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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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Intensity decay of the L-Enk signal as a function of the FG strength at the diffusion time Δdiff of 0.1 (filled circle), 0.5 (filled triangle), and 1.0 s (filled square). Here the signal intensity is evaluated as the peak integral of the aromatic region in Figure 2. Symbols represent the experimental values. The red lines are obtained by fitting Eq. (3) to the experimental values of peak integrals at the respective Δdiff. The blue and the green lines demonstrate the decay curves evaluated by Eqs. (10) and (11), respectively. The dashed black lines are the fitting result of the Stejskal-Tanner plot at the large g limit (g→∞), from which the diffusion coefficients of bound L-Enk, DB are calculated. The results of Δdiff =0.5 and 1.0 s are shifted to negative value by −1.0 and −2.0, respectively.
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f3-7_105: Intensity decay of the L-Enk signal as a function of the FG strength at the diffusion time Δdiff of 0.1 (filled circle), 0.5 (filled triangle), and 1.0 s (filled square). Here the signal intensity is evaluated as the peak integral of the aromatic region in Figure 2. Symbols represent the experimental values. The red lines are obtained by fitting Eq. (3) to the experimental values of peak integrals at the respective Δdiff. The blue and the green lines demonstrate the decay curves evaluated by Eqs. (10) and (11), respectively. The dashed black lines are the fitting result of the Stejskal-Tanner plot at the large g limit (g→∞), from which the diffusion coefficients of bound L-Enk, DB are calculated. The results of Δdiff =0.5 and 1.0 s are shifted to negative value by −1.0 and −2.0, respectively.

Mentions: The result is summarized in Figure 3 at various diffusion times, Δdiff. The circles, triangles, and squares designate the experimental values at Δdiff of 0.1, 0.5, and 1.0 s, respectively. The red lines are obtained by fitting Eq. (3) to the experimental values. Each line well reproduces the observed decay of the signal intensities. The dashed black lines are the fitting results of Eq. (13) to the signal intensity at the large g limit (g→∞). Thus we can evaluate the diffusivity and the rate of the binding and dissociation of L-Enk from Eqs. (3) and (13). It is found that the signal intensity decay is a biexponential function with respect to the FG strength, in contrast to the single exponential decay of L-Enk in water (not shown). The intensities rapidly attenuate at τm2 < 2×1010m−2 s, while slowly at τm2 > 2×1010m−2 s. The non-linear behavior of the signal attenuation is due to the exchange of the peptide molecule between free and bound states that is similar to the 5FU binding to the membrane3.


Kinetics of binding and diffusivity of leucine-enkephalin in large unilamellar vesicle by pulsed-field-gradient 1 H NMR in situ
Intensity decay of the L-Enk signal as a function of the FG strength at the diffusion time Δdiff of 0.1 (filled circle), 0.5 (filled triangle), and 1.0 s (filled square). Here the signal intensity is evaluated as the peak integral of the aromatic region in Figure 2. Symbols represent the experimental values. The red lines are obtained by fitting Eq. (3) to the experimental values of peak integrals at the respective Δdiff. The blue and the green lines demonstrate the decay curves evaluated by Eqs. (10) and (11), respectively. The dashed black lines are the fitting result of the Stejskal-Tanner plot at the large g limit (g→∞), from which the diffusion coefficients of bound L-Enk, DB are calculated. The results of Δdiff =0.5 and 1.0 s are shifted to negative value by −1.0 and −2.0, respectively.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC5036784&req=5

f3-7_105: Intensity decay of the L-Enk signal as a function of the FG strength at the diffusion time Δdiff of 0.1 (filled circle), 0.5 (filled triangle), and 1.0 s (filled square). Here the signal intensity is evaluated as the peak integral of the aromatic region in Figure 2. Symbols represent the experimental values. The red lines are obtained by fitting Eq. (3) to the experimental values of peak integrals at the respective Δdiff. The blue and the green lines demonstrate the decay curves evaluated by Eqs. (10) and (11), respectively. The dashed black lines are the fitting result of the Stejskal-Tanner plot at the large g limit (g→∞), from which the diffusion coefficients of bound L-Enk, DB are calculated. The results of Δdiff =0.5 and 1.0 s are shifted to negative value by −1.0 and −2.0, respectively.
Mentions: The result is summarized in Figure 3 at various diffusion times, Δdiff. The circles, triangles, and squares designate the experimental values at Δdiff of 0.1, 0.5, and 1.0 s, respectively. The red lines are obtained by fitting Eq. (3) to the experimental values. Each line well reproduces the observed decay of the signal intensities. The dashed black lines are the fitting results of Eq. (13) to the signal intensity at the large g limit (g→∞). Thus we can evaluate the diffusivity and the rate of the binding and dissociation of L-Enk from Eqs. (3) and (13). It is found that the signal intensity decay is a biexponential function with respect to the FG strength, in contrast to the single exponential decay of L-Enk in water (not shown). The intensities rapidly attenuate at τm2 < 2×1010m−2 s, while slowly at τm2 > 2×1010m−2 s. The non-linear behavior of the signal attenuation is due to the exchange of the peptide molecule between free and bound states that is similar to the 5FU binding to the membrane3.

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&minus;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&times;10&minus;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 &minus;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