Ionic Strength Effect on the Thermal Unfolding of α-Spectrin Peptides

Abstract

In previous work, we have shown that the ionic strength-mediated differences found for the hydrodynamic dimensions of the human erythrocyte spectrin are not caused by secondary structural changes, but are caused more probably by subtle changes in tertiary interactions (LaBrake, C. C., Wang, L., Keiderling, T. A., and Fung, L. W.-M. (1993) Biochemistry 32, 10296−10302.). The substructure of spectrin has been suggested to be composed largely of triple α-helical bundle structural domains in tandem. In the present study, we used fluorescence and circular dichroism methods to study ionic strength effects on intact spectrin dimers and on recombinant peptides of spectrin domains of different lengths. We observed little ionic strength effect on the thermal unfolding temperature, T_m, values in these systems. However, we found that ionic strength-induced cooperativity in the unfolding processes was similar for the spectrin dimer and for peptides with two or three domains, as measured by entropy changes (ΔS_m). Although single-domain peptides exhibited rather variable ΔS_m values, depending on the specific domain, they showed little salt effects on the ΔS_m values themselves. This suggests that spectrin undergoes subtle ionic strength-induced conformational changes, probably near the interdomain regions of the molecule. These conformational changes may be responsible for the observed hydrodynamic and unfolding properties in intact spectrin under different ionic strength conditions. We suggest that recombinant peptides of various lengths may serve as models for studying the structural flexibility in spectrin.