DIRECT MEASUREMENT OF SMALL LIGAND-INDUCED CONFORMATIONAL-CHANGES IN THE ASPARTATE CHEMORECEPTOR USING EPR

Ligand-binding-induced conformational changes in the Salmonella typhim urium aspartate receptor ware studied using spin-labeling electron par amagnetic resonance. Cysteine residues, introduced by site-directed mu tagenesis at several positions in the aspartate receptor periplasmic d omain, were used to attach covalently a thiol-specific spin label. The electron paramagnetic resonance spectra of these labeled proteins wer e obtained in the presence and absence of the ligand aspartate, and us ed to calculate the distance change between spin labels. The results s upport a model in which transmembrane signaling is executed by a combi ned movement of alpha helix 4 (which leads into transmembrane domain 2 ) relative to alpha helix 1 (connected to transmembrane domain 1), as well as a coming together of the two subunits. Ligand binding causes s pin labels at position 39 and 179 (within one subunit) to move further from each other and spin labels at position 39 and 39' (between two s ubunits) to move closer to each ether. Both of these changes are very small-less than 2.5 Angstrom. No similar changes were detected in any aspartate receptor samples solubilized in detergent, suggesting that t he membrane is required for these conformational changes. This is the first case of physically measured ligand-induced changes in a full-len gth 1-2 transmembrane domain receptor, and the results suggest that ve ry small ligand-induced movements can result in large effects on the a ctivity of downstream proteins.