Md. Abu Khaled

Conformational states of enkephalins in solution

Two pentapeptides with opiate activity, [Met5] enkephalin and [Leu5] enkephalin, were studied by means of PMR, CMR, UV and CD spectroscopies in different solvents and at different concentrations. The primary result which we report is the demonstration of a concentration dependence. Spectral properties which are characteristically used to evaluate conformation are shown to differ at different concentrations. This provides an explanation for conflicting results of previous studies. Two conformational states of enkephalins which are consistent with the data are considered: i) A monomeric form, containing a β-turn with Gly3 and Phe4 at the corners, a 7-atom H-bond and the folding of the Tyr1 aromatic side chain over the molecule stabilized by an interaction of its OH proton with the Gly3 CO. ii) An associated form with an antiparallel cross-β-structure stabilized by four intermolecular H-bonds and with a “head to tail” interaction.

Characterization of micellar-packaged gramicidin A channels.

The effects of heating, on an aqueous gramicidin A lysolecithin system, were examined by carbon-13 nuclear magnetic resonance (13C-NMR), circular dichroism (CD), and sodium-23 nuclear magnetic resonance (23Na-NMR), and the results are collectively interpreted to indicate micellar-packaging of gramicidin channels and cation occupancy in the channel. 13C-NMR of the gramicidin-lysolecithin system demonstrates a decrease in mobility of the micellar lipid on heating which is indicative of incorporation of gramicidin into the hydrophobic core of the micelle. A unique and reproducible CD spectrum is obtained for the heat incorporated state. Sodium-23 spin-lattice relaxation times (T1) demonstrated sodium interaction to be dependent on heat incorporation. The T1 identified interaction is blocked by silver ion which is known to block sodium transport through the channel in lipid bilayer studies. The temperature dependence of the sodium-23 line width defines an exchange process with an activation energy of 6.8 kcal/mole which is essentially the same as the activation energy reported for transport through the channel in lecithin bilayer studies, and the sodium exchange process is blocked by thallium ion which is also known to block sodium transport through the channel.

Nuclear overhauser enhancement demonstration of the type II β-turn in repeat peptides of tropoelastin

Abstract Nuclear Overhauser enhancement (NOE) experiments have been performed with the elastin peptides, namely; HCO-Val1-Pro2-Gly3-Gly4-OMe, t-Boc-Val1-Pro2-Gly3-Val4-Gly5-OMe and t-Boc-Val6-Ala1-Pro2-Gly3-Val4-Gly5-OMe in DMSO-d6. An NOE of approximately 10% was observed between the αCH of Pro2 and the NH of Gly3 involved in the β-turn of all three peptides. This finding shows the close proximity of two aforementioned protons and thus shows the occurrence of Type II β-turn in the repeat elastin peptides. The intermolecular distances are calculated and compared with the distances obtained from other model systems.

The γ-turn as an independent conformational feature in solution

Abstract The γ-turn, an 11-membered hydrogen-bonded ring formed by a 1 → 3 type H-bond, has been observed in chloroform in the tripeptide, t-Boc-Gly1-L-Val2-Gly3-OMe, by proton magnetic resonance spectroscopy. This tripeptide sequence occurs in repeat penta- and hexapeptides of elastin where it also forms a γ-turn. The dihedral angles of γ-turns, which were proposed previously on the bases of theoretical calculations and x-ray diffraction, are compared with the results obtained by proton magnetic resonance. The results reported here demonstrate for the first time that the γ-turn can exist in solution as an independent conformational feature.

Nuclear overhauser enhancement evidence for inverse temperature dependence of hydrophobic side chain proximity in the polytetrapeptide of tropoelastin

Abstract Previous studies on aqueous solutions of HCO-(Val-Pro-Gly-Gly)40-Val-OMe indicated an increase in secondary structure on increasing the temperature implying a concomitant intramolecular hydrophobic association. Nuclear Overhauser enhancement (NOE) studies are reported which explicitly demonstrate an increase in association of γ CH 3 of Val and δ CH 2 of Pro protons on increasing temperature. The analogue where Ala replaces Val does not show this inverse temperature transition. These results provide direct demonstration of the “hydrophobic effect” responsible for inverse temperature transitions in aqueous systems.

Temperature-induced incorporation of gramicidin a into lysolecithin micelles demonstrated by 13C NMR

Lipid-protein interactions play a fundamental role in the organization and function of biological membranes. Gramicidin A, a pentadecapeptide, forms a transmembrane channel by adopting a singlestranded P-helical structure whose helix axis coincides with the channel axis [l-3]. It was proposed [4] that gramicidin A first forms large aggregates at the interface of the lipid bilayer then releases the aggregates that necessarily penetrate the lipid layer in order to form active channels. This aggregation could be viewed as the hydrophobic association of gramicidin A sidechains in an aqueous phase. Being familiar with elevated temperature inducing hydrophobic association in elastin peptides [5,6], it was our purpose to investigate whether or not elevated temperatures could drive the gramicidin A molecules to incorporate into micelles and result in transmembrane channels. Simple 13C spectra of lipid molecules could be utilized to monitor changes in chemical shifts and/or signal intensities of carbon resonances, if such changes were concurrent with the incorporation of gramicidin A molecules in the membrane systems. Therefore, we undertook to study the incorporation of gramicidin A in a micellar system of L-cr-lysolecithin in deuterated water as a function of temperature. The results are reported here.

Nuclear magnetic resonance studies on a cyclic dodecapeptide analogue of a repeat hexapeptide of tropoelastin: evaluation of secondary structure.

The cyclododecapeptide, (Ala1-Pro2-Gly3-Val4-Gly5-Val6)2, was synthesized and its secondary structure was evaluated from extensive studies in dimethyl sulphoxide, trifluoroethanol and water using NMR methods. A selective decoupling technique in 13C-NMR has been utilized in order to assign the C=O carbon resonances. Temperature dependence of the peptide NH protons and the solvent perturbation of the peptide NH and C=O resonances show the occurrence in all solvents of a beta-turn (a 10-membered H-bond between the Val4 NH and Ala1 C=O) and a gamma-turn, an 11-membered H-bond between the Gly3 NH and the Gly5 C=O; and a possible 14-membered H-bond between the Ala1 NH and the Val4 C=O in dimethyl sulphoxide and trifluoroethanol. These secondary structural features are compared with the linear polyhexapeptide and found the the beta-turn and the gamma-turn are the common conformational features of these peptide systems.

Proton magnetic resonance and conformational energy calculations of repeat peptides of tropoelastin: the pentapeptide

The detailed conformation of a repeating pentapeptide segment, HCO-L-Val1-L-Pro2-Gly3-L-Val4-Gly5-OMe, of tropoelastin has been investigated using theoretical conformational energy calculations and 1H n.m.r. studies in CDCl3. Theoretical conformational energy calculations suggest the existence of two broad classes of conformations. One class of conformations (A) is stabilized by a Type II β-turn, involving the Val4 NH and the Val1 CO, a 14-membered hydrogen bonded ring between the Val1 NH and the Val4 CO, and an 11-membered hydrogen bonded system, called a γ-turn, between the Gly3 NH and the Gly5 CO. The second class of conformations (B) is stabilized by the same Type II β-turn and 11-membered hydrogen bonded ring and by a seven-membered hydrogen bonded ring between the Gly5 NH and the Gly3 CO. The theoretical results correlate reasonably well with torsion angles derived from 3JCαH–NH coupling constants obtained in the 1H n.m.r. studies. Temperature dependence and solvent perturbation of NH proton chemical shifts support the above intramolecular hydrogen bonds.

Proton magnetic resonance and conformational energy calculations of repeat peptides of tropoelastin. A permutation of the hexapeptide.

The conformation of a hexapeptide sequence occurring in tropoelastin is discussed from the results obtained using a combined approach of theoretical conformational energy calculations on HCO-Val-Ala-Prb-Gly-OMe and 1h nmr studies on t-Boc-Val-Ala-Pro-Gly-Val-Gly-OMe in a dilute solution of methanol. Both studies have reasonable concurrence with respect to the preferred conformation of the hexapeptide and an analysis of the combined results suggests that the hexapeptide is stabilized by a beta-turn involving the Ala1,iC=O and Val4,iNH groups and a gamma-turn involving Gly5,iC=O and Gly3,iNH groups. A weaker interaction between Gly3,iC=O and Gly5,iNH groups is also found to be possible. Conformational features of the first valyl residue in the sequence Val-Ala-Pro-Gly-Val-Gly and the last valyl residue in Ala-Pro-Gly-Val-Gly-Val are compared and found to have similar torsion angles. The implications of such a similarity are discussed with respect to the conformation of the polyhexapeptide.

Conformational study of the cyclic hexapeptide L-Ala-L-Pro-Gly-L-Val-Gly-L-Val, by nuclear magnetic resonance spectroscopy

The cyclohexapeptide [graphic omitted] has been synthesized and its conformational features have been derived from n.m.r. parameters in chloroform. The nuclear Overhauser effect (n.O.e) has been used to distinguish between the two glycine and valine residues in the molecule. 13C Spin–lattice relaxation times (T1) have been measured and an average T1 value of 189 ms with a range of 158–202 ms for the α-carbon atoms has been obtained, which is indicative of a relatively rigid molecule. The n.O.e. and the geminal coupling constants [2J] and 3J(NH–αCH) have been utilized to obtain an estimation of the torison angles ψ and ϕ. Two β-turns, formed between the Ala1 NH and the Val4(CO (type II′) and the Val4 NH and the Ala1 CO (type II), are proposed. The conformational properties of this cyclic molecule are discussed in relation to the conformational features of its linear counterpart.