Chiral Inversion of Amino Acids in Antiparallel β-Sheets at Interfaces Probed by Vibrational Sum Frequency Generation Spectroscopy

Abstract

A parallel study of protein variants with all (L-), all (D-), or mixed (L-)/(D-) amino acids can be used to assess how backbone architecture versus side chain identity determines protein structure. Here, we investigate the secondary structure and side chain orientation dynamics of the antiparallel β-sheet peptide LK 7 β (Ac-Leu-Lys-Leu-Lys-Leu-Lys-Leu-NH 2 ) composed of all (L-), all (D-), or alternating (L-Leu)/(D-Lys) amino acids. Using interface-selective vibrational sum frequency generation spectroscopy (VSFG), we observe that the alternating (L-)/(D-) peptide lacks a resonant C−H stretching mode compared to the (L-) and (D-) variants and does not form antiparallel β-sheets. We rationalize our observations on the basis of density functional theory calculations and molecular dynamics (MD) simulations of LK 7 β at the air−water interface. Irrespective of the handedness of the amino acids, leucine side chains prefer to orient toward the hydrophobic air phase while lysine side chains prefer the hydrophilic water phase. These preferences dictate the backbone configuration of LK 7 β and thereby the folding of the peptide. Our MD simulations show that the preferred side chain orientations can force the backbone of a single strand of (L-) LK 7 β at the air−water interface to adopt β-sheet Ramachandran angles. However, denaturation of the β-sheets at pH = 2 results in a negligible chiral VSFG amide I response. The combined computational and experimental results lend critical support to the theory that a chiral VSFG response requires macroscopic chirality, such as in β-sheets. Our results can guide expectations about the VSFG optical responses of proteins and should improve understanding of how amino acid chirality modulates the structure and function of natural and de novo proteins at biological interfaces.

N–H Chirality in Folded Peptide LK7β Is Governed by the Cα–H Chirality

“…Therefore, in the folded peptide LK 7 β, the chirality of the C α -H group controls the chirality of the N-H group. Since the LK 7 β peptide with mixed L-and D-amino acids does not fold as recently reported 27,35 and in our own measurement, such one-on-one chiral correspondence between the C α -H group chirality and the N-H chirality in the folded peptide LK 7 β suggests deep connections in the structure of the amino-acids chirality to the chiral structure of folded peptides and proteins. Further studies in this direction may provide clues on understanding of the basic architecture of the folded protein, protein folding processes and the questions of the origin of life.…”

Mirror-image antiparallel β-sheets organize water molecules into superstructures of opposite chirality

“…To gain further insights about the hydration structure around LK 7 β, we performed MD simulations and hydrogen-bond analyses (see Materials and Methods and SI Appendix). LK 7 β was modeled as a protein composed of five β-strands at the vacuum-water interface, as previously reported (11). The MD simulations confirmed that the amphiphilic LK 7 β adopts a stable pleated, antiparallel β-sheet structure (SI Appendix, Figs.…”

“…1 A, Top) of (L-) LK 7 β is centered at 1,620 cm −1 , indicating the formation of antiparallel β-sheet structures. Previously, we showed that the homodyne chiral SFG signal vanishes when the pH of the sample is lowered to ∼2, which denatures the antiparallel β-sheets, or when the LK 7 β peptide is composed of (L-) leucine and (D-) lysine, which disrupts β-sheet formation (11). These experiments demonstrated that the chiral SFG response originates from the folded β-sheet secondary structure.…”

“…is strongly amphiphilic, with the hydrophobic leucine sidechains orienting toward the hydrophobic phase and the polar lysine sidechains orienting toward the hydrophilic phase (11). This sidechain orientation forces the peptide backbone to adopt Ramachandran angles standard for antiparallel β-sheets (11). Hence, it is energetically favorable for the peptide to self-assemble into antiparallel β-sheet structures.…”

“…Indeed, the LK 7 β peptide and its variants in native (L-) form have been commonly used as a model system in surface studies. (2,(8)(9)(10)(11)(12)(15)(16)(17) We obtain the phase-resolved chiral SFG vibrational spectra of (L-) and (D-) LK 7 β in the spectral regions of the O-H/N-H stretching at ∼3,200 cm −1 using H 2 O as solvent and the O-D/N-D stretching at ∼2,400 cm −1 using D 2 O. We also perform the experiments using H 2 18 O and D 2 18 O.…”