Thermodynamic origin of cis/trans isomers of a proline-containing β-turn model dipeptide in aqueous solution: A combined variable temperature 1H-NMR, two-dimensional 1H,1H gradient enhanced nuclear overhauser effect spectroscopy (NOESY), one-dimensional steady-state intermolecular 13C,1H NOE, and molecular dynamics study

The cis/trans conformational equilibrium of the two Ac–Pro isomers of the β‐turn model dipeptide [13C]–Ac–L‐Pro–D‐Ala–NHMe, 98% 13C enriched at the acetyl carbonyl atom, was investigated by the use of variable temperature gradient enhanced 1H‐nmr, two‐dimensional (2D) 1H,1H nuclear Overhauser effect spectroscopy (NOESY), 13C,1H one‐dimensional steady‐state intermolecular NOE, and molecular dynamics calculations. The temperature dependence of the cis/trans Ala(NH) protons are in the region expected for random‐coil peptides in H2O (Δδ/ΔT = −9.0 and −8.9 ppb for the cis and trans isomers, respectively). The trans NH(CH3) proton indicates smaller temperature dependence (Δδ/ΔT ∼ −4.8 ppb) than that of the cis isomer (−7.5 ppb). 2D 1H,1H NOESY experiments at 273 K demonstrate significant NOEs between ProHα—AlaNH and AlaNH—NH(R) for the trans isomer. The experimental NOE data, coupled with computational analysis, can be interpreted by assuming that the trans isomer most likely adopts an ensemble of folded conformations. The C–CONH(CH3) fragment exhibits significant conformational flexibility; however, a low‐energy conformer resembles closely the βII‐turn folded conformations of the x‐ray structure of the related model peptide trans‐BuCO–L‐Pro–Me–D‐Ala–NHMe. On the contrary, the cis isomer adopts open conformations. Steady‐state intermolecular solute–solvent (H2O) 13C,1H NOE indicates that the water accessibility of the acetyl carbonyl carbons is nearly the same for both isomers. This is consistent with rapid fluctuations of the conformational ensemble and the absence of a highly shielded acetyl oxygen from the bulk solvent. Variable temperature 1H‐nmr studies of the cis/trans conformational equilibrium indicate that the trans form is enthalpically favored (ΔH° = −5.14 kJ mole−1) and entropically (ΔS° = −5.47 J · K−1 · mole−1) disfavored relative to the cis form. This demonstrates that, in the absence of strongly stabilizing sequence‐specific interresidue interactions involving side chains and/or charged terminal groups, the thermodynamic difference of the cis/trans isomers is due to the combined effect of intramolecular and intermolecular (hydration) induced conformational changes. © 2000 John Wiley & Sons, Inc. Biopoly 53: 72–83, 2000