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

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

The Δdiff dependence of (a) the diffusion coefficients, (b) the rate constants of the binding and dissociation, and (c) the binding amount of L-Enk at 303K.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC5036784&req=5

f4-7_105: The Δdiff dependence of (a) the diffusion coefficients, (b) the rate constants of the binding and dissociation, and (c) the binding amount of L-Enk at 303K.

Mentions: The diffusivity of the free and the bound peptide is evaluated by fitting Eqs. (3) and (13) to the experimental A(g) in Figure 3. The fitting of Eq. (3) to the experimental value in the whole range of g gives the diffusion coefficient of the free peptide, DF, together with the rate constants kFB and kBF. Next, we calculate the diffusion coefficient of the bound peptide, DB, by using the linear relation of the decay plot (Stejskal-Tanner plot) at g→∞3; see the dashed black lines. The results are summarized in Figure 4a as a function of the time Δdiff. It is found that DF and DB give the values of (2.9±0.1)×10−10 and (5.0±1.4)×10−12m2/s, respectively, at 303K26. The diffusivity of bound L-Enk is almost 60 times as restricted as free L-Enk. Both values are independent of the time, Δdiff, indicating the accuracy of the experiment. DF is in good agreement with the diffusion coefficient of L-Enk in water (DW = (3.3±0.1)×10−10m2/s), and DB corresponds to that of the lipid membrane (DM = (7.3±0.7)×10−12m2/s) obtained simultaneously by the EPC signal27. All results demonstrate that DF and DB are the diffusion coefficients of free and bound L-Enk in the presence of the vesicle.


Kinetics of binding and diffusivity of leucine-enkephalin in large unilamellar vesicle by pulsed-field-gradient 1 H NMR in situ
The Δdiff dependence of (a) the diffusion coefficients, (b) the rate constants of the binding and dissociation, and (c) the binding amount of L-Enk at 303K.
© Copyright Policy
Related In: Results  -  Collection

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

f4-7_105: The Δdiff dependence of (a) the diffusion coefficients, (b) the rate constants of the binding and dissociation, and (c) the binding amount of L-Enk at 303K.
Mentions: The diffusivity of the free and the bound peptide is evaluated by fitting Eqs. (3) and (13) to the experimental A(g) in Figure 3. The fitting of Eq. (3) to the experimental value in the whole range of g gives the diffusion coefficient of the free peptide, DF, together with the rate constants kFB and kBF. Next, we calculate the diffusion coefficient of the bound peptide, DB, by using the linear relation of the decay plot (Stejskal-Tanner plot) at g→∞3; see the dashed black lines. The results are summarized in Figure 4a as a function of the time Δdiff. It is found that DF and DB give the values of (2.9±0.1)×10−10 and (5.0±1.4)×10−12m2/s, respectively, at 303K26. The diffusivity of bound L-Enk is almost 60 times as restricted as free L-Enk. Both values are independent of the time, Δdiff, indicating the accuracy of the experiment. DF is in good agreement with the diffusion coefficient of L-Enk in water (DW = (3.3±0.1)×10−10m2/s), and DB corresponds to that of the lipid membrane (DM = (7.3±0.7)×10−12m2/s) obtained simultaneously by the EPC signal27. All results demonstrate that DF and DB are the diffusion coefficients of free and bound L-Enk in the presence of the vesicle.

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