Cation- and Peptide-Binding Properties of Human Calmodulin-like Skin Protein
| Autor: | Dominique Bernard, Rainer Schmidt, Jos A. Cox, Bruno Mehul, Isabelle Durussel |
|---|---|
| Rok vydání: | 2002 |
| Předmět: |
Calmodulin
Cations Divalent Peptide binding Biochemistry Protein Structure Secondary Naphthalenesulfonates Humans CALMODULIN-LIKE SKIN PROTEIN Magnesium Fluorescent Dyes Skin Binding Sites Transglutaminases biology Chemistry Circular Dichroism Calcium-Binding Proteins Tryptophan Melitten Peptide Fragments Sequence identity Protein Structure Tertiary Spectrometry Fluorescence biology.protein Tyrosine Calcium Electrophoresis Polyacrylamide Gel Spectrophotometry Ultraviolet Protein Binding |
| Zdroj: | Biochemistry. 41:5439-5448 |
| ISSN: | 1520-4995 0006-2960 |
| Popis: | Human CLSP, a new Ca(2+)-binding protein specifically expressed in differentiated keratinocytes, is a 15.9 kDa, four EF-hand containing protein with 52% sequence identity to calmodulin (CaM). The protein binds four Ca(2+) ions at two pairs of sites with [Ca(2+)](0.5) values of 1.2 and 150 microM, respectively. Mg(2+) at millimolar concentrations strongly decreases the affinity for Ca(2+) of the two high-affinity sites, but has no effect on the low-affinity sites. The protein can also bind two Mg(2+) ([Mg(2+)](0.5) = 57 microM) at the sites of high Ca(2+) affinity. Thus, as fast skeletal muscle troponin C (TnC), CLSP possesses two high-affinity Ca(2+)-Mg(2+) mixed sites and two low-affinity Ca(2+)-specific sites. Studies on the isolated recombinant N- (N-CLSP) and C-terminal half domains of CLSP (C-CLSP) revealed that, in contrast to the case of TNC, the high-affinity Ca(2+)-Mg(2+) mixed sites reside in the N-terminal half. The binding of cations modifies the intrinsic fluorescence of the two Tyr residues. Upon Ca(2+) binding, hydrophobicity is exposed at the protein surface that can be monitored with a fluorescent probe. The Ca(2+)-dependency of the two conformational changes is biphasic in the absence of Mg(2+), but monophasic in the presence of 2 mM Mg(2+), both corresponding closely to direct binding of Ca(2+) to CLSP. In the presence of Ca(2+), human CLSP forms a high-affinity 1:1 complex with melittin, a natural peptide considered to be a model for the interaction of CaM with its targets. In the complex, CLSP binds Ca(2+) with high affinity to all four binding sites. Isolated N- and C-CLSP show only a weak interaction with melittin, which is enhanced when both halves are simultaneously presented to the model peptide. |
| Databáze: | OpenAIRE |
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