Two 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 first evidence for a differentiation in the response of calponin to interaction with caltropin versus calmodulin is demonstrated. The binding of caltropin to cleavage and recombinant fragments of calponin was determined 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 cleavage of calponin, residues 2-51 were isolated. This fragment is shown to bind to CaBPs and the affinity for caltropin is slightly higher than that for calmodulin. A carboxyl-terminal truncated mutant of calponin comprising residues 1-228 (CP 1-228) has been produced by recombinant techniques. Analytical ultracentrifugation has shown that CP 1-228, like the parent calponin, is able to bind 2 mol of caltropin per mol of 1-228 in a Ca(2+)-dependent fashion, indicating that there is a second site of interaction between residues 52-228. Temperature denaturation of the carboxyl-terminal truncated fragment compared with whole calponin show that the carboxyl-terminal region does not change the temperature at which calponin melts; however, there is greater residual secondary structure with whole calponin versus the fragment. A second mutant produced through recombinant techniques comprises residues 45-228 and is also able to bind caltropin, thus mapping the location of the second site of interaction to near the actin binding site.
Wills
F L
FL
Department of Biochemistry, University of Alberta, Edmonton, Canada.
McCubbin
W D
WD
Gimona
M
M
Strasser
P
P
Kay
C M
CM
D000199:Actins / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D002135:Calcium-Binding Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002942:Circular Dichroism; D008840:Microfilament Proteins; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D011485:Protein Binding; D011489:Protein Denaturation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000378:metabolism; D013050:Spectrometry, Fluorescence; D014364:Tryptophan / Q000737:chemistry; D014422:Turkeys; D014461:Ultracentrifugation; D000096985:Calponins
D000199:Actins; D002135:Calcium-Binding Proteins; D008840:Microfilament Proteins; D010446:Peptide Fragments; D011993:Recombinant Fusion Proteins; D014364:Tryptophan; D000096985:Calponins