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Kinetics of binding and diffusivity of leucine-enkephalin in large unilamellar vesicle by pulsed-field-gradient 1 H NMR in situ

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ABSTRACT

The kinetics of binding, the diffusivity, and the binding amount of a neuropeptide, leucine-enkephalin (L-Enk) to lipid bilayer membranes are quantified by pulsed-field-gradient (PFG) 1H NMR in situ. The peptide signal is analyzed by the solution of the Bloch equation with exchange terms in the presence of large unilamellar vesicles (LUVs) as confined, but fluid model cell membranes. Even in the case that the membrane-bound and the free states of L-Enk cannot be distinguished in the one-dimensional NMR spectrum, the PFG technique unveils the bound component of L-Enk after the preferential decay of the free component at the high field gradient. In 100-nm diameter LUVs consisting of egg phosphatidylcholine, the rate constants of the peptide binding and dissociation are 0.040 and 0.40 s−1 at 303 K. This means that the lifetime of the peptide binding is of the order from second to ten-second. The diffusivity of the bound L-Enk is 5×10−12m2/s, almost 60 times as restricted as the movement of free L-Enk at 303K. One-tenth of 5mM L-Enk is bound to 40mM LUV. The binding free energy is calculated to be −2.9 kJ/mol, the magnitude close to the thermal fluctuation, 2.5 kJ/mol. The result demonstrates the potential of PFG 1H NMR to quantify molecular dynamics of the peptide binding to membranes.

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1D 1H NMR spectra of 5mM L-Enk in the presence (black) and absence (red) of EPC LUV. The aromatic region of the Tyr and Phe residues of L-Enk is expanded in the inset. Arrows show the EPC signals used for determining the diffusion coefficient of the lipid in membrane.
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f1-7_105: 1D 1H NMR spectra of 5mM L-Enk in the presence (black) and absence (red) of EPC LUV. The aromatic region of the Tyr and Phe residues of L-Enk is expanded in the inset. Arrows show the EPC signals used for determining the diffusion coefficient of the lipid in membrane.

Mentions: As shown in the 1H NMR spectrum (Fig. 1), it is difficult to distinguish the bound and free components of L-Enk; see the detail in section 4.1. In this case, the individual peak height analysis is impossible, and hence the peak integral


Kinetics of binding and diffusivity of leucine-enkephalin in large unilamellar vesicle by pulsed-field-gradient 1 H NMR in situ
1D 1H NMR spectra of 5mM L-Enk in the presence (black) and absence (red) of EPC LUV. The aromatic region of the Tyr and Phe residues of L-Enk is expanded in the inset. Arrows show the EPC signals used for determining the diffusion coefficient of the lipid in membrane.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC5036784&req=5

f1-7_105: 1D 1H NMR spectra of 5mM L-Enk in the presence (black) and absence (red) of EPC LUV. The aromatic region of the Tyr and Phe residues of L-Enk is expanded in the inset. Arrows show the EPC signals used for determining the diffusion coefficient of the lipid in membrane.
Mentions: As shown in the 1H NMR spectrum (Fig. 1), it is difficult to distinguish the bound and free components of L-Enk; see the detail in section 4.1. In this case, the individual peak height analysis is impossible, and hence the peak integral

View Article: PubMed Central - PubMed

ABSTRACT

The kinetics of binding, the diffusivity, and the binding amount of a neuropeptide, leucine-enkephalin (L-Enk) to lipid bilayer membranes are quantified by pulsed-field-gradient (PFG) 1H NMR in situ. The peptide signal is analyzed by the solution of the Bloch equation with exchange terms in the presence of large unilamellar vesicles (LUVs) as confined, but fluid model cell membranes. Even in the case that the membrane-bound and the free states of L-Enk cannot be distinguished in the one-dimensional NMR spectrum, the PFG technique unveils the bound component of L-Enk after the preferential decay of the free component at the high field gradient. In 100-nm diameter LUVs consisting of egg phosphatidylcholine, the rate constants of the peptide binding and dissociation are 0.040 and 0.40 s−1 at 303 K. This means that the lifetime of the peptide binding is of the order from second to ten-second. The diffusivity of the bound L-Enk is 5×10−12m2/s, almost 60 times as restricted as the movement of free L-Enk at 303K. One-tenth of 5mM L-Enk is bound to 40mM LUV. The binding free energy is calculated to be −2.9 kJ/mol, the magnitude close to the thermal fluctuation, 2.5 kJ/mol. The result demonstrates the potential of PFG 1H NMR to quantify molecular dynamics of the peptide binding to membranes.

No MeSH data available.


Related in: MedlinePlus