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.

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Abstract
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 was examined by 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 form human RBC. Experimental EPR lineshapes from soluble and membrane-bound enzymes were analyzed by direct simulation 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 Cys-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 .mu.s. The GAPDHase-band 3 complex apparently exhibits motional freedom relative to the membrane-spanning segment of the band 3 protein or the RBC. The doubly 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.