Conformational change in thrombospondin induced by removal of bound Ca 2+ A spin label approach

Abstract

The effect of removal of Ca 2+ bound to thrombospondin (TSP) on the protein structure in solution has been investigated using ESR spin-label techniques. A maleimide spin label was selectively attached to the free thiol group presumably near the carboxyl-terminal domain in which Ca 2+-binding sites are situated. The ESR spectra of spin-labeled TSP showed that the bound label undergoes a relatively fast rotational motion with an effective rotational correlation time in the nanosecond time regimes. Removal of bound Ca 2+ in TSP by dialyzing spin-labeled TSP from a Ca 2+-containing buffer into an EDTA-containing buffer resulted in an increase in the mobility of the bound label by a factor of 2.3. The data suggest that EDTA chelation of bound Ca 2+ in TSP induces a conformational change of TSP at least near the site of spin labeling.