CONFORMATIONAL STUDY ON PROLINE-CONTAINING TRIPEPTIDES OF RIBONUCLEASE-T(1)

In order to investigate the role of the cis/trans isomerization of the X-Pro peptide bonds in the folding of ribonuclease T1 (RNase T1), con formational free energy calculations using an empirical potential ECEP P/2 and the hydration shell model were carried out on the terminally b locked tripeptides Ac-Tyr-Pro-His-NHMe, Ac-Ser-Pro-Tyr-NHMe, Ac-Trp-Pr o-Ile-NHMe, and Ac-Ser-Pro-Gly-NHMe and on related dipeptides in the u nhydrated and hydrated states. These tripeptides correspond to residue s 38-40, 54-56, 59-61, and 72-74 of native RNase T1, respectively. The conformational entropy computed using a harmonic method was included in the free energy of each minimum in both states. In the hydrated sta te, our results show that, when the X-Pro peptide bond is cis, Ac-Tyr- Pro-His-NHMe has relatively high probabilities of type VI (P(H) = 0.97 ) and I (P(H) = 0.82) beta-bends at Tyr-Pro and Pro-His, respectively, and that Ac-Ser-Pro-Tyr-NHMe adopts a type VI beta-bend at Ser-Pro (P (H) = 0.61). The conformations of type VI beta-bends at X-Pro are cons istent with those observed in the corresponding sequences of native RN ase T1. On the other hand, only Ac-Trp-Pro-Ile-NHMe favors a type I be ta-bend at Pro-Ile (P(H) = 0.49) with a trans peptide bond at Trp-Pro. The calculated low free energy conformations of the tripeptides in th e hydrated state are not superimposed quite well on the crystal struct ures of the corresponding sequences of RNase T1. This implies that lon g-range interactions are of significant importance in stabilizing the conformations of these proline-containing sequences of RNase T1. The f ree energy difference between the trans and cis conformers in water is less for Ac-Tyr-Pro-His-NHMe and Ac-Ser-Pro-Tyr-NHMe than for Ac-Trp- Pro-Ile-NHMe and Ac-Ser-Pro-Gly-NHMe. In addition, the two former trip eptides have higher populations of cis conformers in both states. This indicates that the first two tripeptides have stronger tendencies to form cis-X-Pro peptide bonds than the latter two tripeptides, which is in good agreement with the results on the analysis of proline residue s in protein structures from the Brookhaven Protein Data Bank, and it accounts for why the two former X-Pro peptide bonds are cis in native RNase T1. The calculated activation energy for the trans-to-cis isomer ization of the Tyr-Pro peptide bond in Ac-Tyr-Pro-His-NHMe is the high est among the four tripeptides, and it suggests that the isomerization of the Tyr 38-Pro 39 peptide bond can be a rate-determining step in t he folding or refolding of RNase T1, which is consistent with recent k inetic results. The hydration significantly reduced the probabilities of occurrence of beta-bends for di- and tripeptides but is of no conse quence in the isomerization of X-Pro peptide bonds, which is also in a ccord with the related works reported previously.