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We describe the rational design of a novel class of magnetic resonance imaging contrast agents with engineered proteins (CAi.CD2, i = 1, 2, …, 9) chelated with gadolinium. The design of protein-based contrast agents involves creating high coordination Gd3+ binding sites in a stable host protein using amino acid residues and water molecules as metal coordinating ligands. Designed proteins show strong selectivity for Gd3+ over physiological metal ions such as Ca2+, Zn2+, and Mg2+. These agents exhibit a 20-fold increase in longitudinal and transverse relaxation rate values over the conventional small molecule contrast agents, e.g., Gd-DTPA (diethylene triamine pentaacetic acid), used clinically. Furthermore, they exhibit much stronger contrast enhancement and much longer blood retention time than Gd-DTPA in mice. With good biocompatibility and potential functionalities, these protein contrast agents may be used as molecular imaging probes to target disease markers, extending applications of magnetic resonance imaging (MRI).

Ca 2+ , a signal of life and death, controls numerous cellular processes through interactions with proteins. An effective approach to understanding the role of Ca 2+ is the design of a Ca 2+ -binding protein with predicted structural and functional properties. To design de novo Ca 2+ -binding sites in proteins is challenging due to the high coordination numbers and the incorporation of charged ligand residues, in addition to Ca 2+ -induced conformational change. Here, we demonstrate the successful design of a Ca 2+ -binding site in the non-Ca 2+ -binding cell adhesion protein CD2. This designed protein, Ca.CD2, exhibits selectivity for Ca 2+ versus other di- and monovalent cations. In addition, La 3+ (K d 5.0 μM) and Tb 3+ (K d 6.6 μM) bind to the designed protein somewhat more tightly than does Ca 2+ (K d 1.4 mM). More interestingly, Ca.CD2 retains the native ability to associate with the natural target molecule. The solution structure reveals that Ca.CD2 binds Ca 2+ at the intended site with the designed arrangement, which validates our general strategy for designing de novo Ca 2+ -binding proteins. The structural information also provides a close view of structural determinants that are necessary for a functional protein to accommodate the metal-binding site. This first success in designing Ca 2+ -binding proteins with desired structural and functional properties opens a new avenue in unveiling key determinants to Ca 2+ binding, the mechanism of Ca 2+ signaling, and Ca 2+ -dependent cell adhesion, while avoiding the complexities of the global conformational changes and cooperativity in natural Ca 2+ -binding proteins. It also represents a major achievement toward designing functional proteins controlled by Ca 2+ binding.

Calcium ions play key roles as structural components in biomineralization and as a second messenger in signaling pathways. We have introduced a de novo designed calcium-binding site into the framework of a non-calcium-binding protein, domain 1 of CD2. The resulting protein selectively binds calcium over magnesium with calcium-binding affinity comparable to that of natural extracellular calcium-binding proteins (K(d) of 50 microM). This experiment is the first successful metalloprotein design that has a high coordination number (seven) metal-binding site constructed into a beta-sheet protein. Our results demonstrate the feasibility of designing a single calcium-binding site into a host protein, taking into account only local properties of a calcium-binding site obtained by a survey of natural calcium-binding proteins and chelators. The resulting site exhibits strong metal selectivity, suggesting that it should now be feasible to understand and manipulate signaling processes by designing novel calcium-modulated proteins with specifically desired functions and to affect their stability.

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.

To establish an approach to obtain the site-specific calcium binding affinity of EF-hand proteins, we have successfully designed a series of model proteins, each containing the EF-hand calcium-binding loop 3 of calmodulin, but with increasing numbers of Gly residues linking the loop to domain 1 of CD2. Structural analyses, using different spectroscopic methods, have shown that the host protein is able to retain its native structure after insertion of the 12-residue calcium-binding loop and retains a native thermal stability and thermal unfolding behavior. In addition, calcium binding to the engineered CD2 variants does not result in a significant change from native CD2 conformation. The CD2 variant with two Gly linkers has been shown to have the strongest metal binding affinity to Ca(II) and La(III). These experimental results are consistent with our molecular modeling studies, which suggest that this protein with the engineered EF-loop has a calmodulin-like calcium binding geometry and backbone conformation. The addition of two Gly linkers increases the flexibility of the inserted EF-loop 3 from calmodulin, which is essential for the proper binding of metal ions.