A carbon-13 nuclear magnetic resonance study of the molecular dynamics of methionine-enkephalin and .alpha.-endorphin in aqueous solution

'H spectra at 270 MHz of the Met-enkephalin pentapeptide (Tyr-Gly-Gly-Phe-Met) in aqueous solution, as a function of pH and of temperature, are reported.The analysis of the chemical shifts, line widths, coupling constants and rotamer populations around x1 and x2, suggests the existence of three conformational regions in the pH range 1-10, due to some rearrangements of the first two amino acids, Tyr-1 and Gly-2. A first conformational transition occurs corresponding to the N-terminus titration, with an exchange rate between conformational states which is not too fast. The second transition occurs concomitantly with the phenolic group titration. The conformational states of the cationic and zwitterionic forms are probably very similar and are characterized by thc abscncc of intramolccular hydrogcn bonds and of head-to-tail intcractions. A bent structure, with the Tyr-1 ring facing the Gly-2 could contribute to the equilibrium of the conformational states of Met-enkephalin in the pH range 8 -10. This structure could be destabilized as a consequence of the phenolic group deprotonation.The Met-5 and Phe-4 residues appear to have little sensitivity to the conformational transitions of the backbone and both show a settling of the side chains corresponding to the carboxyl group titration, thus showing a reciprocal steric hindrance.The temperature coefficients of the chemical shifts of amide protons at acidic pH are in the range of solvated peptides. In the temperature range 0 -75 "C, the 3 J ,~~ coupling constants were independent of temperature.Met-enkephalin (Tyr-Gly-Gly-Phe-Met), an endogenous pentapeptide that acts on opiate receptors, has recently been the subject of several papers, both experimental and theoretical, aimed at establishing the relationship between structure and activity. Its possible conformations were studied by optical spectroscopy [l], X-ray of related fragments [2], semiempirical conformational calculations [3 -61 and NMR spectroscopy [7-121, 13C and 'H relaxation measurements, both in (C2H3)2S0 and in 2H20 [7, lo], exclude a random-coil structure and agree in showing a rigid backbone, with possible rotational freedom of the Met-5 and Phe-4 side chains and a higher degree of hindrance for the Tyr-1 side chain. Recent proton magnetic resonance measurements in water, (C2H3)2S0 and a mixture of these two [13-151, have apparently In fact, because of its high polarity, water might destabilize intramolecular H bonds and cause unfolded structures with amides exposed to the solvent, while (C2H3)2S0 might protect the intramolecular H bonds and then the folded structures.In order to contribute to the clarification of these results, we have undertaken a systematic study of the spectral characteristics of Met-enkephalin as a function of pH and of temperature in water, this being the most suitable solvent for the reproduction of the physiological environment. Even though the study of Met-enkephalin in other solvents gives information on the stability of the energetically possible conformational states, from...

Section: Resultssupporting

confidence: 94%

Section: Glycine-2 and Glycine-3mentioning

confidence: 99%

270‐MHz ¹H Nuclear‐Magnetic‐Resonance Study of Met‐enkephalin in Water

Zetta

Cabassi

Tomatis

et al. 1979

“…Since TT1 = ndvli2, T1 can be estimated directly from the linewidths. Activation energies for rotational processes [53] were determined from T , values using Arrhenius expression l/T1 = l/Tl"exp (EIRT),…”

Section: Results and Discussion "0-nmr And Ftir Data On Peptide -Hzo mentioning

confidence: 99%

Hydration of the Gly2 and Gly3 peptide oxygens of [Leu5]‐enkephalin in aqueous solution as revealed by the combined use of ¹⁷O‐NMR and Fourier‐transform infrared spectroscopy

Gerothanassis

Birlirakis

Karayannis

et al. 1992

Solvent-induced and temperature-induced ' 0 chemical shifts of , LeuSI-enkephalin and solvent-induced spectral modifications of the amide-I' stretching vibrations of [1-13C-Gly2, Leu51-enkephalin and [1-13C-Gly2, Leu51-enkephalin are reported and correlated with the spectroscopic characteristics of model amides. It is demonstrated that both Gly2 and Gly3 peptide oxygens are motionally equivalent and form solvation species which are essentially monohydrated in aqueous solution, contrary to several simple amides and model peptides in which water largely forms dihydrates. It is shown that the combined use of 170-NMR and Fourier transform infrared is a unique methodology for studying the hydration state of specific peptide oxygens in peptide hormones.Peptides and proteins in biological systems are usually surrounded by a predominantly aqueous solvent. Their small size, large dipole moment and high capacity for forming hydrogen bonds means that the water molecules have unique effects on peptide and protein conformations [l -31. Hydrogen-bonding interactions between the oxygen atom of the peptide groups and the molecules of water undoubtedly contribute to the overall conformational stability of peptides and proteins in aqueous medium. The knowledge of these solvent interactions is of importance for any conformational study since it is known that biomolecules often change their conformation as the solvent is varied.As a first step towards the solution of hydration of peptides and proteins, numerous experimental and theoretical investigations on the most probable sites for binding of the water molecules have been undertaken on amides [4-lo]. These molecules are considered as the simplest representatives of the peptide bond; therefore, the determination of the possible monohydrated and multihydrated species has been considered as crucial in gauging the magnitude of specific hydration of more complex peptides and proteins. Furthermore, several theoretical calculations of model peptides [11 -201 and numerous X-ray structural investigations of several peptide hydrates and proteins [21-281 revealed thermodynamical and geometrical aspects of peptide-backbone hydration.

“…Usually the I3C chemical shift of one amino acid is only slightly affected by its position in the sequence of a peptide. In the case of peptides I and 11, whatever the solvent used, the I3C chemical shifts are very different from those found in Met-enkephalin or Leu-enkephalin [15,16,18,19]. Furthermore, the chemical shift sepa-rations between each corresponding proton or carbon in the cis and truns forms of peptides I and I1 are very large in comparison to those found in thyroliberin (< Glu-His-Pro-NHz) [32].…”

Section: Resultsmentioning

confidence: 99%

“…The nuclear Overhauser enhancement effects are only 60 % of the maximal value indicating that, at 68 MHz, the relaxation occurs outside the 'extreme narrowing limit' (ohf I 1). For peptides of the size of I, several studies have shown that rotational diffusion is highly anisotropic whatever the conformation (extended or folded) adopted by the peptide [18,40]. However, despite these limitations, several very interesting conclusions can be made.…”

Section: I3c Relaxation Measurementsmentioning

confidence: 99%

Studies of the Conformational Behaviour and Preferential Interactions with Opiate Receptors of the cis and trans Forms of [dMet2, Pro5]Enkephalin and [dMet2, Pro5]Enkephalinamide by 1H and 13C NMR, Theoretical Calculations and 13C Relaxation Measurements

Roques¹,

Garbay‐Jaureguiberry²,

Bajusz³

et al. 2005

[DMet', Pro5]Enkephalin (I) and [DMet', Pro']enkephalinamide (11), two of the more potent opioid peptides, have been studied by 'H and I3C N M R and by theoretical calculations. Their biological activity is tentatively related to their conformational behaviour. A cis S trans equilibrium around the Phe-Pro bond is brought about by the terminal prolyl residue. The cisjtrans ratios lie around 50j50 for peptide I (55 cis in dimethylsulfoxide, 35 o/, cis in water) and around 20/80 for peptide 11, the more potent compound, in both the solvents. Almost all the signals, both in 'H and I3C spectra, give separate resonances for the cis and trans rotamers, in dimethylsulfoxide as well as in water. A temperature variation allows one to determine the rotational barrier (AG;, z 81.5 kJ/mol) for the cis $ trans interconversion. The analysis of the vicinal coupling constants and theoretical calculations suggests that Tyr-DMet-Gly-Phe-Pro exists as a class of highly puckered conformations with short end-to-end distances at r = 0.4 nm and r = 0.6 nm for the trans and cis configurations respectively. The populations of the side-chain rotamers are predominantly trans/gauche (tg) for the tyrosine residue. This feature, which is quite general in enkephalins, is related to the crucial role of the tyramine moiety for opioid activity. The orientation of the Phe-4 side-chain is independent of the spatial disposition of the following prolyl residue. pH titrations provide evidence for the existence of head-to-tail interactions. Hence, in ('H6)dimethylsulfoxide, and to a lesser extent in 'HzO, the titration of the Pro-5 residue is clearly felt on the Tyr-1 moiety, whereas the deprotonation of this latter residue leads to changes in the chemical shifts of the pyrrolidine ring protons. The B protons of the Tyr-1 and Phe-4 residues are strongly magnetically nonequivalent; the opposite is true for Met-enkephalin where they show the same chemical shifts below pH 6. The titration effects suggest that no strong conformational change occurs during the amino or carboxyl group titration and indicate an important steric hindrance on the N M R time scale for the two aromatic side chains.The 13C NMR spectra of peptide I in ('Hg)dimethylsulfoxide and in a mixture of ('H6)dimethylsulfoxidej'H20 (50/50) exhibit for almost all the resonances well-separated signals, corresponding to the cis and trans conformers. The large changes in I3C chemical shifts occurring Qn the backbone and side-chain carbons during the titration steps confirm the through-space effects already deduced from the proton titration curves. Hence, several C-a (Met-2, Phe-4) and C-p (Met-2, Pro-5) atoms are affected by both the titrations of the two terminal parts of the peptide. Such effects were not observed in Met-enkephalin or Leu-enkephalin. The I3C relaxation times for the cis and trans rotamers were compared, in ('H6)dimethylsulfoxide, under strictly the same conditions. All the NTl values are small and lie in the same range both for the carbons of the peptide backbone and for the ca...