Measurement of 15N relaxation in the detergent-solubilized tetrameric KcsA potassium channel.
A set of TROSY-HNCO (tHNCO)-based 3D experiments is presented for measuring (15)N relaxation parameters in large, membrane-associated proteins, characterized by slow tumbling times and significant spectral overlap. Measurement of backbone (15)N R (1), R (1rho), (15)N-{(1)H} NOE, and (15)N CSA/dipolar cross correlation is demonstrated and applied to study the dynamic behavior of the homotetrameric KcsA potassium channel in SDS micelles under conditions where this channel is in the closed state. The micelle-encapsulated transmembrane domain, KcsA(TM), exhibits a high degree of order, tumbling as an oblate ellipsoid with a global rotational correlation time, tau(c) = 38 +/- 2.5 ns, at 50 degrees C and a diffusion anisotropy, Dparallel/Dperpendicular = 0.79+/-0.05, corresponding to an aspect ratio a/b >/= 1.4. The N- and C-terminal intracellular segments of KcsA exhibit considerable internal dynamics (S (2) values in the 0.2-0.45 range), but are distinctly more ordered than what has been observed for unstructured random coils. Relaxation behavior in these domains confirms the position of the C-terminal helix, and indicates that in SDS micelles, this amphiphilic helix does not associate into a stable homotetrameric helical bundle. The relaxation data indicate the absence of elevated backbone dynamics on the ps-ns time scale for the 5-residue selectivity filter, which selects K(+) ions to enter the channel.
Chill
Jordan H
JH
Laboratory of Chemical Physics, NIDDK, National Institutes of Health, Building 5, Room 126, 9000 Rockville Pike, Bethesda, Maryland 20892, USA.
Louis
John M
JM
Baber
James L
JL
Bax
Ad
A
D016880:Anisotropy; D001426:Bacterial Proteins / Q000737:chemistry*; D003902:Detergents / Q000737:chemistry*; D008823:Micelles; D009587:Nitrogen Isotopes / Q000737:chemistry*; D019906:Nuclear Magnetic Resonance, Biomolecular; D015221:Potassium Channels / Q000737:chemistry*; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry*; D012995:Solubility
D001426:Bacterial Proteins; D003902:Detergents; D008823:Micelles; D009587:Nitrogen Isotopes; D015221:Potassium Channels; C107790:prokaryotic potassium channel; D012967:Sodium Dodecyl Sulfate