Assessment of monoclonal antibody glycosylation: a comparative study using HRMS, NMR, and HILIC-FLD.

Monoclonal antibodies (mAbs) represent the largest class of therapeutic protein drug products. mAb glycosylation produces a heterogeneous, analytically challenging distribution of glycoforms that typically should be adequately characterized because glycosylation-based product quality attributes (PQAs) can impact product quality, immunogenicity, and efficacy. In this study, two products were compared using a panel of analytical methods. Two high-resolution mass spectrometry (HRMS) workflows were used to analyze N-glycans, while nuclear magnetic resonance (NMR) was used to generate monosaccharide fingerprints. These state-of-the-art techniques were compared to conventional analysis using hydrophilic interaction chromatography (HILIC) coupled with fluorescence detection (FLD). The advantages and disadvantages of each method are discussed along with a comparison of the identified glycan distributions. The results demonstrated agreement across all methods for major glycoforms, demonstrating how confidence in glycan characterization is increased by combining orthogonal analytical methodologies. The full panel of methods used represents a diverse toolbox that can be selected from based on the needs for a specific product or analysis.

Human amniotic MSCs-mediated anti-inflammation of CD206hiIL-10hi macrophages alleviates isoproterenol-induced ventricular remodeling in mice.

BACKGROUND: Human amniotic mesenchymal stem cells (hAMSCs) derived from amniotic membrane have multilineage differentiation, immunosuppressive, and anti-inflammation which makes them suitable for the treatment of various diseases. OBJECTIVE: This study aimed to explore the therapeutic effect and molecular mechanism of hAMSCs in ventricular remodeling (VR). METHODS: hAMSCs were characterized by a series of experiments such as flow cytometric analysis, immunofluorescence, differentiative induction and tumorigenicity. Mouse VR model was induced by isoproterenol (ISO) peritoneally, and the therapeutic effects and the potential mechanisms of hAMSCs transplantation were evaluated by echocardiography, carboxy fluorescein diacetate succinimidyl ester (CFSE) labeled cell tracing, histochemistry, qRT-PCR and western blot analysis. The co-culturing experiments were carried out for further exploring the mechanisms of hAMSCs-derived conditioned medium (CM) on macrophage polarization and fibroblast fibrosis in vitro. RESULTS: hAMSCs transplantation significantly alleviated ISO-induced VR including cardiac hypertrophy and fibrosis with the improvements of cardiac functions. CFSE labeled hAMSCs kept an undifferentiated state in heart, indicating that hAMSCs-mediated the improvement of ISO-induced VR might be related to their paracrine effects. hAMSCs markedly inhibited ISO-induced inflammation and fibrosis, seen as the increase of M2 macrophage infiltration and the expressions of CD206 and IL-10, and the decreases of CD86, iNOS, COL3 and αSMA expressions in heart, suggesting that hAMSCs transplantation promoted the polarization of M2 macrophages and inhibited the polarization of M1 macrophages. Mechanically, hAMSCs-derived CM significantly increased the expressions of CD206, IL-10, Arg-1 and reduced the expressions of iNOS and IL-6 in RAW264.7 macrophages in vitro. Interestingly, RAW264.7-CM remarkably promoted the expressions of anti-inflammatory factors such as IL-10, IDO, and COX2 in hAMSCs. Furthermore, the CM derived from hAMSCs pretreated with RAW264.7-CM markedly inhibited the expressions of fibrogenesis genes such as αSMA and COL3 in 3T3 cells. CONCLUSION: Our results demonstrated that hAMSCs effectively alleviated ISO-induced cardiac hypertrophy and fibrosis, and improved the cardiac functions in mice, and the underlying mechanisms might be related to inhibiting the inflammation and fibrosis during the ventricular remodeling through promoting the polarization of CD206hiIL-10hi macrophages in heart tissues. Our study strongly suggested that by taking the advantages of the potent immunosuppressive and anti-inflammatory effects, hAMSCs may provide an alternative therapeutic approach for prevention and treatment of VR clinically.

Tuning Protein Dynamics to Sense Rapid Endoplasmic-Reticulum Calcium Dynamics.

Multi-scale calcium (Ca2+ ) dynamics, exhibiting wide-ranging temporal kinetics, constitutes a ubiquitous mode of signal transduction. We report a novel endoplasmic-reticulum (ER)-targeted Ca2+ indicator, R-CatchER, which showed superior kinetics in vitro (koff ≥2×103  s-1 , kon ≥7×106  M-1 s-1 ) and in multiple cell types. R-CatchER captured spatiotemporal ER Ca2+ dynamics in neurons and hotspots at dendritic branchpoints, enabled the first report of ER Ca2+ oscillations mediated by calcium sensing receptors (CaSRs), and revealed ER Ca2+ -based functional cooperativity of CaSR. We elucidate the mechanism of R-CatchER and propose a principle to rationally design genetically encoded Ca2+ indicators with a single Ca2+ -binding site and fast kinetics by tuning rapid fluorescent-protein dynamics and the electrostatic potential around the chromophore. The design principle is supported by the development of G-CatchER2, an upgrade of our previous (G-)CatchER with improved dynamic range. Our work may facilitate protein design, visualizing Ca2+ dynamics, and drug discovery.

NMR Spectroscopy for Protein Higher Order Structure Similarity Assessment in Formulated Drug Products.

Peptide and protein drug molecules fold into higher order structures (HOS) in formulation and these folded structures are often critical for drug efficacy and safety. Generic or biosimilar drug products (DPs) need to show similar HOS to the reference product. The solution NMR spectroscopy is a non-invasive, chemically and structurally specific analytical method that is ideal for characterizing protein therapeutics in formulation. However, only limited NMR studies have been performed directly on marketed DPs and questions remain on how to quantitively define similarity. Here, NMR spectra were collected on marketed peptide and protein DPs, including calcitonin-salmon, liraglutide, teriparatide, exenatide, insulin glargine and rituximab. The 1D 1H spectral pattern readily revealed protein HOS heterogeneity, exchange and oligomerization in the different formulations. Principal component analysis (PCA) applied to two rituximab DPs showed consistent results with the previously demonstrated similarity metrics of Mahalanobis distance (DM) of 3.3. The 2D 1H-13C HSQC spectral comparison of insulin glargine DPs provided similarity metrics for chemical shift difference (Δδ) and methyl peak profile, i.e., 4 ppb for 1H, 15 ppb for 13C and 98% peaks with equivalent peak height. Finally, 2D 1H-15N sofast HMQC was demonstrated as a sensitive method for comparison of small protein HOS. The application of NMR procedures and chemometric analysis on therapeutic proteins offer quantitative similarity assessments of DPs with practically achievable similarity metrics.

Text Mining-Based Drug Discovery in Osteoarthritis.

Background: Osteoarthritis (OA) is a chronic and degenerative joint disease, which causes stiffness, pain, and decreased function. At the early stage of OA, nonsteroidal anti-inflammatory drugs (NSAIDs) are considered the first-line treatment. However, the efficacy and utility of available drug therapies are limited. We aim to use bioinformatics to identify potential genes and drugs associated with OA. Methods: The genes related to OA and NSAIDs therapy were determined by text mining. Then, the common genes were performed for GO, KEGG pathway analysis, and protein-protein interaction (PPI) network analysis. Using the MCODE plugin-obtained hub genes, the expression levels of hub genes were verified using quantitative real-time polymerase chain reaction (qRT-PCR). The confirmed genes were queried in the Drug Gene Interaction Database to determine potential genes and drugs. Results: The qRT-PCR result showed that the expression level of 15 genes was significantly increased in OA samples. Finally, eight potential genes were targetable to a total of 53 drugs, twenty-one of which have been employed to treat OA and 32 drugs have not yet been used in OA. Conclusions: The 15 genes (including PTGS2, NLRP3, MMP9, IL1RN, CCL2, TNF, IL10, CD40, IL6, NGF, TP53, RELA, BCL2L1, VEGFA, and NOTCH1) and 32 drugs, which have not been used in OA but approved by the FDA for other diseases, could be potential genes and drugs, respectively, to improve OA treatment. Additionally, those methods provided tremendous opportunities to facilitate drug repositioning efforts and study novel target pharmacology in the pharmaceutical industry.

Cyclopentadienone Derivative Dimers as Tunable Photoswitches.

A series of photoswitchable cyclopentadienone derivative dimers bearing bromo, thienyl, 4-(dimethylamino)phenyl, 3-pyridinyl, 4-nitrophenyl and cyano groups was designed and facilely synthesized. Photoswitching properties such as the photoconversions in the photostationary state (PSS), the thermal kinetics and thermal half-lives of photoisomers were systematically investigated. These photoswitches show high fatigue resistance and large photoconversions in the PSS. This work proves that the photoswitching properties of photoswitches based on cyclopentadienone dimers can be tuned by substitution groups and also pave the way to functionalize the cyclopentadienone derivative dimer-based photoswitch, which is important for its future applications.

Rapid subcellular calcium responses and dynamics by calcium sensor G-CatchER.

The precise spatiotemporal characteristics of subcellular calcium (Ca2+) transients are critical for the physiological processes. Here we report a green Ca2+ sensor called "G-CatchER+" using a protein design to report rapid local ER Ca2+ dynamics with significantly improved folding properties. G-CatchER+ exhibits a superior Ca2+ on rate to G-CEPIA1er and has a Ca2+-induced fluorescence lifetimes increase. G-CatchER+ also reports agonist/antagonist triggered Ca2+ dynamics in several cell types including primary neurons that are orchestrated by IP3Rs, RyRs, and SERCAs with an ability to differentiate expression. Upon localization to the lumen of the RyR channel (G-CatchER+-JP45), we report a rapid local Ca2+ release that is likely due to calsequestrin. Transgenic expression of G-CatchER+ in Drosophila muscle demonstrates its utility as an in vivo reporter of stimulus-evoked SR local Ca2+ dynamics. G-CatchER+ will be an invaluable tool to examine local ER/SR Ca2+ dynamics and facilitate drug development associated with ER dysfunction.

Minor N-Glycan Mapping of Monoclonal Antibody Therapeutics Using Middle-Down NMR Spectroscopy.

The N-glycosylation pattern of Asn-297 may have impacts on monoclonal antibody (mAb) drug plasma clearance, antibody-dependent cell mediated cytotoxicity (ADCC), and complement-dependent cytotoxicity (CDC). Notably, the changes in the relative abundance of certain minor glycans, like the afucosylation, high-mannose, or galactosylation are known to change mAb properties and functions. Here, a middle-down NMR spectroscopy based analytical procedure was applied to assess the composition and structure of glycans on adalimumab and trastuzumab without glycan cleavage from the mAbs. The anomeric 2D 1H-13C spectra showed distinct patterns that could be used to profile and differentiate mAb glycan compositions. Specifically, the anomeric C1/H1 resonances from N-acetylglucosamine (GlcNAc2 and -5) and mannose (Man4) were identified as characteristic peaks for key glycan anomeric linkages and branching states. They were also utilized for measuring the relative abundance of minor glycans of total afucosylation (aFuc%), high mannose (HM%), and branch specific galactosylation (Gal1-3% and Gal1-6%). The obtained total aFuc% value of 11-12% was similar between the two mAbs; however, trastuzumab had significantly lower level of high mannose and a higher level of galactosylation than adalimumab. Overall, the 2D-NMR measurements provided functionally relevant mAb glycan composition and structure information. The method was deemed fit-for-purpose for assessment of these mAb quality attributes and involved fewer chemical preparation steps than the classical approaches that cleave glycans prior to making measurements.

Changes in the phospholipid molecular species in water-boiled salted duck during processing based on shotgun lipidomics.

Knowledge of the changes in the phospholipid molecular species during processing is helpful to understanding the complicated mechanisms of lipid degradation and transformation. The shotgun lipidomics strategy was utilized to analyze the phospholipid (PL) molecular species in raw Pekin duck and the subsequent dynamic changes that occur during the processing of water-boiled salted duck (WSD). Only 110 PL molecular species have been identified in raw duck meat, while a total of 119 PL molecular species were identified during processing, including 33 phosphatidylcholines, 22 phosphatidylethanolamines, 13 phosphatidylglycerols, 18 phosphatidylinositols and 33 phosphatidylserines. Most of the content of PL molecular species gradually decreased during processing, while the content of most of the lysophospholipids (LPLs) increased. After reaching a maximum, the LPLs were obviously reduced during the 3 d of dry-ripening. The results showed that processing techniques, such as dry-curing, dry-ripening and boiling, had a significant effect on the changes in the PLs in WSD. We further screened 10 PL molecular markers, which can be used to distinguish different operating units.

Cocrystallization of Imide-Fused Corannulene Derivatives and C60 : Guest-Induced Conformational Switching and 1:1 Segregated Packing.

A pair of interconvertible stereoisomers of imide-fused corannulene derivatives was mixed with C60 , which resulted in cocrystallization into a 1:1 segregated packing motif through concave-convex π-π interactions. Only one conformation was observed in the cocrystal owing to guest-induced conformational switching. The 1D assemblies of the complex showed promising applications in organic electronics.

Rapid B1 field mapping at 3 T using the 180° signal null method with extended flip angle.

PURPOSE: To extend the null signal method (NSM) for B1 mapping to 3 T magnetic resonance imaging (MRI). BACKGROUND: The NSM operates in the steady state regime and exploits the linearity of the spoiled gradient recalled echo (SPGR) signal around the 180° flip angle (FA). Using linear regression, B1 maps are derived from three SPGR images acquired at different FAs with a short repetition time. While the conventional NSM allows accurate mapping of B1 for moderate B1 variation, we observed that this method fails for the larger B1 variations typical of high-field MRI. METHODS: We analyzed the effect of the FA range of the acquired SPGR images on B1 determination using the NSM for 3 T MRI through extensive numerical and in vivo analyses. B1 maps derived from the extended angle-range NSM (EA-NSM) were calculated and compared to those derived from the conventional, more restricted angle range, NSM, and to those derived from the reference, but much more time-consuming, double angle method (DAM). Furthermore, we investigated the compatibility of EA-NSM B1 mapping and the half-scan and SENSE reconstruction methods for accelerating acquisition time. RESULTS: Our results show that the use of the conventional FA range leads to substantial inaccuracies in B1 determination. Both numerical and in vivo analyses demonstrate that expanding the FA range of the acquired SPGR images substantially improves the accuracy of B1 maps. Furthermore, B1 maps derived from EA-NSM were demonstrated to be quantitatively comparable to those derived from the lengthy DAM protocol. We also found that B1 maps derived from SPGR images using the EA-NSM and imaging acceleration methods were comparable to those derived from images acquired without acceleration. Finally, the use of half scanning combined with SENSE reconstruction permits whole-brain B1 mapping in ~1 min. CONCLUSIONS: The EA-NSM permits accurate, fast, and practical B1 mapping in a 3 T clinical setting.

Direct visualization of interaction between calmodulin and connexin45.

Calmodulin (CaM) is an intracellular Ca2+ transducer involved in numerous activities in a broad Ca2+ signaling network. Previous studies have suggested that the Ca2+/CaM complex may participate in gap junction regulation via interaction with putative CaM-binding motifs in connexins; however, evidence of direct interactions between CaM and connexins has remained elusive to date due to challenges related to the study of membrane proteins. Here, we report the first direct interaction of CaM with Cx45 (connexin45) of γ-family in living cells under physiological conditions by monitoring bioluminescence resonance energy transfer. The interaction between CaM and Cx45 in cells is strongly dependent on intracellular Ca2+ concentration and can be blocked by the CaM inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W7). We further reveal a CaM-binding site at the cytosolic loop (residues 164-186) of Cx45 using a peptide model. The strong binding (Kd ∼ 5 nM) observed between CaM and Cx45 peptide, monitored by fluorescence-labeled CaM, is found to be Ca2+-dependent. Furthermore, high-resolution nuclear magnetic resonance spectroscopy reveals that CaM and Cx45 peptide binding leads to global chemical shift changes of 15N-labeled CaM, but does not alter the size of the structure. Observations involving both N- and C-domains of CaM to interact with the Cx45 peptide differ from the embraced interaction with Cx50 from another connexin family. Such interaction further increases Ca2+ sensitivity of CaM, especially at the N-terminal domain. Results of the present study suggest that both helicity and the interaction mode of the cytosolic loop are likely to contribute to CaM's modulation of connexins.

Dynamic nuclear polarization facilitates monitoring of pyruvate metabolism in Trypanosoma brucei.

Dynamic nuclear polarization provides sensitivity improvements that make NMR a viable method for following metabolic conversions in real time. There are now many in vivo applications to animal systems and even to diagnosis of human disease. However, application to microbial systems is rare. Here we demonstrate its application to the pathogenic protozoan, Trypanosoma brucei, using hyperpolarized 13C1 pyruvate as a substrate and compare the parasite metabolism with that of commonly cultured mammalian cell lines, HEK-293 and Hep-G2. Metabolic differences between insect and bloodstream forms of T. brucei were also investigated. Significant differences are noted with respect to lactate, alanine, and CO2 production. Conversion of pyruvate to CO2 in the T. brucei bloodstream form provides new support for the presence of an active pyruvate dehydrogenase in this stage.

Glycosylation Alters Dimerization Properties of a Cell-surface Signaling Protein, Carcinoembryonic Antigen-related Cell Adhesion Molecule 1 (CEACAM1).

Human carcinoembryonic antigen-related cell adhesion molecule 1 (C?/Au: EACAM1) is a cell-surface signaling molecule involved in cell adhesion, proliferation, and immune response. It is also implicated in cancer angiogenesis, progression, and metastasis. This diverse set of effects likely arises as a result of the numerous homophilic and heterophilic interactions that CEACAM1 can have with itself and other molecules. Its N-terminal Ig variable (IgV) domain has been suggested to be a principal player in these interactions. Previous crystal structures of the ß-sandwich-like IgV domain have been produced using Escherichia coli-expressed material, which lacks native glycosylation. These have led to distinctly different proposals for dimer interfaces, one involving interactions of ABED ß-strands and the other involving GFCC'C″ ß-strands, with the former burying one prominent glycosylation site. These structures raise questions as to which form may exist in solution and what the effect of glycosylation may have on this form. Here, we use NMR cross-correlation measurements to examine the effect of glycosylation on CEACAM1-IgV dimerization and use residual dipolar coupling (RDC) measurements to characterize the solution structure of the non-glycosylated form. Our findings demonstrate that even addition of a single N-linked GlcNAc at potential glycosylation sites inhibits dimer formation. Surprisingly, RDC data collected on E. coli expressed material in solution indicate that a dimer using the non-glycosylated GFCC'C″ interface is preferred even in the absence of glycosylation. The results open new questions about what other factors may facilitate dimerization of CEACAM1 in vivo, and what roles glycosylation may play in heterophylic interactions.

Fast kinetics of calcium signaling and sensor design.

Fast calcium signaling is regulated by numerous calcium channels exhibiting high spatiotemporal profiles which are currently measured by fluorescent calcium sensors. There is still a strong need to improve the kinetics of genetically encoded calcium indicators (sensors) to capture calcium dynamics in the millisecond time frame. In this review, we summarize several major fast calcium signaling pathways and discuss the recent developments and application of genetically encoded calcium indicators to detect these pathways. A new class of genetically encoded calcium indicators designed with site-directed mutagenesis on the surface of beta-barrel fluorescent proteins to form a pentagonal bipyramidal-like calcium binding domain dramatically accelerates calcium binding kinetics. Furthermore, novel genetically encoded calcium indicators with significantly increased fluorescent lifetime change are advantageous in deep-field imaging with high light-scattering and notable morphology change.

Effect of Ca²âº on the steady-state and time-resolved emission properties of the genetically encoded fluorescent sensor CatchER.

We previously designed a calcium sensor CatchER (a GFP-based Calcium sensor for detecting high concentrations in the high calcium concentration environment such as ER) with a capability for monitoring calcium ion responses in various types of cells. Calcium binding to CatchER induces the ratiometric changes in the absorption spectra, as well as an increase in fluorescence emission at 510 nm upon excitation at both 395 and 488 nm. Here, we have applied the combination of the steady-state and time-resolved optical methods and Hydrogen/Deuterium isotope exchange to understand the origin of such calcium-induced optical property changes of CatchER. We first demonstrated that calcium binding results in a 44% mean fluorescence lifetime increase of the indirectly excited anionic chromophore. Thus, CatchER is the first protein-based calcium indicator with the single fluorescent moiety to show the direct correlation between the lifetime and calcium binding. Calcium exhibits a strong inhibition on the excited-state proton transfer nonadiabatic geminate recombination in protic (vs deuteric) medium. Analysis of CatchER crystal structures and the MD simulations reveal the proton transfer mechanism in which the disrupted proton migration path in CatchER is rescued by calcium binding. Our finding provides important insights for a strategy to design calcium sensors and suggests that CatchER could be a useful probe for FLIM imaging of calcium in situ.

Direct determination of multiple ligand interactions with the extracellular domain of the calcium-sensing receptor.

Numerous in vivo functional studies have indicated that the dimeric extracellular domain (ECD) of the CaSR plays a crucial role in regulating Ca(2+) homeostasis by sensing Ca(2+) and l-Phe. However, direct interaction of Ca(2+) and Phe with the ECD of the receptor and the resultant impact on its structure and associated conformational changes have been hampered by the large size of the ECD, its high degree of glycosylation, and the lack of biophysical methods to monitor weak interactions in solution. In the present study, we purified the glycosylated extracellular domain of calcium-sensing receptor (CaSR) (ECD) (residues 20-612), containing either complex or high mannose N-glycan structures depending on the host cell line employed for recombinant expression. Both glycosylated forms of the CaSR ECD were purified as dimers and exhibit similar secondary structures with ∼ 50% α-helix, ∼ 20% ß-sheet content, and a well buried Trp environment. Using various spectroscopic methods, we have shown that both protein variants bind Ca(2+) with a Kd of 3.0-5.0 mm. The local conformational changes of the proteins induced by their interactions with Ca(2+) were visualized by NMR with specific (15)N Phe-labeled forms of the ECD. Saturation transfer difference NMR approaches demonstrated for the first time a direct interaction between the CaSR ECD and l-Phe. We further demonstrated that l-Phe increases the binding affinity of the CaSR ECD for Ca(2+). Our findings provide new insights into the mechanisms by which Ca(2+) and amino acids regulate the CaSR and may pave the way for exploration of the structural properties of CaSR and other members of family C of the GPCR superfamily.

The hemodynamically-regulated vascular microenvironment promotes migration of the steroidogenic tissue during its interaction with chromaffin cells in the zebrafish embryo.

BACKGROUND: While the endothelium-organ interaction is critical for regulating cellular behaviors during development and disease, the role of blood flow in these processes is only partially understood. The dorsal aorta performs paracrine functions for the timely migration and differentiation of the sympatho-adrenal system. However, it is unclear how the adrenal cortex and medulla achieve and maintain specific integration and whether hemodynamic forces play a role. METHODOLOGY AND PRINCIPAL FINDINGS: In this study, the possible modulation of steroidogenic and chromaffin cell integration by blood flow was investigated in the teleostean counterpart of the adrenal gland, the interrenal gland, in the zebrafish (Danio rerio). Steroidogenic tissue migration and angiogenesis were suppressed by genetic or pharmacologic inhibition of blood flow, and enhanced by acceleration of blood flow upon norepinephrine treatment. Repressed steroidogenic tissue migration and angiogenesis due to flow deficiency were recoverable following restoration of flow. The regulation of interrenal morphogenesis by blood flow was found to be mediated through the vascular microenvironment and the Fibronectin-phosphorylated Focal Adhesion Kinase (Fn-pFak) signaling. Moreover, the knockdown of krüppel-like factor 2a (klf2a) or matrix metalloproteinase 2 (mmp2), two genes regulated by the hemodynamic force, phenocopied the defects in migration, angiogenesis, the vascular microenvironment, and pFak signaling of the steroidogenic tissue observed in flow-deficient embryos, indicating a direct requirement of mechanotransduction in these processes. Interestingly, epithelial-type steroidogenic cells assumed a mesenchymal-like character and downregulated ß-Catenin at cell-cell junctions during interaction with chromaffin cells, which was reversed by inhibiting blood flow or Fn-pFak signaling. Blood flow obstruction also affected the migration of chromaffin cells, but not through mechanosensitive or Fn-pFak dependent mechanisms. CONCLUSIONS AND SIGNIFICANCE: These results demonstrate that hemodynamically regulated Fn-pFak signaling promotes the migration of steroidogenic cells, ensuring their interaction with chromaffin cells along both sides of the midline during interrenal gland development.

Structural basis for a hand-like site in the calcium sensor CatchER with fast kinetics.

Calcium ions, which are important signaling molecules, can be detected in the endoplasmic reticulum by an engineered mutant of green fluorescent protein (GFP) designated CatchER with a fast off-rate. High resolution (1.78-1.20 Å) crystal structures were analyzed for CatchER in the apo form and in complexes with calcium or gadolinium to probe the binding site for metal ions. While CatchER exhibits a 1:1 binding stoichiometry in solution, two positions were observed for each of the metal ions bound within the hand-like site formed by the carboxylate side chains of the mutated residues S147E, S202D, Q204E, F223E and T225E that may be responsible for its fast kinetic properties. Comparison of the structures of CatchER, wild-type GFP and enhanced GFP confirmed that different conformations of Thr203 and Glu222 are associated with the two forms of Tyr66 of the chromophore which are responsible for the absorbance wavelengths of the different proteins. Calcium binding to CatchER may shift the equilibrium for conformational population of the Glu222 side chain and lead to further changes in its optical properties.