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Rational design of a conformation‐switchable Ca 2+ ‐ and Tb 3+ ‐binding protein without the use of multiple coupled metal‐binding sites
Author(s) -
Li Shunyi,
Yang Wei,
Maniccia Anna W.,
Barrow Jr Doyle,
Tjong Harianto,
Zhou HuanXiang,
Yang Jenny J.
Publication year - 2008
Publication title -
the febs journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.981
H-Index - 204
eISSN - 1742-4658
pISSN - 1742-464X
DOI - 10.1111/j.1742-4658.2008.06638.x
Subject(s) - cooperativity , chemistry , conformational change , cooperative binding , protein folding , biophysics , binding site , dissociation constant , protein structure , crystallography , protonation , protein design , folding (dsp implementation) , plasma protein binding , stereochemistry , biochemistry , ion , biology , receptor , organic chemistry , electrical engineering , engineering
Ca 2+ , as a messenger of signal transduction, regulates numerous target molecules via Ca 2+ ‐induced conformational changes. Investigation into the determinants for Ca 2+ ‐induced conformational change is often impeded by cooperativity between multiple metal‐binding sites or protein oligomerization in naturally occurring proteins. To dissect the relative contributions of key determinants for Ca 2+ ‐dependent conformational changes, we report the design of a single‐site Ca 2+ ‐binding protein (CD2.trigger) created by altering charged residues at an electrostatically sensitive location on the surface of the host protein rat Cluster of Differentiation 2 (CD2). CD2.trigger binds to Tb 3+ and Ca 2+ with dissociation constants of 0.3 ± 0.1 and 90 ± 25 μ m , respectively. This protein is largely unfolded in the absence of metal ions at physiological pH, but Tb 3+ or Ca 2+ binding results in folding of the native‐like conformation. Neutralization of the charged coordination residues, either by mutation or protonation, similarly induces folding of the protein. The control of a major conformational change by a single Ca 2+ ion, achieved on a protein designed without reliance on sequence similarity to known Ca 2+ ‐dependent proteins and coupled metal‐binding sites, represents an important step in the design of trigger proteins.