INFLUENCE OF SAMPLE PH ON THE CONFORMATIONAL BACKBONE DYNAMICS OF A PSEUDOTRIPEPTIDE (H-TYR-TIC-PSI[CH2-NH]PHE-OH) INCORPORATING A REDUCED PEPTIDE-BOND - AN NMR INVESTIGATION

In the present paper we investigate the influence of sample pH on the conformational and dynamical properties of the pseudotripeptide H-Tyr- Tic psi[CH2-NH]Phe-OH (TIP[psi]; Tic: 1,2,3,4-tetrahydroisoquinoline-3 -carboxylic acid) using various one-and two-dimensional nmr techniques in conjunction with molecular modeling. Studies were conducted at thr ee different pH levels corresponding to the zwitterionic peptide conta ining a formal positive charge (pH 3.1), the deprotonated molecule (pH 9.1), and a situation at neutral pH (pH 7.2) involving both protonate d and deprotonated states of the reduced peptide bond. Analysis of the one-dimensional H-1-nmr spectra reveals that in solution TIP[psi] is in slow dynamic exchange between conformations containing cis and tran s configurations of the Tyr-Tic bond. An nmr pH dependence study of th e cis:trans ratio indicated that the exchange process was governed by the protonation state of the reduced bond amine. From the nmr data, re duced peptide bond pK(a) values of 6.5 and 7.5 were determined for the cis and trans conformers, respectively. It was concluded that conform ations containing alpha trans Tyr-Tic bond are stabilized at low pH by an intramolecular hydrogen bond between the Tyr carbonyl and the redu ced peptide bond protonated amine. This observation was corroborated b y molecular mechanics investigations that revealed low energy trans st ructures compatible with nmr structural data, and furthermore, were co nsistently characterized by the existence of a strong N+ H ... O=C int eraction closing a seven-membered cycle. The dynamics of cis-trans iso merization about the Tyr-Tic peptide bond were probed by nmr exchange experiments. The selective presaturation of exchanging resonances carr ied out at several temperatures between 50 and 70 degrees C allowed th e determination of isomerization rate constants as well as thermodynam ic activation parameters. Delta G(not equal) values were in close agre ement with the cis --> trans energy barrier found in X-Pro peptide fra gments (similar to 83 kJ/mol). A large entropic barrier determined for the trans --> cis conversion of TIP[psi] (5.7 JK(-1) mol(-1) at pH 3. 1;6.5 JK(-1) mol(-1) at PH 9.1) is discussed in terms of decreased sol vent molecular ordering around the conformers possessing alpha trans T yr-Tic bond. Evidence that the neutral form of the reduced peptide bon d gains rigidity upon protonation was obtained from relaxation measure ments in the rotating frame. T-1p measurements of several protons in t he vicinity for the reduced peptide bond were made as a function of sp in-lock field. Quantitative analysis of the relaxation data indicated that chemical shift fluctuations in the 10(-1)-10(-5) s range were mor e pronounced in the case of deprotonated TIP[psi]. Results of molecula r dynamics simulations in addition to (3)J(alpha beta) coupling consta nt measurements support the experimentally observed greater flexibilit y in the C-terminal region of TIP[psi]. (C) 1995 John Wiley & Sons, In c.