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 three different pH levels corresponding to the zwitterionic peptide containing a formal positive charge (pH 3.1), the deprotonated molecule (pH 9.1), and a situation at neutral pH (pH 7.2) involving both protonated and deprotonated states of the reduced peptide bond. Analysis of the one-dimensional1H-nmr spectra reveals that in solution TIP[psi] is in slow dynamic exchange between conformations containing cis and trans configurations of the Tyr-Tic bond. An nmr pH dependence study of the cis:trans ratio indicated that the exchange process was governed by the protonation state of the reduced bone amine. From the nmr data, reduced peptide bond pK alpha values of 6.5 and 7.5 were determined for the cis and trans conformers, respectively. It was concluded that conformations containing a trans Tyr-Tic bond are stabilized at low pH by an intramolecular hydrogen bond between the Tyr carbonyl and the reduced peptide bond protonated amine. This observation was corroborated by molecular mechanics investigations that revealed low energy trans structures compatible with nmr structural data, and furthermore, were consistently characterized by the existence of a strong N+ H ... O = C interaction closing a seven-membered cycle. The dynamics of cis-trans isomerization about the Tyr-Tic peptide bond were probed by nmr exchange experiments. The selective presaturation of exchanging resonances carried out at several temperatures between 50 and 70 degrees C allowed the determination of isomerization rate constants as well as thermodynamic activation parameters. delta G not equal to values were in close agreement with the cis-->trans energy barrier found in X-Pro peptide fragments (approximately 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 J K-1 mol-1 at pH 9.1) is discussed in terms of decreased solvent molecular ordering around the conformers possessing a trans Tyr-Tic bond. Evidence that the neutral form of the reduced peptide bond gains rigidity upon protonation was obtained from relaxation measurements in the rotating frame. T1 rho measurements of several protons in the vicinity of the reduced peptide bond were made as a function of spin-lock field.(ABSTRACT TRUNCATED AT 400 WORDS)
Carpenter
K A
KA
Laboratory of Chemical Biology and Peptide Research, Clinical Research Institute of Montreal, Quebec, Canada.
Wilkes
B C
BC
Schiller
P W
PW
D000595:Amino Acid Sequence; D006863:Hydrogen-Ion Concentration; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D010084:Oxidation-Reduction; D010455:Peptides / Q000737:chemistry*; D011487:Protein Conformation
D010455:Peptides