Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Synthetic Calcium-Binding Peptides 175
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Synthetic Calcium-Binding Peptides
Gary S. Shaw
1. Introduction
The architecture of many calcium-binding proteins makes them exceptional
candidates for synthetic peptide approaches. In particular, synthetic peptides
have provided a wealth of insight into the calcium-binding properties and architecture
of proteins in the EF-hand family of calcium-binding proteins. In this
group of proteins, which includes members such as troponin C, calmodulin, and
S100B, the calcium-binding sites are formed from contiguous stretches of about
30-residues (1). This sequence forms a helix-loop-helix structural motif whereby
coordination of calcium occurs in a 12-residue loop region centered within the
motif. The contiguous nature of the calcium-binding sites and their modular
assembly is shown in Fig. 1 for the muscle protein troponin C. Extensive use of
the synthetic peptide approach has allowed a detailed examination of the
importance of particular residues at the chelating positions to be addressed (2,3).
Further, synthetic peptides from calcium-binding sites III and IV in troponin C
have shown that the two-site domain is the integral building block of EF-hand
calcium-binding proteins (4,5). Similar approaches using synthetic peptides have
been used to study the assembly of calcium-binding sites in S100B (6) and
calbindin D 28k (7). Similar studies would have been relatively difficult to examine
by other methods, such as site-directed mutagenesis. In addition, synthetic
peptides have been used to study the calcium binding properties and threedimensional
structures of a variety of Gla-containing proteins (8).
Synthetic peptides offer several advantages for structure-function studies of
calcium-binding proteins. First, they allow rapid production of a peptide typically
in less than 1 wk, from idea to synthesis to purified peptide. Second, the
efficiency of the method allows very high amounts of purified peptide, to be
obtained, generally >25 mg per synthesis. Third, the method is sufficiently
From: Methods in Molecular Biology, vol. 173:
Calcium-Binding Protein Protocols, Vol. 2: Methods and Techniques
Edited by: H. J. Vogel © Humana Press Inc., Totowa, NJ
175