Metal-binding studies for a de novo designed calcium-binding protein.
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 K(d) of 21 microM, than natural calcium-binding proteins with a similar Greek key scaffold.
Department of Chemistry, Georgia State University, Atlanta, GA 30303, USA.
Anna L
AL
Wilkins
Yiming
Y
Ye
Wei
W
Yang
Hsiau-Wei
HW
Lee
Zhi-ren
ZR
Liu
Jenny J
JJ
Yang
D000596:Amino Acids / Q000737:chemistry; D001665:Binding Sites; D001667:Binding, Competitive; D002135:Calcium-Binding Proteins / Q000737:chemistry / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer; D007700:Kinetics; D007811:Lanthanum / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D017434:Protein Structure, Tertiary; D013725:Terbium / Q000737:chemistry / Q000378:metabolism*
D000596:Amino Acids; D002135:Calcium-Binding Proteins; D013725:Terbium; D007811:Lanthanum