Protein Engineering, Vol. 15, No. 7, 571-574,
July 2002
© 2002 Oxford University Press
Metal-binding studies for a de novo designed calcium-binding protein
1 Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA 2 Department of Animal and Dairy Sciences, Auburn University, Auburn, AL 36849, USA
To understand the key determinants in calcium-binding affinity, a calcium-binding site with pentagonal bipyramid geometry was designed into a non-calcium-binding protein, domain 1 of CD2. This metal-binding protein has five mutations with a net charge in the coordination sphere of –5 and is termed DEEEE. Fluorescence resonance energy transfer was used to determine the metal-binding affinity of DEEEE to the calcium analog terbium. The addition of protein concentration to Tb(III) solution results in a large enhancement of Tb(III) fluorescence due to energy transfer between terbium ions and aromatic residues in CD2-D1. In addition, both calcium and lanthanum compete with terbium for the same desired metal binding pocket. Our designed protein exhibits a stronger affinity for Tb(III), with a Kd of 21 µM, than natural calcium-binding proteins with a similar Greek key scaffold.