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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 oligomeriza-tion in naturally occurring proteins. | ễFEBS Journal Rational design of a conformation-switchable Ca2 - and Tb3 -binding protein without the use of multiple coupled metal-binding sites Shunyi Li1 2 Wei Yang3 Anna W. Maniccia1 Doyle Barrow Jr1 Harianto Tjong4 Huan-Xiang Zhou4 and Jenny J. Yang1 1 Department of Chemistry Center of Drug Design and Biotechnology Georgia State University Atlanta GA USA 2 College of Life Sciences Hubei University Wuhan China 3 Changchun Institute of Applied Chemistry Chinese Academy of Sciences Changchun Jilin China 4 Department of Physics and Institute of Molecular Biophysics and Schoolof ComputationalScience Florida State University Tallahassee FL USA Keywords Ca2 binding conformational change protein design Tb3 binding trigger Correspondence J. J. Yang 50 Decatur Street Atlanta GA 30303 USA Fax 1 404 413 5551 Tel 1 404 413 5520 E-mail chejjy@langate.gsu.edu Received 14 May 2008 revised 10 July 2008 accepted 12 August 2008 doi 10.1111 j.1742-4658.2008.06638.x Ca2 as a messenger of signal transduction regulates numerous target molecules via Ca2 -induced conformational changes. Investigation into the determinants for Ca2 -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 Ca2 -dependent conformational changes we report the design of a single-site Ca2 -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 Tb3 and Ca2 with dissociation constants of 0.3 0.1 and 90 25 M respectively. This protein is largely unfolded in the absence of metal ions at physiological pH but Tb3 or Ca2 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. .
