Vinyl ketone reagents for covalent protein modification. Nitroxide derivatives suited to rotational diffusion studies by saturation transfer electron spin resonance, using membrane-bound Na,K-ATPase as an example.
The reactivity of a series of substituted vinyl ketone nitroxides with an integral membrane protein, the Na,K-ATPase, is described. Increasing the electrophilicity of the conjugated double bond enhances reactivity markedly, with some spin labels showing higher reactivity than the conventionally used maleimide derivatives. The spectroscopic characteristics of the spin-labeled protein are also better suited for motional analysis by the saturation transfer electron spin resonance (STESR) method than with previous labeling procedures. The rotational correlation time, deduced from STESR experiments, is in the same range (100-300 microseconds) irrespective of the vinyl ketone derivative used, and the rotational mobility corresponds to an (alpha beta)2 or higher oligomer of the membrane-bound Na,K-ATPase.
Esmann
M
M
Institute of Biophysics, University of Aarhus, Denmark.
Hankovszky
H O
HO
Hideg
K
K
Pedersen
J A
JA
Marsh
D
D
D000255:Adenosine Triphosphate / Q000378:metabolism; D004058:Diffusion; D004578:Electron Spin Resonance Spectroscopy; D004789:Enzyme Activation; D004800:Enzymes, Immobilized; D007659:Ketones / Q000737:chemistry*; D007700:Kinetics; D008565:Membrane Proteins / Q000737:chemistry*; D008566:Membranes / Q000201:enzymology; D009589:Nitrogen Oxides / Q000737:chemistry; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism; D013113:Spin Labels; D013997:Time Factors; D014753:Vinyl Compounds / Q000737:chemistry*
D004800:Enzymes, Immobilized; D007659:Ketones; D008565:Membrane Proteins; D009589:Nitrogen Oxides; D013113:Spin Labels; D014753:Vinyl Compounds; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase