Slow diffusion of macromolecular assemblies by a new pulsed field gradient NMR method.
The translational diffusion coefficient of an integral membrane protein/surfactant complex has been measured using a novel pulsed field gradient NMR method. In this new approach, the information about the localization of the molecules is temporarily stored in the form of longitudinal magnetization of isotopes with long spin-lattice relaxation times. This allows one to increase the duration of the diffusion interval by about 1 order of magnitude. Unlike standard proton NMR methods using pulsed field gradients and stimulated echoes, the new method can be applied to macromolecular assemblies with diffusion coefficients well below 10(-10) m(2) s(-1), corresponding to masses in excess of 25 kDa in aqueous solution at room temperature. The method was illustrated by application to a water-soluble complex of tOmpA, the hydrophobic transmembrane domain of bacterial outer membrane protein A, with the detergent octyl-tetraoxyethylene (C(8)E(4); overall mass of complex approximately 45 kDa). The diffusion coefficient was found to be D = (4.99 +/- 0.07) x 10(-11) m(2) s(-1), consistent with measurements by size exclusion chromatography and by ultracentrifugation. The method has also been applied to a solution of recombinant human tRNA(3)(Lys), which has a molecular mass of 24 kDa, and the diffusion coefficient D = (1.05 +/- 0.015) x 10(-10) m(2) s(-1).
Ferrage
Fabien
F
Département de Chimie, associé au CNRS, Ecole Normale Supérieure, 24 rue Lhomond, F-75231 Paris Cedex 05, France.
Zoonens
Manuela
M
Warschawski
Dror E
DE
Popot
Jean-Luc
JL
Bodenhausen
Geoffrey
G
D001425:Bacterial Outer Membrane Proteins / Q000737:chemistry*; D004058:Diffusion; D004926:Escherichia coli / Q000737:chemistry; D005030:Ethylenes / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular / Q000379:methods*; D017434:Protein Structure, Tertiary; D013501:Surface-Active Agents / Q000737:chemistry*
D001425:Bacterial Outer Membrane Proteins; D005030:Ethylenes; D013501:Surface-Active Agents; C078167:OMPA outer membrane proteins