BIOPHYSICS
Online ISSN : 1349-2942
ISSN-L : 1349-2942
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Kinetics of binding and diffusivity of leucine-enkephalin in large unilamellar vesicle by pulsed-field-gradient 1H NMR in situ
Noriyuki YoshiiTomomi EmotoEmiko Okamura
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2011 Volume 7 Pages 105-111

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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 5×10–12 m2/s, almost 60 times as restricted as the movement of free L-Enk at 303 K. One-tenth of 5 mM L-Enk is bound to 40 mM 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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© 2011 THE BIOPHYSICAL SOCIETY OF JAPAN
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