2 DOMAINS OF INTERACTION WITH CALCIUM-BINDING PROTEINS CAN BE MAPPED USING FRAGMENTS OF CALPONIN

Native calponin is able to bind 2 mol of calcium binding protein (CaBP ) per mole calponin. This study extends this observation to define the 2 domains of interaction, one of which is near the actin binding site , and the other in the amino-terminal region of calponin. Also, the fi rst evidence for a differentiation in the response of calponin to inte raction with caltropin versus calmodulin is demonstrated. The binding of caltropin to cleavage and recombinant fragments of calponin was det ermined by 3 techniques: tryptophan fluorescence of the fragments, CD measurements to determine secondary structure changes, and analytical ultracentrifugation. In order to delineate the sites of interaction, 3 fragments of calponin have been studied. From a cyanogen bromide clea vage of calponin, residues 2-51 were isolated. This fragment is shown to bind to CaBPs and the affinity for caltropin is slightly higher tha n that for calmodulin. A carboxyl-terminal truncated mutant of calponi n comprising residues 1-228 (CP 1-228) has been produced by recombinan t techniques. Analytical ultracentrifugation has shown that CP 1-228, like the parent calponin, is able to bind 2 mol of caltropin per mol o f 1-228 in a Ca2+-dependent fashion, indicating, that there is a secon d site of interaction between residues 52-228. Temperature denaturatio n of the carboxyl-terminal truncated fragment compared with whole calp onin show that the carboxyl-terminal region does not change the temper ature at which calponin melts; however, there is greater residual seco ndary structure with whole calponin versus the fragment. A second muta nt produced through recombinant techniques comprises residues 45-228 a nd is also able to bind caltropin, thus mapping the location of the se cond site of interaction to near the actin binding site.