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Open Access Research article

Inverse tuning of metal binding affinity and protein stability by altering charged coordination residues in designed calcium binding proteins

Anna W Maniccia1¤, Wei Yang2¤, Julian A Johnson1, Shunyi Li1, Harianto Tjong3, Huan-Xiang Zhou3, Lev A Shaket1 and Jenny J Yang1*

Author Affiliations

1 Department of Chemistry, Center for Drug Design and Biotechnology, Georgia State University, Atlanta, GA 30303, USA

2 Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Renmin Road 5625, Changchun, Jilin 130022, PR China

3 Department of Physics and Institute of Molecular Biophysics and School of Computational Science, Florida State University, Tallahassee, Florida 32306, USA


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PMC Biophysics 2009, 2:11 doi:10.1186/1757-5036-2-11

Published: 21 December 2009

Abstract

Ca2+ binding proteins are essential for regulating the role of Ca2+ in cell signaling and maintaining Ca2+ homeostasis. Negatively charged residues such as Asp and Glu are often found in Ca2+ binding proteins and are known to influence Ca2+ binding affinity and protein stability. In this paper, we report a systematic investigation of the role of local charge number and type of coordination residues in Ca2+ binding and protein stability using de novo designed Ca2+ binding proteins. The approach of de novo design was chosen to avoid the complications of cooperative binding and Ca2+-induced conformational change associated with natural proteins. We show that when the number of negatively charged coordination residues increased from 2 to 5 in a relatively restricted Ca2+-binding site, Ca2+ binding affinities increased by more than 3 orders of magnitude and metal selectivity for trivalent Ln3+ over divalent Ca2+ increased by more than 100-fold. Additionally, the thermal transition temperatures of the apo forms of the designed proteins decreased due to charge repulsion at the Ca2+ binding pocket. The thermal stability of the proteins was regained upon Ca2+ and Ln3+ binding to the designed Ca2+ binding pocket. We therefore observe a striking tradeoff between Ca2+/Ln3+ affinity and protein stability when the net charge of the coordination residues is varied. Our study has strong implications for understanding and predicting Ca2+-conferred thermal stabilization of natural Ca2+ binding proteins as well as for designing novel metalloproteins with tunable Ca2+ and Ln3+ binding affinity and selectivity.

PACS codes: 05.10.-a