Structural and motional changes in glyceraldehyde-3-phosphate dehydrogenase upon binding to the band-3 protein of the erythrocyte membrane examined with [15N,2H]maleimide spin label and electron paramagnetic resonance.
Binding of the glycolytic enzyme, glyceraldehyde-3-phosphate dehydrogenase [GAPDHase; D-glyceraldehyde-3-phosphate:NAD+ oxidoreductase (phosphorylating EC 1.2.1.12], to the cytoplasmic segment of band-3 protein in the erythrocyte (RBC) membrane has been examined by electron paramagnetic resonance (EPR) and saturation transfer EPR (ST-EPR) spectroscopies. GAPDHase, which was isolated from rabbit muscle and labeled with the resolution-enhancing deuterated N-(15N-1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)maleimide spin label ([15N,2H]MSL), showed the same binding specificity for the transmembrane band-3 protein of human erythrocyte membranes as reported for unlabeled GAPDHase from human RBC. Experimental EPR lineshapes from soluble and membrane-bound enzymes were analyzed by direct stimulation of spectra and indicated a structural alteration of the bound GAPDHase in the vicinity of the spin label, which was attached covalently to the active-site cysteine-149 residue. A rigorous theoretical analysis of the ST-EPR spectra of soluble and membrane-bound enzyme is presented and utilized in conjunction with model system analysis to demonstrate that the motion of membrane-bound GAPDHase could be characterized by an effective isotropic rotational correlation time of 20 microseconds. This indicated that the GAPDHase--band-4 complex exhibits motional freedom relative to the membrane-spanning segment of the band-3 protein or the RBC. The double substituted spin label [15N,2H]MSL affords gains in sensitivity and resolution that permit studies of membrane-bound enzymes at physiological levels and quantitative simulations of the EPR and ST-EPR lineshapes with reasonable computation times.
D001457:Anion Exchange Protein 1, Erythrocyte; D001798:Blood Proteins / Q000378:metabolism*; D004578:Electron Spin Resonance Spectroscopy; D004910:Erythrocyte Membrane / Q000378:metabolism*; D004912:Erythrocytes / Q000378:metabolism*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000378:metabolism*; D006801:Humans; D008565:Membrane Proteins / Q000378:metabolism*; D009038:Motion; D011487:Protein Conformation; D012995:Solubility; D013113:Spin Labels; D014783:Viscosity
D001457:Anion Exchange Protein 1, Erythrocyte; D001798:Blood Proteins; D008565:Membrane Proteins; D013113:Spin Labels; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases