We describe a family of calcium indicators for magnetic resonance imaging (MRI), formed by combining a powerful iron oxide nanoparticle-based contrast mechanism with the versatile calciumsensing protein calmodulin and its targets. Calcium-dependent protein-protein interactions drive particle clustering and produce up to 5-fold changes in T2 relaxivity, an indication of the sensors' potency. A variant based on conjugates of wild-type calmodulin and the peptide M13 reports concentration changes near 1 M Ca 2؉ , suitable for detection of elevated intracellular calcium levels. The midpoint and cooperativity of the response can be tuned by mutating the protein domains that actuate the sensor. Robust MRI signal changes are achieved even at nanomolar particle concentrations (<1 M in calmodulin) that are unlikely to buffer calcium levels. When combined with technologies for cellular delivery of nanoparticulate agents, these sensors and their derivatives may be useful for functional molecular imaging of biological signaling networks in live, opaque specimens. magnetic resonance ͉ T2 relaxation ͉ signal transduction ͉ molecular imaging ͉ neuroimaging C alcium ions (Ca 2ϩ ) have been a favorite target in molecular imaging studies because of the important role of calcium as a second messenger in cellular signaling pathways. Fluorescent calcium sensors are used widely in optical imaging, both at the cellular level and at the cell population level. Calcium-sensitive dyes have recently been used in conjunction with laser scanning microscopy to follow neural network activity in small, threedimensional brain areas (1) and to characterize patterns of interaction among cells in developing vertebrate embryos (2). Because of the scattering properties of dense tissue, highresolution optical approaches like these are usually limited to superficial regions of specimens and to restricted fields of view (3). To probe calcium dynamics more globally in living systems, a different imaging modality must be used.Magnetic resonance imaging (MRI) is an increasingly accessible technique for imaging opaque subjects at fairly high spatial resolution, and MRI studies of calcium dynamics could, in principle, complement optical approaches by offering both greatly expanded coverage and depth penetration in vivo (4). Calcium isotopes are unsuitable for direct imaging by magnetic resonance, so attempts to sensitize MRI to calcium have focused around the use of molecular imaging agents. Fluorinated derivatives of the bivalent cation chelator 1,2-bis(2-aminophenoxy)ethane-N,N,NЈ,NЈ-tetraacetic acid (BAPTA) have permitted calcium measurements in vivo by 19 F MRI but only at Ϸ10 Ϫ5 the sensitivity of standard MRI methods (5, 6). Two potentially more powerful proton T1 relaxation-promoting contrast agents were subsequently introduced. The paramagnetic ion manganese (Mn 2ϩ ) mimics calcium by entering cells through calcium channels. Because it accumulates much faster than it is removed, Mn 2ϩ produces an ''integral'' of calcium signaling history that can be d...

Calcium-saturated calmodulin (CaM) binds and influences the activity of a varied collection of target proteins in most cells. This promiscuity underlies CaM's role as a shared participant in calciumdependent signal transduction pathways, but imposes a handicap on popular CaM-based calcium biosensors, which display an undesired tendency to cross-react with cellular proteins. Designed CaM/ target pairs that retain high affinity for one another, but lack affinity for wild-type CaM and its natural interaction partners, would therefore be useful as sensor components, and possibly also as elements of "synthetic" cellular signaling networks. Here we have adopted a rational approach to creating suitably modified CaM/target complexes by using computational design methods to guide parallel site-directed mutagenesis of both binding partners. A hierarchical design procedure was applied to suggest a small number of complementary mutations on CaM and on a peptide ligand derived from skeletal muscle light chain kinase (M13). Experimental analysis showed that the procedure was successful in identifying CaM and M13 mutants with novel specificity for one another. Importantly, the designed complexes retained affinity comparable to the wild-type CaM/M13 complex. These results represent a step toward the creation of CaM and M13 derivatives with specificity fully orthogonal to the wild-type proteins, and show that qualitatively accurate predictions may be obtained from computational methods applied simultaneously to two proteins involved in multiple linked binding equilibria. Figure 1A). (2,3) Calcium-loaded CaM binds to isolated target sequences with affinity comparable to its complexes with the intact proteins, and with dissociation constants often in the nanomolar range. With few exceptions, CaM ligands tend to be highly basic, presenting lysine and arginine residues that form salt-bridge networks with negatively-charged and polar amino-acid side chains bordering the binding cleft on CaM ( Figure 1B). In addition, many CaM targets appear to be anchored by a key pair of bulky groups spaced apart by 2.5 or 3.5 helical turns. (4) Deletion or mutation of these anchor residues dramatically reduces CaMbinding affinity. (5,6) Considerable interest in CaM/peptide interactions has revolved around potential applications in biotechnology. CaM-affinity chromatography (7) The ability to understand and control determinants of binding specificity at the CaM/target interface is important both for biotechnological applications and for appreciation of how CaM transduces metabolic signals via multiple protein interaction networks. Many studies have probed the contributions of both the charged and hydrophobic anchor residues to CaM binding affinity, (5,6,(19)(20)(21)(22)) but relatively few have sought to manipulate specificity through purposeful mutagenesis of one or both binding partners. A step in this direction was taken by Mayo and colleagues,(23,24) and involved the integration of computational and experimental methods to bias CaM spe...