Dynamic properties of the backbone of an integral membrane polypeptide measured by ²H-NMR

Abstract

The ²H-NMR spectrum of the exchangeable hydrogens of the synthetic amphiphilic polypeptide, lys₂-gly-leu₂₄-lys₂-ala-amide, was measured for the solid peptide at room temperature and, as a function of temperature, for the peptide incorporated into hydrated dipalmitoylphosphatidylcholine (DPPC) bilayers. This study is a prototype of a similar class of experiments which can be carried out on integral membrane proteins to characterize, quantitatively, the dynamic properties of integral membrane proteins. At temperatures below the DPPC gel-liquid crystalline phase transition, the ²H NMR spectrum was very similar to that of the solid peptide indicating that the peptide was immobilized in the lipid bilayer on the time scale (≈10^-5 s) of the ²H-NMR measurements. The ²H-NMR spectrum above the phase transition corresponded to that expected from a peptide in the α-helical conformation reorienting rapidly about the symmetry axis of the α-helix. Measurements of the quadrupolar echo relaxation time, T _2e , gave a quantitative measure of the correlation time, τ _c , for this motion. The value of τ _c decreased rapidly with increasing temperature as the fraction of DPPC molecules in the liquid crystalline phase increased, reaching a value of 2×10^-7s above the phase transition. The observation of a characteristic minimum in T _2e as the temperature was raised provided a definitive, quantitative interpretation of the T _2e measurements. Using the known geometry of the peptide and the theory of uniaxial rotational diffusion, a value of η=1.1 poise was obtained for the effective viscosity of the membrane in close agreement with values obtained previously from transient linear dichroism measurements.