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Research Database PMU-SQQUID

Two domains of interaction with calcium binding proteins can be mapped using fragments of calponin.
Wills, FL; McCubbin, WD; Gimona, M; Strasser, P; Kay, CM;
Protein Sci. 1994; 3(12): 2311-2321.
Originalarbeiten (Zeitschrift)

PMU-Authors

Gimona Mario
Strasser Peter

Abstract

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.


Useful keywords (using NLM MeSH Indexing)

Actins/metabolism

Animals

Binding Sites

Calcium-Binding Proteins/chemistry*

Calcium-Binding Proteins/genetics

Calcium-Binding Proteins/metabolism

Circular Dichroism

Microfilament Proteins

Peptide Fragments/chemistry

Peptide Fragments/metabolism

Protein Binding

Protein Denaturation

Protein Structure, Secondary

Protein Structure, Tertiary*

Recombinant Fusion Proteins/chemistry

Recombinant Fusion Proteins/metabolism

Spectrometry, Fluorescence

Tryptophan/chemistry

Turkeys

Ultracentrifugation


Find related publications in this database (Keywords)

CALMODULIN
CALPONIN
CALTROPIN
SMOOTH MUSCLE
THIN FILAMENT