1. Bioactivity of peptide analogs of the neutrophil chemoattractant, N-acetyl-proline-glycine-proline
J L Haddox, R R Pfister, D D Muccio, M Villain, C I Sommers, M Chaddha, G M Anantharamaiah, W J Brouillette, L J DeLucas Invest Ophthalmol Vis Sci. 1999 Sep;40(10):2427-9.
Purpose: The release of N-acetyl-proline-glycine-proline (PGP), a chemoattractant resulting from direct alkaline hydrolysis of corneal proteins, is believed to be the initial trigger for neutrophil invasion into the alkali-injured cornea. The purpose of this study is twofold: (1) to compare the activity of N-acetyl-PGP with the bioactivities of other similar synthetic peptides in an effort to uncover information about this chemoattractant molecule, and (2) to test these peptide analogs as potential antagonists of N-acetyl-PGP. Methods: The polarization assay was used to measure the potential chemotactic response of human neutrophils to peptides. Bioactivity was expressed as the peptide concentration required to produce 50% neutrophil polarization (EC50). Antagonist activity was expressed as the peptide concentration required to produce 50% inhibition (ID50) of polarization activated by N-acetyl-PGP. Results: Peptide bioactivities (EC50) were ranked as follows: APGPR (0.34 mM) > N-acetyl-PGP (0.5 mM) > N-(PGP)4-PGLG (3 mM) = t-Boc-PGP (3 mM) > N-acetyl-PG (3.4 mM) > N-methyl-PGP (15 mM) = PGP (15 mM) > peptides without detectable activity (t-Boc-PGP-OMe, N-acetyl-P, PG, PGG, GP, GG and gly-pro-hyp). Peptides with no detectable bioactivity were tested as potential antagonists of neutrophil polarization induced by N-acetyl-PGP. Gly-Pro-Hyp inhibited N-acetyl-PGP activation of polarization at 20 mM (ID50). No other synthetic peptide demonstrated a capacity for inhibition. Conclusions: The minimum requirement to elicit bioactivity was the presence of PGP alone or derivatives of PG in which the N-terminal proline is blocked. Using this approach, active and inactive mimetic peptides of N-acetyl-PGP were produced. The most active peptide, APGPR, was equal to or slightly greater than N-acetyl-PGP, suggesting that more potent analogs might be designed. Gly-pro-hyp was the only inactive peptide analog to inhibit the chemoattractant.
2. 1H- and 13C-NMR investigations on cis-trans isomerization of proline peptide bonds and conformation of aromatic side chains in H-Trp-(Pro)n-Tyr-OH peptides
J Poznański, A Ejchart, K L Wierzchowski, M Ciurak Biopolymers. 1993 May;33(5):781-95. doi: 10.1002/bip.360330507.
1H and 13C high-resolution nmr spectra of cationic, zwitterionic, and anionic forms of the peptides: H-Trp-(Pro)n-Tyr-OH, n = 0-5, and H-Trp-Pro-OCH3 were obtained in D2O solution. Analysis of H alpha (Pro1), H alpha (Trp), C gamma (Pro), H epsilon (Tyr), and H delta (Trp) resonances provided evidence for the presence of two predominant backbone isomers: the all-trans one and another with the Trp-Pro peptide bond in cis conformation; the latter constituted about 0.8 molar fraction of the total peptide (n > 1) concentration. Relative content of these isomers varied in a characteristic way with the number of Pro residues and the ionization state of the peptides. The highest content of the cis (Trp-Pro) isomer, 0.74, was found in the anionic form of H-Trp-Pro-Tyr-OH; it decreased in the order of: anion >> zwitterion approximately cation, and with the number of Pro residues to reach the value of 0.42 in the cationic form of H-Trp-(Pro)5-Tyr-OH. Isomerization equilibria about Pro-Pro bond(s) were found to be shifted far (> or = 0.9) in favor of the trans conformation. Interpretation of the measured vicinal coupling constants J alpha-beta' and J alpha-beta" for C alpha H-C beta H2 proton systems of Trp and Tyr side chains in terms of relative populations of g+, g-, and t staggered rotamers around the chi 1 dihedral angle indicated that in all the peptides studied (a) rotation of Trp indole ring in cis (Trp-Pro) isomers is strongly restricted, and (b) rotation of Tyr phenol ring is relatively free. The most preferred chi 1 rotamer of Trp (0.8-0.9 molar fraction) was assigned as the t one on the basis of a large value of the vicinal coupling constant between the high-field H beta and carbonyl carbon atoms of Trp, estimated for the cis (Pro1) form of H-Trp-Pro-Tyr-OH from a 1H, 13C correlated spectroscopy 1H-detected multiple quantum experiment. This indicates that cis<-->trans equilibrium in the Trp-Pro fragment is governed by nonbonding interactions between the pyrrolidine (Pro) and indole (Trp) rings. A molecular model of the terminal cis Trp-Pro dipeptide fragment is proposed, based on the presented nmr data and the results of our molecular mechanics modeling of low-energy conformers of the peptides, reported elsewhere.
3. Thermodynamic origin of cis/trans isomers of a proline-containing beta-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
A Troganis, I P Gerothanassis, Z Athanassiou, T Mavromoustakos, G E Hawkes, C Sakarellos Biopolymers. 2000 Jan;53(1):72-83. doi: 10.1002/(SICI)1097-0282(200001)53:13.0.CO;2-5.
The cis/trans conformational equilibrium of the two Ac-Pro isomers of the beta-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 (delta delta/delta T = -9.0 and -8.9 ppb for the cis and trans isomers, respectively). The trans NH(CH3) proton indicates smaller temperature dependence (delta delta/delta T approximately -4.8 ppb) than that of the cis isomer (-7.5 ppb). 2D 1H,1H NOESY experiments at 273 K demonstrate significant NOEs between ProH alpha-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 beta II-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 (delta H degree = -5.14 kJ mole-1) and entropically (delta S degree = -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.
