Flexibility of the alpha-spectrin N-terminus by EPR and fluorescence polarization

The structure and flexibility of the biologically important alpha-spectrin amino terminal region was examined by the use of fluorescence and EPR spect roscopy. The region studied has been previously demonstrated to be essentia l for the alpha-spectrin:beta-spectrin association of the tetramerization s ite. Appropriate spectroscopic probe moieties were coupled to this region i n a recombinant fragment of human erythroid alpha-spectrin. There was good agreement between the EPR and fluorescence techniques in most of this regio n. Mobility determinations indicated that a portion of the region was relat ively immobilized. This is significant, since although predictive methods h ave indicated that this region should be alpha-helical, previous experiment al evidence obtained on smaller synthetic peptides had indicated that this region was disordered. Observed rigidity appears to be incompatible with su ch a disordered state, and has important ramifications for the flexibility of this molecule that is so integral to its role in stabilizing erythrocyte membranes.