A fraction of band 3 protein, the major transmembrane protein of erythrocyte membranes, is held to the cytoskeletal protein spectrin via noncovalent interactions with the protein ankyrin (band 2.1). In this study, trypsin was used under defined conditions to selectively proteolyze ankyrin and thereby destroy the band 3-ankyrin linkage on the cytoplasmic side of erythrocyte ghost membranes. Electron paramagnetic resonance (EPR) spectroscopy, in conjunction with selective spin labeling methods, was used to monitor conformational changes occurring in cytoskeletal proteins or cell-surface carbohydrates as a result of this treatment. Treatment of RBC ghosts with TPCK-trypsin for 5 s at 0 degrees C caused an approx. 56% increase in the relevant EPR parameter of a maleimide spin label bound to spectrin (P < 0.004), indicative of increased segmental motion of the spin label and decreased protein-protein interactions. Analysis of the apparent rotational correlation time parameter tau of a spin label covalently and selectively bound to terminal sialic acid residues of glycophorin showed no significant effect from trypsin treatment. However, tau of spin label covalently and specifically bound to terminal galactose residues of cell-surface glycoconjugates of band 3 and other transmembrane glycoproteins significantly decreased with tryptic uncoupling of the ankyrin linkage (P < 0.005). These results suggest a marked conformational alteration in both cytoskeletal and transmembrane proteins as a result of uncoupling from ankyrin. Spermine (N,N'-bis(3-aminopropyl)tetramethylenediamine), a naturally occurring polyamine known to strengthen cytoskeletal protein-protein interactions (Wyse and Butterfield (1988) Biochim. Biophys. Acta 941, 141-149), was used to partially reverse the trypsin-induced cytoskeletal alterations. Addition of 2 mM spermine to ghosts previously treated with trypsin increased cytoskeletal protein-protein interactions as indicated by EPR (P < 0.002). SDS-PAGE was used to confirm the integrity of spectrin, band 3, and band 4.1 in all experiments. The results are discussed with reference to transmembrane signaling mechanisms and membrane-associated pathologies.