title: Inverse tuning of metal binding affinity and protein stability by altering charged coordination residues in designed calcium binding proteins.
abstract: Ca(2+ )binding proteins are essential for regulating the role of Ca(2+ )in cell signaling and maintaining Ca(2+ )homeostasis. Negatively charged residues such as Asp and Glu are often found in Ca(2+ )binding proteins and are known to influence Ca(2+ )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 Ca(2+ )binding and protein stability using de novo designed Ca(2+ )binding proteins. The approach of de novo design was chosen to avoid the complications of cooperative binding and Ca(2+)-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 Ca(2+)-binding site, Ca(2+ )binding affinities increased by more than 3 orders of magnitude and metal selectivity for trivalent Ln(3+ )over divalent Ca(2+ )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 Ca(2+ )binding pocket. The thermal stability of the proteins was regained upon Ca(2+ )and Ln(3+ )binding to the designed Ca(2+ )binding pocket. We therefore observe a striking tradeoff between Ca(2+)/Ln(3+ )affinity and protein stability when the net charge of the coordination residues is varied. Our study has strong implications for understanding and predicting Ca(2+)-conferred thermal stabilization of natural Ca(2+ )binding proteins as well as for designing novel metalloproteins with tunable Ca(2+ )and Ln(3+ )binding affinity and selectivity.PACS codes: 05.10.-a.
authors.affiliation: Department of Chemistry, Center for Drug Design and Biotechnology, Georgia State University, Atlanta, GA 30303, USA.
authors.forename: Anna Wilkins
authors.initials: AW
authors.lastname: Maniccia
authors.affiliation: Changchun Institute of Applied Chemistry, Chinese Academy of Sciences, Renmin Road 5625, Changchun, Jilin 130022, PR China.
authors.forename: Wei
authors.initials: W
authors.lastname: Yang
authors.affiliation: Department of Chemistry, Center for Drug Design and Biotechnology, Georgia State University, Atlanta, GA 30303, USA.
authors.forename: Julian A
authors.initials: JA
authors.lastname: Johnson
authors.affiliation: Department of Chemistry, Center for Drug Design and Biotechnology, Georgia State University, Atlanta, GA 30303, USA.
authors.forename: Shunyi
authors.initials: S
authors.lastname: Li
authors.affiliation: Department of Physics and Institute of Molecular Biophysics and School of Computational Science, Florida State University, Tallahassee, Florida 32306, USA.
authors.forename: Harianto
authors.initials: H
authors.lastname: Tjong
authors.affiliation: Department of Physics and Institute of Molecular Biophysics and School of Computational Science, Florida State University, Tallahassee, Florida 32306, USA.
authors.forename: Huan-Xiang
authors.initials: HX
authors.lastname: Zhou
authors.affiliation: Department of Chemistry, Center for Drug Design and Biotechnology, Georgia State University, Atlanta, GA 30303, USA.
authors.forename: Lev A
authors.initials: LA
authors.lastname: Shaket
authors.affiliation: Department of Chemistry, Center for Drug Design and Biotechnology, Georgia State University, Atlanta, GA 30303, USA.
authors.forename: Jenny J
authors.initials: JJ
authors.lastname: Yang