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Metalloproteins have long been recognized as key determinants of endogenous contrast in magnetic resonance imaging (MRI) of biological subjects. More recently, both natural and engineered metalloproteins have been harnessed as biotechnological tools to probe gene expression, enzyme activity, and analyte concentrations by MRI. Metalloprotein MRI probes are paramagnetic and function by analogous mechanisms to conventional gadolinium or iron oxide-based MRI contrast agents. Compared with synthetic agents, metalloproteins typically offer worse sensitivity, but the possibilities of using protein engineering and targeted gene expression approaches in conjunction with metalloprotein contrast agents are powerful and sometimes definitive strengths. This review summarizes theoretical and practical aspects of metalloprotein-based contrast agents, and discusses progress in the exploitation of these proteins for molecular imaging applications.
Metalloproteins are essential to life. Roughly a third of proteins are associated with metals, most frequently magnesium, zinc, iron, and manganese, in order from most to least abundant . Metalloprostheses enable many of these proteins to play important roles in biological processes, prominently including photosynthesis, respiration, and various oxidation reduction reactions . A particularly impressive example, the cytochrome b6f from plants and photosynthetic bacteria, contains four types of heme, a magnesium porphyrin, and an [2Fe-2S] cluster all in a single supramolecular assembly designed to drive proton gradient formation using energy from light absorbed by the metal complexes . Both optical and electronic properties of the metal centers thus contribute to a reaction that ultimately gives rise to the nutrients used by most organisms on the planet. A far simpler but also famously vital metalloprotein is hemoglobin (Hb). Hb contains four polypeptides, each bound to a heme group. Oxygen binding to the metal centers induces a change in the coordination geometry, which in turn drives a global conformation change that favors further oxygen binding . Via this mechanism, the chemistry of metal-ligand interactions governs the ability of Hb to bind and release oxygen in physiologically appropriate concentration ranges.
Biophysical properties of metalloproteins and protein-metal interactions have led to a number of biotechnological applications. The strength and specificity of metal chelation by polypeptides gives rise to the well-known Ni2+ affinity purification method used to isolate polyhistidine-tagged proteins . Catalytic oxidation of chlorinated alkanes by a diiron active center in methane monooxygenase from Methylosinus trichosporium OB3b has been used for bioremediation of contaminated groundwater [5,6]. A redox-active metalloprotein, azurin, has been used as a biotransistor  and component of a protein-based biomemory device . Metalloproteins are also a basis for monitoring physiology in living animals. The most famous example is provided again by Hb, which due to the oxygen-dependent spectral properties of its heme groups, and the relative transparency of tissue to wavelengths differentially absorbed by oxy- and deoxy-Hb, is the basis for vital signs monitoring in virtually every hospital in the world .
It has been recognized more recently that magnetic properties of metalloproteins also provide important biotechnological capabilities. Many metalloproteins contain ions with unpaired electrons, rendering them paramagnetic and detectable or manipulable by magnetic tools. Accompanying the excitement around optogenetics  has been a particular interest in using metalloproteins such as ferritin (Ft) to provide magnetic “handles” on cell function. In a recent example, implanted cells overexpressing a Ft derivative were induced by application of oscillating magnetic fields to secrete insulin in mice . There is also a suggestion that protein-catalyzed paramagnetic metal accumulation in cells could be used for magnetic pull-down assays [12,13]. Finally, paramagnetic metalloproteins have been used as contrast agents for magnetic resonance imaging (MRI). Here, the prospect of finding MRI-detectable analogs to green fluorescent protein (GFP) has been an important inspiration; a hope is that suitable proteins could be used as gene reporters and sensors analogous to the various fluorescent and luminescent proteins that have transformed research in cell biology over the past two decades.
Although the majority of MRI contrast agents have historically been based on small organic molecules such as Gd3+ chelates [14,15], metalloprotein MRI contrast agents present a variety of advantages, each of which may be important in distinct contexts. Thanks to the advancement of molecular biology techniques, proteins are much easier to synthesize and modify than organic molecules; numerous protein engineering techniques may be applied to tune metalloprotein properties for MRI (reviewed in ). Due to their larger size, protein-based contrast agents are retained in the blood pool for a longer time than small molecule agents, allowing for longer imaging times in some types of experiments [17,18]. In some cases, metalloproteins can indeed be targeted and expressed using gene delivery methods, a la GFP [19,20]. Such reporters generate relatively static contrast, but can allow particular types of cells to be tracked in vivo over time [21–23]. By furthermore sensitizing metalloprotein contrast agents to analytes [24,25], additional information about physiologically relevant signals can be obtained. Development of such environmentally-sensitive agents is underway but largely in its infancy. In this article, we discuss the characteristics of metalloprotein-based MRI contrast agents and review recent progress in the development and applications of magnetically active proteins in these new spheres of investigation.
In proton MRI , the form of MRI most commonly applied in laboratories and clinics, populations of nuclear spins arising from hydrogen nuclei primarily in water are perturbed and monitored to generate images. At thermal equilibrium in a strong magnetic field (B0), the water proton spins align weakly with the applied field and give rise to a net “longitudinal” magnetization aligned with B0. This magnetization is unobservable, but can be detected following application of radiofrequency energy pulses which tilt the magnetization vector off of the B0 axis and give rise to a nonzero “transverse” magnetization component. After excitation, the transverse magnetization component decays away with a time constant T2 (the transverse relaxation time) and the overall magnetization returns to thermal equilibrium with a time constant T1 (the longitudinal relaxation time). The shorter T1 is, the more frequently an MRI signal can be repeatedly measured per unit time; areas of a specimen with short T1 therefore give rise to a larger average MRI signal. Conversely, the shorter T2 is, the more rapidly the observable component of magnetization disappears, and the lower the MRI signal becomes.
Paramagnetic species influence contrast in MRI by reducing the T1 and T2 relaxation times [27,28] (Figure 1). In both cases, accelerated relaxation arises from coupling between the magnetic dipole of the contrast agent and the nuclear spins of water molecules that interact with the agent through bonds (“inner sphere” interactions) or through space (“outer sphere”). Relaxation rates R1 (= 1/T1) and R2 (= 1/T2) are generally linear with contrast agent concentration. The slopes of these relationships are referred to as the T1 and T2 relaxivities, r1 and r2, respectively, which measure the strength of the contrast agent and are expressed in units of mM−1s−1. Greater relaxivity is beneficial to an MRI contrast agent, because the agent can then be applied at lower doses or to greater effect at any given concentration. Both r1 and r2 vary strongly with B0 and depend on physical parameters including the electron spin number (S) or magnetic moment of the contrast agent, the number of coordinated inner sphere water molecules (q), the time constant for inner sphere water exchange (τM), and the rotational correlation time of the agent (τR). Inner sphere contributions to relaxivity are described by the theory of Solomon, Bloembergen, and Morgan [29–31], and apply to metalloproteins with adjustments to account for slow rotation in macromolecules . Outer sphere contributions are described by related theories applicable to mononuclear  and particulate [34–36] metal complexes. Determinants of relaxivity are summarized in the appendix to this article, and are also thoroughly discussed in a number of secondary references [14,37–39]. Relaxivity determinants are important not only because they explain how contrast agents may be optimized, but also because they provide potential mechanisms for designing MRI-detectable sensors.
Most MRI contrast agents or sensors, including paramagnetic proteins, tend to affect T1- weighted MRI scans more than T2-weighted scans, or vice versa, and are correspondingly referred to as T1 or T2 agents. T1 agents have an r1/r2 ratio of 1–2 and generally contain one or a small number of paramagnetic ions. Classical T1 agents are exemplified by complexes of Gd3+ with small chelators like diethylenetriaminepenaacetic acid (DTPA) [40,41], and are the most commonly applied agents in clincial MRI; analogous proteins can include porphyrin prostheses or directly bound metal ions (Figure 1A). At typical field strengths for clinical or preclinical MRI (> 1 T), most T1 agents have r1 values from 1–10 mM−1s−1. In biological samples with T1 values typically near 1 s, T1 agents need to be applied at concentrations near 100 μM to induce substantial contrast effects; 100 μM of a contrast agent with r1 = 5 mM−1s−1, for instance, would induce an MRI signal increase of ~20% against a background with a T1 of 1 s under conditions of optimal signal to noise ratio. Inner sphere r1 contributions are usually particularly important for T1 agents, and T1-based sensors typically undergo analyte-dependent changes in the parameters that most affect inner sphere relaxation mechanisms, such as q and τR.
T2 agents have r2/r1 ratio greater than ~10 (a necessary condition because T2 values are much shorter than T1 values in vivo) and are best exemplified by superparamagnetic nanoparticles (SPNs) [42–44], contrast agents that incorporate discrete crystalline domains that exhibit highly cooperative magnetic behavior. A biosynthetic analog to SPNs is ferritin (Ft) , an iron storage protein that accumulates minerals in a 12 nm shell like structure formed from 24 polypeptide chains (Figure 1B). SPN T2 agents produce high r2/r1 ratio because they become magnetized by the B0 field and create microscopic magnetic perturbations experienced by diffusing water molecules in solution. These perturbations affect T2 relaxation more than T1, particularly for particles with highly magnetic mineral cores over ~3 nm in diameter and at high B0 strengths (> 1 T), where SPN magnetizations tend toward an asymptotic “saturation” point . The physics of the interaction between diffusing protons and SPNs also leads to a strong dependence of r2 on R2/D, where R is the mineral core radius and D is the solvent self-diffusion constant (see appendix); SPNs with larger size shorten T2 more effectively, up to a so-called static dephasing limit  near ~50 nm for typical SPNs. Most SPN contrast agents incorporate iron oxide; synthetic iron-containing SPNs usually have r2 values of 50–500 (mM Fe)−1s−1. Given typical background T2 values near 100 ms in tissue, synthetic SPNs applied at concentrations of 1–10 μM Fe can produce substantial effects under optimal T2-weighted imaging conditions. For instance, an agent with r2 = 200 mM−1s−1 could produce a ~20% decrease in MRI signal at a concentration of 10 μM Fe. Because of the dependence of r2 on particle size for SPN agents, sensors can be constructed by coupling analyte concentration to the clustering of these particles [47,48], which results in de facto size changes .
Protein contrast agents have distinct advantages and disadvantages compared with conventional synthetic contrast agents in terms of relaxivity. In most cases, proteins are handicapped by incorporating metal ions with electronic properties that are suboptimal for R1 or R2 enhancement . Naturally-occurring paramagnetic proteins tend to contain Cu2+, Mn2+, Mn3+, Fe2+, or Fe3+ ions, ranging in spin number from 1/2 to 5/2. On the other hand, Gd3+ ions used in most synthetic small molecule agents provide S = 7/2. Since relaxivity values are approximately proportional to S(S+1), the maximum relaxivity of a gadolinium agent is about twice what would be achieved in principle with a transition metal-containing protein with otherwise equivalent relaxivity parameters. Electronic relaxation times T1e and T2e have a great influence on inner sphere relaxivities and also tend to be less advantageous (shorter) for transition metals than for gadolinium. Short electronic relaxation times limit relaxivity when they are less than the molecular motion timescale τR (~10 ns), as has been reported for some metalloproteins. A further limitation on the relaxivity of metalloproteins, compared with small metal complexes, can arise from the relative inaccessibility of protein-coordinated metal ions to outer sphere water molecules. At field strengths above 1.5 T, outer sphere interactions account for a sizeable fraction of the r1 of typical small molecule contrast agents [37,51], a contribution that could be reduced due to steric effects in a macromolecule.
The limitations of metalloprotein contrast agents are partially offset by properties that are predicted to benefit relaxivity. At moderate magnetic field strengths and for molecules with sufficiently long electronic relaxation times, inner sphere relaxivity tends to be greater for molecules with longer τR . Approximate τR values can be estimated by the Stokes-Einstein relationship  and are proportional to molecular size; values of 10 ns or above are typical of proteins (> 20 kD), whereas small molecules generally have τR less than 1 ns. Water structure around metalloproteins may also be conducive to greater relaxivity. Many paramagnetic proteins provide more than one water-accessible coordination site per metal ion (q > 1), compared with q = 1 for conventional Gd3+-containing contrast agents; this is a substantial benefit because inner sphere relaxivity scales with q. Although complexes with higher q bind metal ions less tightly, the potential health risk from complex dissociation is mitigated by the fact that naturally abundant transition metals are far less toxic than Gd3+ at low doses. The presence of additional bound water molecules associated with metalloproteins, including “second sphere” waters near but not directly coordinated to paramagnetic ions, may confer a further relaxivity gain . A final relaxivity-related advantage of protein contrast agents over some small molecule agents is their relative solubility. Most cytosolic or secreted metalloproteins are evolved to remain in solution or interact with well-defined ligands; this limits the potential for biological environments to substantially degrade relaxivity or analyte sensing capabilities by adversely affecting water proton interaction parameters.
The preceding discussion of advantages and disadvantages relates most directly to synthetic vs. metalloprotein T1 agents, but analogous criteria differentiate synthetic T2 agents (SPNs) from Ft as well. Synthetic iron oxide SPN contrast agents contain magnetite or maghemite, magnetic materials with saturation magnetization (MS) values of 92–100 or 60–80 emu/g, respectively , whereas Ft naturally contains a hydrated iron oxide called ferrihydrite, which has a reported MS of only 0.9–1.2 emu/g . Because the expected T2 effects of iron oxide cores are proportional to their magnetization, the relaxivity of Ft at saturating B0 is in principle only about 1% that of synthetic iron oxides for equivalent core sizes. The situation with ferrihydrite-loaded Ft may be more complex, however, as its r2 appears to increase linearly with field at least up to 11.7 T, as opposed to reaching a saturating value around 1 T like most SPNs . Further, it has been shown that Ft r2 is more directly dependent on stored iron concentration than on core size per se , suggesting an important role for inner sphere relaxivity mechanisms akin to those of conventional paramagnetic, as opposed to superparamagnetic, contrast agents. Practical advantages of Ft compared with synthetic SPNs include its regular structure and predictable size, both of which can aid in characterization or engineering the relaxivity of Ft-based contrast agents.
The first substantial investigation of magnetic properties in a metalloprotein centered on hemoglobin, the oxygen transporting heme protein in blood. Pauling and Coryell reported in a 1936 paper that hemoglobin is paramagnetic in the absence of ligand but diamagnetic when bound to oxygen . This discovery eventually led to the development of blood oxygenation level dependent (BOLD) technique for functional neuroimaging (fMRI) studies , which is by far the most commonly exploited example of metalloprotein-induced contrast in MRI. In the BOLD effect, T2-related signal changes are produced by variations in the oxygenation of hemoglobin in blood vessels. Although deoxyhemoglobin can act as a contrast agent through direct interactions with water molecules, its dominant contribution in the BOLD effect is to change the overall magnetic susceptibility of erythrocytes and blood vessels in their entirety, converting these structures into cellular-scale “contrast agents” that alter MRI signal by outer sphere effects analogous to those of SPN agents . The BOLD effect is enabled by the large concentration of hemoglobin, ~150 g/L in whole blood , giving rise to ~4 mM Fe, which ensures that even a relatively low percentage of deoxygenation translates into a formidable deoxyhemoglobin concentration. In BOLD fMRI, brain activity-induced increases in the blood supply to affected areas induces transient drops in the concentrations of deoxyhemoglobin, which are detectable as localized MRI signal increases [63–65]. BOLD imaging has been applied to detect hemodynamic changes in other tissues as well [66,67], and a BOLD-like effect produced by paramagnetic deoxymyoglobin in muscle can also be used to monitor aspects of muscular physiology .
More recently, interest in exploiting natural metalloproteins for MRI contrast has revolved largely around the potential for expressed paramagnetic proteins to act as gene reporters. An early effort to express myoglobin in transgenic mice did not produce substantial MRI contrast changes , but contrast changes have been achieved by overexpressing Ft in cells and animals. Ft has been reported to bind up to ~4500 Fe atoms per 24-mer , providing a stoichiometric advantage of over a hundred, compared with heme proteins, in terms of sheer iron accumulation. Although many of the iron atoms in Ft are organized into “antiferromagnetic” ferrihydrite domains , the limited per-iron relaxivity of Ft is still compensated for by the number of atoms that can be stored. The effect of overexpressing Ft was first demonstrated in C6 glioma cells transfected with murine heavy chain Ft (one of two isoforms, heavy and light, expressed in mammals) . A 2005 study then showed for the first time that ectopic (adenovirus driven) Ft overexpression leads to detectable T2 contrast in rodent brains , and later papers reported T2 effects in transgenic mice  and transplanted cells in vivo [21–23].
A requirement for efficacy of the Ft reporter approach is that highly regulated endogenous iron dynamics must be effectively perturbed by overexpression of the metalloprotein without toxic effects on cells; this criterion might be satisfied to varying extents in different tissues or cell types. Indeed, as potential gene reporters, metalloproteins in general are limited by the kinetics of metal ion transport and processing. Iron uptake by cells, for instance, depends on the import rate and availability of extracellular iron sources, and can require exposure times on the order of hours to achieve saturation [75–77]. To address the potentially restrictive role of iron import machinery, one study proposed co-expressing Ft with the transferrin receptor, a natural participant in cellular iron transport . A conceptually related strategy involves using overexpression of paramagnetic metal ion transporters themselves to induce MRI contrast; contrast changes in rodents have been reported using the magnetotactic bacterial protein MagA  and the mammalian divalent cation transporter DMT1 , but it is not clear which specific metalloproteins might be involved in binding the metal ions once they have entered cells.
One of the greatest strengths of protein contrast agents with respect to small molecules is in the area of biomolecular target detection, the objective of “molecular imaging.” The large surface area and numerous functional groups presented by protein interfaces render these macromolecules especially suited to binding potential ligands with high affinity and specificity. Although the potential utility of natural metalloproteins for MRI is far from fully explored, however, it is unusual to be able to find a naturally occurring paramagnetic molecule that spontaneously fits the demands of a particular biosensing application. Here another strength of proteins—their amenability to engineering approaches—comes into play. Whereas modifying small molecule agents often requires complicated synthetic schemes, proteins may be engineered using simple DNA-level changes or straightforward post-translational approaches .
The simplest examples of how proteins may be modified to facilitate analyte detection include the use of bioconjugation strategies to attach protein targeting domains to metal compounds, creating metal-protein hybrids with desirable binding and relaxivity properties. This approach was first applied to attach Gd3+ ions to antibodies esterified to DTPA or similar chelating groups [81–83], in the expectation that the resulting complexes could be used to home in on antigens in vivo and facilitate their visualization by MRI. A recent achievement using this type of approach involved the modification of the neuronal tract tracer cholera toxin subunit B with Gd3+-tetraazacyclododecanetetraacetic acid (Gd-DOTA) complexes . The resulting molecules were injected into rat brains, where they were taken up by neurons and transported to distal areas connected to the injection site, exposing patterns of connectivity in the brain. Protein bioconjugation strategies have also been extensively applied in combination with SPNs, which provide substantially greater contrast per metal atom than paramagnetic T1 agents . Individual SPN particles can be attached to multiple targeting proteins, adding the potential for improved targeting through avidity effects. SPN-protein conjugates have been produced to target genetically-expressed reporters , markers of apoptosis , vascular pathology , and cancer cells [88,89]. Analyte sensors have also been actuated by proteins that bring about reversible clustering of SPNs in the presence of various targets [90,91]. In one instance, a calcium ion sensor was made by attaching SPNs to two proteins that bind to each other in the presence but not the absence of Ca2+ . Calcium-dependent SPN aggregation was observed and response properties could be tuned by further engineering of the protein domains.
Canonical metalloproteins have also been engineered to act as MRI probes. Heme proteins are particularly attractive bases for MRI sensors because of their high metal affinity and diverse ligand binding functionality, but their properties typically need to be adjusted. For instance, Hb is the quintessential metalloprotein-based sensor , but its use as an explicit probe for measuring tissue oxygen pressure (pO2) by MRI is hindered by the fact that its natural binding midpoint (EC50) is near a pO2 of 8 mmHg, below concentrations normally found in tissue. Crosslinking Hb with glutaraldehyde improves its stability and shifts to the EC50 to 38 mmHg , allowing it to sense physiological relevant pO2 levels in tissue with a Δr2 of ~7 mM−1s−1 at 14.1 T . In a far more generalizable example of heme protein engineering for MRI, a bacterial cytochrome P450 heme domain (BM3h) that normally binds unsaturated fatty acids was retuned through the process of directed evolution  to bind and sense neural signaling molecules  in which binding of the analytes competes with inner sphere water to bring about a change in q (Figure 2A). Over five rounds of random mutagenesis and selection based on an optical titration screen, BM3h variants were obtained with micromolar affinity for dopamine, a neurotransmitter involved in reward-related signaling in the brain. These sensors had Δr1 of ~1 mM−1s−1 at 4.7 T (Figure 2B), a modest change, but one that nevertheless permitted detection of stimulus-induced dopamine release in rats. The same directed evolution strategy could also be applied to generate sensors for other targets, such as the neurotransmitter serotonin .
Ft is an obvious platform for engineering MRI probes because of its similarity to SPNs and the evidence that endogenous [95–97] or ectopic [73,74] Ft expression affects MRI contrast in vivo. The most natural way to engineer Ft without disrupting its metal storage functionality is to “display” targeting moieties on the protein surface. In one example of this approach, Uchida et al. introduced a tumor targeting peptide, RGD-4C, at the N-terminus of human heavy chain Ft and confirmed that the engineered protein (RGD4C-Fn) can still mineralize iron oxide in its cavity . RGD4C-Fn was shown to bind melanoma cells better than unmodified Ft. The solvent exposed Ft N-terminus can also be modified to create responsive MRI contrast agents. Following this strategy, Shapiro et al. developed a protein kinase A (PKA) activity sensor based on Ft, which produces aggregation-dependent T2-weighted MRI contrast changes analogous to those obtained using synthetic SPNs . The PKA sensor contains two populations of engineered Ft particles, one fused with the kinase inducible domain (KID) of the transcription factor CREB, and the other with KIX domain of the protein CBP. Upon phosphorylation of the KID domain by PKA, KID and KIX domains interact with each other  and induce clustering of KID-Ft with KIX-Ft, increasing the r2 of the sensor (Figure 2C–D). These sensors have not yet been applied in vivo, but may have the potential to allow genetically targeted functional imaging of cell or tissue types because of the all-protein nature of the Ft-based sensors and the important role of kinases in mediating cell signaling processes. Responsive Ft derivatives have also been constructed by chemically crosslinking Ft to reversible polymerizing domains .
The chief limitation of engineered metalloproteins for targeting, sensing, and gene reporting applications in MRI is their low relaxivity. For this reason, there is great interest in engineering metalloproteins with higher r1 and r2 than natural proteins. One approach to this problem is to mutate metalloprotein polypeptide sequences and examine effects on relaxivity. In one instance of this approach, a library of P450 BM3h domains selected for neurotransmitter affinity was examined for r1 variation . A range of values from 0.7 to 1.9 mM−1s−1 at 4.7 T was found among mutants that differed primarily in residues near the ligand binding site proximal to the heme. The shapes of so-called nuclear magnetic relaxation dispersion (NMRD) curves, which plot relaxation rate as a function of B0 field strength, combined with X-ray crystallographic analysis, suggested that r1 differences arose from minor variations among multiple relaxivity determinants, with subtle changes in the metal-proton distance for inner sphere water (2.6–3.0 Å) having greatest effect on r1 (Figure 3). Unfortunately it was not possible to verify the inferred distance changes at the resolution of the crystal structures, but it could be possible in the future to apply rational design principles to bring about similar changes. The NMRD results more generally indicate that nontrivial enhancement of metalloprotein relaxivity is possible through mutagenesis and screening-based approaches. A gain in the MRI contrast induced by human Ft expression has also been achieved by amino-acid level changes. Functionality of mammalian Ft is normally quite sensitive to the balance between the Ft heavy and light chains. Iordanova et al. fused H and L chains together and showed that the resulting chimera improved relaxation rates in U2OS cells were improved by roughly 50% . Although it is unclear whether any difference in r2 per iron or per Ft was achieved, cells harboring the construct appeared to contain significantly more iron than cells expressing Ft heavy and light chains separately.
Some of the most severe limits on metalloprotein relaxivity come from the characteristics of naturally bound metal ions themselves, and in particular from the spin numbers (S ≤ 5/2) of bound transition metal ions. For this reason, a second strategy for creating metalloproteins with enhanced relaxivity is to engineer the metal content of proteins, rather than simply modifying their polypeptide sequences. This strategy was applied to BM3h, which normally contains a low spin (S = 1/2) Fe3+ ion . By substituting the native ferric heme with a Mn3+ protoporphyrin complex (S = 2), a gain in relaxivity by a factor of 2.5 was obtained. In principle, the strategy should have resulted in an eight-fold improvement in r1, all else being equal, suggesting that further relaxivity enhancements might be possible by using mutagenesis approaches in parallel to metal substitution. Non-native Mn3+ incorporation into BM3h was made possible within bacterial cells by coexpressing BM3h with a porphyrin transporter called ChuA. This facilitates large scale production of metal-substituted protein in bacteria, as well as possible applications requiring intracellular compartmentalization of the Mn3+-containing protein variants.
Metal substitution has also been possible with Ft, which can be engineered to contain mineral species with higher magnetization than the natural ferrihydrite core material. The most impressive example of this approach has been the creation of “magnetoferritin”, an Ft complex in which magnetite is mineralized in the protein shell [104–106]. This can be accomplished by incubating purified apoferritin under controlled pH and oxygenation conditions in the presence of iron salts, and results in particles with a reported r2 of 78 (mM Fe)−1s−1 at 1.5 T and 37 °C, comparable to synthetic SPNs and two orders of magnitude higher than the r2 of ferrihydrite-loaded Ft. Yet higher relaxivity has been reported following mineralization of gadolinium oxide (r2 = 240 mM−1s−1 at 1.5 T)  in Ft variants, and moderate T1 relaxivity has been reported following mineralization of manganese oxyhydroxide (r1 = 6 mM−1s−1 at 0.47 T) . This strategy has also been adapted to convert viral capsids into protein-based MRI contrast agents [109–111]. Finally, Ft has also been used as a compartment for entrapment of soluble Gd-DOTA-related T1 agents . This approach takes advantage of the protein’s shell structure and availability of exchangeable protons at the protein surface, but not its mineral nucleation capabilities per se. Very high r1 values, near 80 mM−1s−1, were reported at 0.47 T. A variant of this strategy was also used to entrap Mn2+ ions in a Ft variant engineered to contain metal ions better than native Ft ; documented r1 and r2 values were 10 mM−1s−1 and 74 mM−1s−1, respectively, at 3 T. A disadvantage of most metal substitution approaches for Ft and other proteins is that they cannot be implemented in cells expressing a metalloprotein, with examples like the BM3h/ChuA strategy being occasional exceptions. Metal substituted proteins could nevertheless be useful contrast reagents for exogenous application in MRI experiments, however.
A different technique for engineering metalloprotein contrast agents is to graft metal binding functionality onto proteins or polypeptides that do not normally bind metals. One version of this route was taken with the gadolinium compound MS-325, which was designed to bind noncovalently to serum albumin in the bloodstream . The resulting complex has higher relaxivity at B0 values because of the long τR of the albumin protein. Karfeld et al. took a somewhat different approach by conjugating a large number of gadolinium chelators covalently via lysine side chains in a family of engineered repetitive polypeptides [114,115]. The authors found that the amino acid sequence of the polypeptide could be modified to produce substantial changes in T1 relaxivity. A parent sequence with ~22 kD molecular weight bearing eight Gd3+ per molecule displayed a per gadolinium r1 of 8.8 mM−1s−1 at 1.4 T, but bioconjugates with denser spacing of conjugation sites and more Gd3+ ions per polypeptide achieved r1 values up to 12.1 mM−1s−1. Relaxivities of up to 14.6 mM−1s−1 could also be obtained by increasing the molecular weight of the complexes up to ~80 kD.
Polypeptide contrast agents that chelate lanthanides without the need for bioconjugation have also been devised. In one example, an immunoglobulin domain was mutated to introduce multiple acidic sidechains in a cluster at the protein surface . One variant formed a Gd3+ complex with a reported r1 = 117 mM−1s−1 at 1.5 T, and with an apparent Kd < 1 pM and q = 2. Several efforts to construct artificial metalloprotein complexes have made use of the EF hand motif [117,118], a sequence derived from calcium binding proteins such as parvalbumin. Caravan et al. inserted an EF hand motif into a DNA-binding helix-loop-helix motif to create a chimeric DNA-sensing contrast agent . The Gd3+ complex has reported r1 values of 21 and 42 mM−1s−1 at 1.4 T in the absence and presence of DNA, respectively. In another study, the inherent promiscuity of metal binding by the EF hand motif was used to generate a sensor that functions by calcium-dependent displacement of Mn2+ ions associated with the protein calmodulin . Longitudinal relaxivity values of 11 and 8 mM−1s−1 at 4.7 T were reported in the absence and presence of 1 mM Ca2+, respectively. To improve the affinity and specificity of EF hand motifs for lanthanide binding, another group used a luminescence assay to screen libraries of troponin-derived peptides for Tb3+ binding [121,122]. T1 relaxivities were subsequently measured from Gd3+ complexes of several variants of an optimized peptide sequence . Values of up to 5.9 mM−1s−1 at 14 T were observed, despite an apparent q = 0 for the sequence with the highest relaxivity. Several of the lanthanide binding tags also retained r1 values of 2.3–5.0 mM−1s−1 when fused to the small protein ubiquitin, and crystallographic analysis of the fusion proteins reveals that the Gd3+-binding peptide domains are capable of forming q = 1 chelate complexes similar to Gd-DOTA (Figure 4). Although r1 values of these domains do not exceed those of conventional MRI contrast agents, the ability to incorporate high affinity Gd3+-binding moieties into genetically encodable proteins may facilitate application of protein engineering techniques to further enhance relaxivity or target analytes of interest.
The continued development and exploitation of metalloprotein contrast agents for MRI poses unique opportunities in the fields of molecular imaging and protein engineering. It is still uncertain what applications protein-based MRI probes will be most successful for, but utility for measuring macromolecular biokinetics and sensing a variety of ligands in vivo seems within reach. Only metalloproteins that do not require chemical modification or artificial transmetallation are applicable as endogenously expressed reporters, but efforts to improve relaxivity of these agents may ultimately yield valuable biotechnological tools, perhaps alongside recently-engineered diamagnetic proteins detectable by chemical exchange saturation contrast . Further analysis of structure-activity relationships in metalloprotein contrast agents will be interesting from a basic chemical perspective, and could also guide design of synthetic contrast agents with enhanced properties. In each of these contexts, the array of powerful genetic techniques for engineering and expressing proteins will be a tremendous benefit, and helps make continued research in this area a potential rewarding investment.
The authors were supported by NIH grants DP2-OD002114, R01-DA028299, and R01-NS076462, DARPA grant W911NF-10-0059, and a grant from the Simons Center for the Social Brain at MIT.
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