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.
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.
Kovacs
James M
JM
Department of Biochemistry and Molecular Genetics, University of Colorado at Denver and Health Sciences Center, Aurora, CO 80045, USA.
Mant
Colin T
CT
Kwok
Stanley C
SC
Osguthorpe
David J
DJ
Hodges
Robert S
RS
D000596:Amino Acids / Q000737:chemistry*; D002851:Chromatography, High Pressure Liquid; D002942:Circular Dichroism; D057927:Hydrophobic and Hydrophilic Interactions; D008958:Models, Molecular; D009842:Oligopeptides / Q000737:chemistry* / Q000302:isolation & purification*; D011487:Protein Conformation; D013237:Stereoisomerism
D000596:Amino Acids; D009842:Oligopeptides