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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.

No MeSH data available.


PFG 1H NMR spectra of the aromatic region of L-Enk in the presence of LUV at 303K. Here, the 16 FG strengths g = 0.05, 0.15, 0.225, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.91T/m are applied (from top to bottom) with the pulse width of 0.004 s at the diffusion time, 0.1 s.
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f2-7_105: PFG 1H NMR spectra of the aromatic region of L-Enk in the presence of LUV at 303K. Here, the 16 FG strengths g = 0.05, 0.15, 0.225, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.91T/m are applied (from top to bottom) with the pulse width of 0.004 s at the diffusion time, 0.1 s.

Mentions: The line broadening and the peak shift in Figure 1 imply that L-Enk is bound to the membrane. The bound component of L-Enk is, however, hardly separated from free L-Enk in the 1D NMR spectrum. To separate the bound component from the peptide signal, we apply the PFG technique. The criterion is that the signal assignable to the free peptide preferentially decays with increasing the field gradient (FG) strength to unveil the bound component at the high FG2. In Figure 2 is demonstrated how the signal of L-Enk is attenuated by applying the magnetic field gradient. It is evident that all signals decay in intensity with increasing the FG strength (from top to bottom). Instead, the broad components are uncovered. The intensities of the broad components are less attenuated up to the FG strength of 0.91T/m (the bottom). From these results, the broad components are considered to be bound L-Enk with its motion slowed down in the viscous membrane environment21,25. The situation is quite similar to the 5FU binding reported previously2.


Kinetics of binding and diffusivity of leucine-enkephalin in large unilamellar vesicle by pulsed-field-gradient 1 H NMR in situ
PFG 1H NMR spectra of the aromatic region of L-Enk in the presence of LUV at 303K. Here, the 16 FG strengths g = 0.05, 0.15, 0.225, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.91T/m are applied (from top to bottom) with the pulse width of 0.004 s at the diffusion time, 0.1 s.
© Copyright Policy
Related In: Results  -  Collection

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

f2-7_105: PFG 1H NMR spectra of the aromatic region of L-Enk in the presence of LUV at 303K. Here, the 16 FG strengths g = 0.05, 0.15, 0.225, 0.30, 0.35, 0.40, 0.45, 0.50, 0.55, 0.60, 0.65, 0.70, 0.75, 0.80, 0.85, 0.91T/m are applied (from top to bottom) with the pulse width of 0.004 s at the diffusion time, 0.1 s.
Mentions: The line broadening and the peak shift in Figure 1 imply that L-Enk is bound to the membrane. The bound component of L-Enk is, however, hardly separated from free L-Enk in the 1D NMR spectrum. To separate the bound component from the peptide signal, we apply the PFG technique. The criterion is that the signal assignable to the free peptide preferentially decays with increasing the field gradient (FG) strength to unveil the bound component at the high FG2. In Figure 2 is demonstrated how the signal of L-Enk is attenuated by applying the magnetic field gradient. It is evident that all signals decay in intensity with increasing the FG strength (from top to bottom). Instead, the broad components are uncovered. The intensities of the broad components are less attenuated up to the FG strength of 0.91T/m (the bottom). From these results, the broad components are considered to be bound L-Enk with its motion slowed down in the viscous membrane environment21,25. The situation is quite similar to the 5FU binding reported previously2.

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.