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Research article

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

Anna Wilkins Maniccia^1* , Wei Yang^2* , Julian A Johnson¹ , Shunyi Li¹ , Harianto Tjong³ , Huan-Xiang Zhou³ , Lev A Shaket¹ and Jenny J Yang¹

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

author email corresponding author email* Contributed equally

PMC Biophysics 2009, 2:11doi: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