertical dimension represents conformational transitions between folded (F) and unfolded (U) states, while the horizontal dimension represents a continuous transition from quantum nuclei (q) to classical nuclei (c) through mass scaling. Boxes circled with ring-polymer beads represent systems with quantum nuclei, and Da means the active hydrogens in the system are deuterated. The double arrows represent changes in the folding free energies: the green double arrow represent the change in the folding free energy from classical nuclei to quantum nuclei, and the red double arrow represent the change folding free energy upon deuteration of the active hydrogens. (b) Thermodynamic cycle illustrating the partitioning of the experimentally-observed isotope effects on protein stability in solution into intrinsic and solvation contributions. The blue and yellow shading of the peptides represent Ha and Da states, respectively. The rectangles with transparent, blue and yellow background indicate vacuum, H2O and D2O environments, respectively. The experimentally-observed total free-energy change from H2O solvent to D2O solvent ($\Delta \Delta F_{\mathrm{U}\leftarrow \mathrm{F}}^{\mathrm{D}_{\mathrm{a}}\leftarrow \mathrm{H}_{\mathrm{a}}}\mathrm{( sol)}$) can be decomposed into two components: (1) the intrinsic peptide isotope effects $\Delta \Delta F_{\mathrm{U}\leftarrow \mathrm{F}}^{\mathrm{D}_{\mathrm{a}}\leftarrow \mathrm{H}_{\mathrm{a}}}$, and (2) the differential solvation free energy $\Delta \Delta \Delta F_{\mathrm{sol} ,\ \mathrm{U}\leftarrow \mathrm{F}}^{\mathrm{D}_{\mathrm{a}}\leftarrow \mathrm{H}_{\mathrm{a}}}$.
• Figure 3: Summary of NQEs on the stability ($\Delta \Delta F_{\mathrm{U}\leftarrow \mathrm{F}}^{\mathrm{q}\leftarrow \mathrm{c}}$ per residue) across all studied peptide systems. Bright green bars with black error bars represent PIMD simulation results, and dim green bars show harmonic approximation results. Positive value indicates NQEs destabilize the peptide's folded state relative to the unfolded state, while negative value indicates stabilization of the folded state due to NQEs.
• Figure 4: Decomposition of the NQEs on the folding free energies in the harmonic limit into contributions from the following modes: NH/OH stretching, CH stretching, C=O stretching, NH/OH bending, and Other/Collective modes. Each panel presents results for a different peptide system studied. Negative values indicate NQEs stabilize the folded state relative to the unfolded state, while positive values indicate the opposite. Blue background highlights HB vibrational modes, which are emphasized in the conventional CQE model, while pink background highlights non-HB modes.
• Figure 5: H/D isotope effects on peptide conformational stability. (a) Folding free energy change upon deuteration of the active hydrogens ($\Delta \Delta F_{\mathrm{U}\leftarrow \mathrm{F}}^{\mathrm{D_a}\leftarrow \mathrm{H_a}}$) calculated from PIMD for the six peptides. The range of experimental total isotope effects is shown in shaded region, with literature values marked for different protein/peptide systems: (i) Equine heart cytochrome c krantz2000d, (ii) Cross-linked GCN4 krantz2000d, (iii) Hen egg lysozyme Efimova_Biopolymers_2007_v85_p264, (iv) Bovine serum albumin Efimova_Biopolymers_2007_v85_p264, (v) Rat CD2 domain 1 Parker_Biochemistry_1997_v36_p5786, (vi) SH3 domain Stadmiller_ProteinSciPublProteinSoc_2018_v27_p1710. (b) Contributions of each individual active H that forms a stable HB in the folded conformer, plotted against trajectory-averaged HB length (heavy-atom separation distance). Points are colored to distinguish backbone (purple) from side-chain (yellow) HBs. Marker shapes represent different peptides: diamond for ALA-10 (helix), square for ALA-10 (hairpin), downward triangle for frag-1, upward triangle for frag-2, leftward triangle for frag-3, and rightward triangle for frag-4.