Quantitation of the nearest-neighbour effects of amino acid side-chains that restrict conformational freedom of the polypeptide chain using reversed-phase liquid chromatography of synthetic model peptides with l- and d-amino acid substitutions

Abstract

Side-chain backbone interactions (or "effects") between nearest neighbours may severely restrict the conformations accessible to a polypeptide chain and thus represent the first step in protein folding. We have quantified nearest-neighbour effects (i to i+1) in peptides through reversed-phase liquid chromatography (RP-HPLC) of model synthetic peptides, where L- and D-amino acids were substituted at the N-terminal end of the peptide sequence, adjacent to a L-Leu residue. These nearest-neighbour effects (expressed as the difference in retention times of L- and D-peptide diastereomers at pHs 2 and 7) were frequently dramatic, depending on the type of side-chain adjacent to the L-Leu residue, albeit such effects were independent of mobile phase conditions. No nearest-neighbour effects were observed when residue i is adjacent to a Gly residue. Calculation of minimum energy conformations of selected peptides supported the view that, whether a L- or D-amino acid is substituted adjacent to L-Leu, its orientation relative to this bulky Leu side-chain represents the most energetically favourable configuration. We believe that such energetically favourable, and different, configurations of L- and D-peptide diastereomers affect their respective interactions with a hydrophobic stationary phase, which are thus quantified by different RP-HPLC retention times. Side-chain hydrophilicity/hydrophobicity coefficients were generated in the presence of these nearest-neighbour effects and, despite the relative difference in such coefficients generated from peptides substituted with L- or D-amino acids, the relative difference in hydrophilicity/hydrophobicity between different amino acids in the L- or D-series is maintained. Overall, our results demonstrate that such nearest-neighbour effects can clearly restrict conformational space of an amino acid side-chain in a polypeptide chain.

Intrinsic amino acid side‐chain hydrophilicity/hydrophobicity coefficients determined by reversed‐phase high‐performance liquid chromatography of model peptides: Comparison with other hydrophilicity/hydrophobicity scales

“…Ideally, the distribution of each amino acid residue of the total residues present should be equal. In addition, any peptide secondary structure (induced by the hydrophobic environment of RP-HPLC 9-13) and/or conformational constraints caused by nearest-neighbor effects 14 may also affect measured coefficients, particularly in such a small sample size. Wilce et al 15, 16 noted that at least 100 retention time entries of different randomly selected peptides are required to sample the contribution of an amino acid side-chain in a suitably large number of peptide sequences.…”

Requirements for prediction of peptide retention time in reversed-phase high-performance liquid chromatography: Hydrophilicity/hydrophobicity of side-chains at the N- and C-termini of peptides are dramatically affected by the end-groups and location

“…Thus, nearest-neighbour effects during RP-HPLC imply sequence-dependent variability of peptide retention behaviour but independent of a defined secondary structure (β-turn, β-sheet or α-helical). Attempts to quantify such nearest-neighbour effects have been rare, although our laboratory has made a recent initial quantification of such effects by observation of the differences in observed retention times of L-and D-peptide diastereomers, where the model peptides varied only in the L-or D-amino acid substituted adjacent to a bulky leucine residue [34]. In addition, we have also developed a set of intrinsic hydrophilicity/hydrophobicity coefficients based on our model peptide approach where amino acid substitutions were made at the N-terminal end of the model peptide adjacent to a glycine residue, ensuring the complete absence of any conformational restraints on the substituted amino acid [35].…”

MALDI TOF/TOF-Based Approach for the Identification of d- Amino Acids in Biologically Active Peptides and Proteins

“…In principle, RP-HPLC can provide separation of epimeric peptides; their resolution, however, depends on the hydrophobicities of the d - residue and its nearest neighbors. 30 In consequence, the observation of a peptide mass appearing in several HPLC peaks may hint toward the presence of epimeric forms albeit other explanations may be likely as well. Moreover, a false positive epimer identification may occur during the highly sensitive MALDI-MS analysis because of detection of traces of the all- l peptide tailing after its actual peak, which often elutes prior to the epimer.…”