A novel yellow fluorescent protein of recombinant apoPholasin with dehydrocoelenterazine.

Pholasin is classified as a photoprotein and comprises apoPholasin (an apoprotein of pholasin) and an unknown prosthetic group as the light-emitting source. The luminescence reaction of pholasin is triggered by reactive oxygen species. Recombinant apoPholasin was recently expressed as a fusion protein of glutathione S-transferase (GST-apoPholasin) and purified from E. coli cells. By incubating non-fluorescent dehydrocoelenterazine (dCTZ, dehydrogenated form of CTZ) with GST-apoPholasin, the complex of GST-apoPholasin and dCTZ (GST-apoPholasin/dCTZ complex) was formed immediately and showed bright yellow fluorescence (λmax = 539 nm, excited at 430 nm). Unexpectedly, the fluorescent chromophore of the GST-apoPholasin/dCTZ complex was identified as non-fluorescent dCTZ. The luminescence intensity of the GST-apoPholasin/dCTZ complex was increased in a catalase-H2O2 system, but not in sodium hypochlorite.

Transferrin and thyroid hormone converge in the control of myelinogenesis.

Myelination is a concerted mechanism tightly regulated in the brain. Although several factors are known to participate during this process, the complete sequence of events is far from being fully elucidated. Separate effects of apotransferrin (aTf) and thyroid hormone (TH) are well documented on rat myelin formation. TH promotes the maturation of oligodendrocyte progenitors (OPCs) into myelinating oligodendrocytes (OLGs), while aTf is able to induce the commitment of neural stem cells (NSCs) toward the oligodendroglial linage and favors OLG maturation. We have also demonstrated that Tf mRNA exhibited a seven-fold increase in hyperthyroid animals. These observations have led us to hypothesize that both factors may interplay during oligodendrogenesis. To assess the combined effects of aTf and TH on proper myelination in the rat brain, Tf expression and oligodendroglial maturation were evaluated at postnatal days 10 (P10) and 20 (P20) in several experimental groups. At P10, an up-regulation of both Tf mRNA and protein, as well as myelination, was found in hyperthyroid animals, while a decrease in Tf mRNA levels and myelin formation was detected in the hypothyroid group. At P20, no differences were found either in Tf mRNA or protein levels between hyperthyroid and control (Ctrol) rats, although differences in OLG differentiation remained. Also at P20, hypothyroid animals showed decreased Tf mRNA and protein levels accompanied with a less mature myelinating phenotype. Moreover, TH and aTf differentially regulate the expression of KLF9 transcription factor as well as TRα and TRß at P10 and P20. Our results suggest that TH is necessary early in OLG development for aTf action, as exogenous aTf administration was unable to counteract the effect of low TH levels in the hypothyroid state in all the time points analyzed. Furthermore, the fact that hyperthyroidism induced an increase in Tf expression and aTf-dependent regulation of TRα strongly suggests that Tf could be involved in some of TH later effects on OLG maturation. Here we describe the possible relationship between TH and aTf and its implication in oligodendrogenesis.

Expression of recombinant full-length plant phytochromes assembled with phytochromobilin in Pichia pastoris.

We have successfully developed a system to produce full-length plant phytochrome assembled with phytochromobilin in Pichia pastoris by co-expressing apophytochromes and chromophore biosynthetic genes, heme oxygenase (HY1) and phytochromobilin synthase (HY2) from Arabidopsis. Affinity-purified phytochrome proteins from Pichia cells displayed zinc fluorescence indicating chromophore attachment. Spectroscopic analyses showed absorbance maximum peaks identical to in vitro reconstituted phytochromobilin-assembled phytochromes, suggesting that the co-expression system is effective to generate holo-phytochromes. Moreover, mitochondria localization of the phytochromobilin biosynthetic genes increased the efficiency of holophytochrome biosynthesis. Therefore, this system provides an excellent source of holophytochromes, including oat phytochrome A and Arabidopsis phytochrome B.

Safety assessment of the calcium-binding protein, apoaequorin, expressed by Escherichia coli.

Calcium-binding proteins are ubiquitous modulators of cellular activity and function. Cells possess numerous calcium-binding proteins that regulate calcium concentration in the cytosol by buffering excess free calcium ion. Disturbances in intracellular calcium homeostasis are at the heart of many age-related conditions making these proteins targets for therapeutic intervention. A calcium-binding protein, apoaequorin, has shown potential utility in a broad spectrum of applications for human health and well-being. Large-scale recombinant production of the protein has been successful; enabling further research and development and commercialization efforts. Previous work reported a 90-day subchronic toxicity test that demonstrated this protein has no toxicity by oral exposure in Sprague-Dawley rodents. The current study assesses the allergenic potential of the purified protein using bioinformatic analysis and simulated gastric digestion. The results from the bioinformatics searches with the apoaequorin sequence show the protein is not a known allergen and not likely to cross-react with known allergens. Apoaequorin is easily digested by pepsin, a characteristic commonly exhibited by many non-allergenic dietary proteins. From these data, there is no added concern of safety due to unusual stability of the protein by ingestion.

Mucin5B expression by lung alveolar macrophages is increased in long-term smokers.

This study investigated the expression of MUC5B by AMs in the lungs of cigarette smokers and nonsmokers. We analyzed MUC5B expression by measuring the levels of apomucin and mRNA in human BALF cells from 50 subjects (20 nonsmokers, 17 patients with CB, and 13 patients with COPD). apoMUC5B was observed in BALF mononuclear cells in 60% of all subjects, but a significantly higher frequency of apoMUC5B(+) cells was found in subjects with CB (95% CI, 4.5-24.9) or COPD (95% CI, 6.2-39.6) than in nonsmokers (95% CI, 0.5-2.5). apoMUC5B(+) mononuclear cells showed strong expression of CD163, confirming their identity as AMs. MUC5B mRNA expression was detected by ISH in AMs of subjects investigated, and real-time qPCR analysis confirmed MUC5B mRNA expression. In conclusion, MUC5B is expressed in a subset of lung AMs and long-term cigarette smoking may increase the level of MUC5B produced by these cells.

Thiol redox requirements and substrate specificities of recombinant cytochrome c assembly systems II and III.

The reconstitution of biosynthetic pathways from heterologous hosts can help define the minimal genetic requirements for pathway function and facilitate detailed mechanistic studies. Each of the three pathways for the assembly of cytochrome c in nature (called systems I, II, and III) has been shown to function recombinantly in Escherichia coli, covalently attaching heme to the cysteine residues of a CXXCH motif of a c-type cytochrome. However, recombinant systems I (CcmABCDEFGH) and II (CcsBA) function in the E. coli periplasm, while recombinant system III (CCHL) attaches heme to its cognate receptor in the cytoplasm of E. coli, which makes direct comparisons between the three systems difficult. Here we show that the human CCHL (with a secretion signal) attaches heme to the human cytochrome c (with a signal sequence) in the E. coli periplasm, which is bioenergetically (p-side) analogous to the mitochondrial intermembrane space. The human CCHL is specific for the human cytochrome c, whereas recombinant system II can attach heme to multiple non-cognate c-type cytochromes (possessing the CXXCH motif.) We also show that the recombinant periplasmic systems II and III use components of the natural E. coli periplasmic DsbC/DsbD thiol-reduction pathway. This article is part of a Special Issue entitled: Biogenesis/Assembly of Respiratory Enzyme Complexes.

Glycine cleavage enzyme complex: molecular cloning and expression of the H-protein cDNA from cultured human skin fibroblasts.

The human H-protein is one of four essential components (H-, L-, P-, and T-proteins) of the mammalian glycine cleavage enzyme complex and its function is involved in the pathogenesis and diagnosis of glycine encephalopathy. A transcript corresponding to the glycine cleavage H-protein functional gene was isolated from cultured human skin fibroblasts along with a transcript for a putative processed pseudogene on chromosome 2q33.3. Sequence analysis of the fibroblast H-protein functional gene transcript showed complete identity to that reported from human liver. The H-protein cDNA was subsequently cloned with a hexahistidine affinity tag in the Pichia pastoris plasmid vector pPICZαA and recombined into the yeast genome downstream of the alcohol oxidase promoter for methanol-induced expression. The recombinant H-protein was secreted into the culture medium and purified to homogeneity using a one-step nickel-nitrilotriacetic acid resin column. Approximately 4 mg of homogeneous H-protein was obtained from 1 L of culture medium. Since the attachment of a lipoic acid prosthetic group is required for H-protein function, we have expressed and purified E. coli lipoate protein ligase and succeeded in lipoylating H-protein, converting the apo-H-protein to the functional holo-H-protein. A lipoamide dehydrogenase assay was performed to confirm that the apo-H-protein was inactive, whereas the holo-H-protein was approximately 2.3-fold more active than free lipoic acid as a hydrogen donor in driving the reaction. The availability of copious amounts of human recombinant H-protein by using Pichia pastoris expression and affinity purification will facilitate the elucidation of the structure and function of the H-protein and its relationship to the P-, T-, and L-proteins in the glycine cleavage enzyme complex. In view of the fact that there is no detectable glycine cleavage enzyme activity in human skin fibroblasts, we speculate that a plausible function of the H-protein is to interact with the L-protein, which is also part of the l-ketoglutarate dehydrogenase complex present in fibroblasts.

Visualization, characterization and modulation of calcium signaling during the development of slow muscle cells in intact zebrafish embryos.

Intact zebrafish embryos were used as an in vivo animal model to investigate the role of Ca2+ signaling during the differentiation of slow muscle cells (SMCs) within forming skeletal muscle. Transgenic zebrafish were generated using an a-actin promoter that targeted apoaequorin expression specifically to muscle cells. Two distinct Ca2+ signaling periods (CSPs) were visualized in the developing SMCs: between ~17.5-19.5 hours post-fertilization (hpf) and after ~23 hpf, separated by a ~3.5 h Ca2+ signaling quiet period. Further spatial characterization of these Ca2+ signals using confocal fluorescent microscopy and calcium green-1 dextran as a reporter, indicated that the earlier CSP displayed distinct nuclear and cytoplasmic components, whereas the later CSP was predominantly cytoplasmic. Both CSPs consisted of a series of oscillating Ca2+ waves generated at distinct frequencies, while the earlier CSP also displayed a slow rise then fall in the Ca2+ baseline-level. Imaging of cyclopamine- and forskolin-treated wild-type, or smo-/- mutant embryos, where SMCs do not form, confirmed the specific cell population generating the signals. Treating embryos with antagonists indicated that both IP3Rs and RyRs are responsible for generating the temporal characteristics of the Ca2+ signaling signature, and that the latter plays a necessary role in SMC differentiation and subsequent myotome patterning. Together, these data support and extend the proposition that specific spatiotemporal patterns of spontaneous Ca2+ signals might be used for different as well as combinatorial regulation of both nuclear and cytosolic signal transduction cascades, resulting in myofibrillogenesis in SMCs as well as myotome patterning.

Techniques to study specific cell-surface receptor-mediated cellular vitamin A uptake.

STRA6 is a multitransmembrane domain protein that was recently identified as the cell-surface receptor for plasma retinol-binding protein (RBP), the vitamin A carrier protein in the blood. STRA6 binds to RBP with high affinity and mediates cellular uptake of vitamin A from RBP. It is not homologous to any known receptors, transporters, and channels, and it represents a new class of membrane transport protein. Consistent with the diverse physiological functions of vitamin A, STRA6 is widely expressed in diverse adult organs and throughout embryonic development. Mutations in human STRA6 that abolish its vitamin A uptake activity cause severe pathological phenotypes in many human organs including the eye, brain, lung, and heart. This chapter describes functional assays for STRA6 in live cells and on cellular membranes. These assays can be employed to study the mechanism of this new membrane transport mechanism and its roles in the physiology and pathology of many organs.

Reconstitution of blue fluorescent protein from recombinant apoaequorin and synthetic coelenteramide.

Blue fluorescent protein of aequorin (BFP) is a complex of Ca(2+)-bound apoaequorin with coelenteramide and is a bifunctional protein, which shows blue fluorescence and the luminescence activity like a luciferase. To reconstitute synthetic BFP (syn-BFP) from apoaequorin and coelenteramide, we established new synthetic route of coelenteramide and prepared highly purified recombinant aequorin using the histidine-tagged secretion system in Escherichia coli cells. As a result, we succeeded in reconstituting syn-BFP quantitatively and the fluorescence and luminescence properties of syn-BFP were identical to that of BFP obtained from aequorin.

Chapter 14 Nucleotide-dependent iron-sulfur cluster biogenesis of endogenous and imported apoproteins in isolated intact mitochondria.

Iron-sulfur [Fe-S] clusters are cofactors of proteins involved in electron transfer, enzyme catalysis, radical generation, sulfur donation, and signal transduction. Biogenesis of [Fe-S] clusters is mediated by numerous conserved proteins present in E. coli and in mitochondria of eukaryotic cells such as yeast and humans. Although a completely reconstituted system for study of this process does not yet exist, isolated intact mitochondria are capable of synthesizing new [Fe-S] clusters when supplied with a few ingredients. Here we describe methods for studying the biogenesis of [Fe-S] clusters in intact mitochondria. In these assays, metabolically active mitochondria isolated from a wild-type Saccharomyces cerevisiae strain are incubated with (35)S-cysteine. The (35)S is rapidly (approximately 15 min) and efficiently incorporated by physiologic pathways into newly formed [Fe-S] clusters and inserted into target proteins. Proteins labeled with [Fe-(35)S] clusters are then separated by native polyacrylamide gel electrophoresis followed by autoradiography, thereby allowing direct visualization and quantitation. Both endogenous (Aco1p aconitase) and newly imported (Yah1p ferredoxin) apoproteins can be used as substrates. [Fe-S] cluster biogenesis in isolated intact mitochondria is greatly enhanced by the addition of nucleotides (GTP and ATP) and requires hydrolysis of both. A major advantage of the methods described here is that neither in vivo overexpression of target substrates nor enrichment by immunoprecipitation is necessary to detect radiolabeled proteins. It is also not necessary to perform these assays under anaerobic conditions, because intact mitochondria are capable of protecting newly formed [Fe-S] clusters from oxidative damage.

Soluble protein expression in E. coli cells using IgG-binding domain of protein A as a solubilizing partner in the cold induced system.

We constructed a cold induced expression vector in Escherichia coli cells that consists of a histidine tag sequence for nickel chelate affinity purification, IgG-binding domain of protein A (ZZ-domain) and the multiple cloning sites. The role of ZZ-domain as a solubilizing partner at 15 degrees C was demonstrated by expressing the imidazopyrazinone-type luciferases of Renilla, Oplophorus, Gaussia, and Vargula (Cypridina) as well as the calcium-binding photoproteins and firefly luciferase. The fused protein with ZZ-domain was expressed efficiently as a soluble form in the cytoplasm of E. coli cells at low temperature.

Folding on the assembly line.

Deciphering the mechanism of folding of newly synthesized proteins in the cell is a major challenge because of the large size and multiplicity of molecular components involved and the asynchrony of biosynthesis. Fluorescently labeled ribosome-bound nascent chains of a defined length were prepared and subjected to dynamic fluorescence depolarization spectroscopy measurements. Nanosecond anisotropy decay correlation times of proteins' nascent chains at different stages of polypeptide elongation were determined for the first time. Striking dependence of the chain dynamics on the stages of elongation was observed and revealed chain length dependence of folding on the ribosome.

Chain dynamics of nascent polypeptides emerging from the ribosome.

Very little is known about the conformation of polypeptides emerging from the ribosome during protein biosynthesis. Here, we explore the dynamics of ribosome-bound nascent polypeptides and proteins in Escherichia coli by dynamic fluorescence depolarization and assess the population of cotranslationally active chaperones trigger factor (TF) and DnaK. E. coli cell-free technology and fluorophore-linked E. coli Met-tRNA f Met enable selective site-specific labeling of nascent proteins at the N-terminal methionine. For the first time, direct spectroscopic evidence captures the generation of independent nascent chain motions for a single-domain protein emerging from the ribosome (apparent rotational correlation time approximately 5 ns), during the intermediate and late stages of polypeptide elongation. Such motions are detected only for a sequence encoding a globular protein and not for a natively unfolded control, suggesting that the independent nascent chain dynamics may be a signature of folding-competent sequences. In summary, we observe multicomponent, severely rotationally restricted, and strongly chain length/sequence-dependent nascent chain dynamics.

A micropapillary pattern is predictive of a poor prognosis in lung adenocarcinoma, and reduced surfactant apoprotein A expression in the micropapillary pattern is an excellent indicator of a poor prognosis.

A micropapillary pattern is defined as papillary tufts without a fibrovascular core and is known to be a factor that indicates a poor prognosis in numerous cancers. However, their role in lung adenocarcinoma has not been investigated widely. In 185 cases of small-size lung adenocarcinoma (< or =3 cm), cases with a micropapillary pattern ratio of more than 1% (analyzed by NIH image) were defined as micropapillary pattern positive. Correlations between the micropapillary pattern and clinicopathological factors were investigated and immunohistochemical expression of mucin and various antigens was examined in regions with and without micropapillary patterns. Micropapillary pattern-positive tumors (micropapillary pattern ratio > or =1%) were observed in 11.4% of cases (21/185) and the micropapillary pattern ratio correlated with TNM stage (P=0.0002), lymphatic invasion (P=0.0002) and lymph node metastasis (P=0.03). Disease-free interval (P<0.0002) and survival (P=0.027) were significantly shorter for micropapillary pattern-positive patients, and micropapillary pattern-positive stage IA cases also had a significantly shorter disease-free interval (P<0.0001). MUC1 was expressed strongly across the surface of the micropapillary structure, whereas MUC4 tended to show lower expression in the micropapillary pattern. It was noteworthy that the disease-free interval in patients with high surfactant apoprotein A expression was significantly better than in patients with low surfactant apoprotein A expression (P=0.03), and no recurrence or death occurred in patients with high surfactant apoprotein A expression. Our results show that the micropapillary pattern ratio correlates with lymphatic invasion and lymph node metastasis, and that a high micropapillary pattern ratio leads to a poor prognosis. High MUC1 expression on the surface is an important characteristic of a micropapillary pattern, and reduced surfactant apoprotein A expression in the micropapillary pattern may be an excellent indicator for poor prognosis in small-size lung adenocarcinoma.

Formation of fluorescent proteins by the attachment of phycoerythrobilin to R-phycoerythrin alpha and beta apo-subunits.

Formation of fluorescent proteins was explored after incubation of recombinant apo-subunits of phycobiliprotein R-phycoerythrin with phycoerythrobilin chromophore. Alpha and beta apo-subunit genes of R-phycoerythrin from red algae Polisiphonia boldii were cloned in plasmid pET-21d(+). Hexahistidine-tagged alpha and beta apo-subunits were expressed in Escherichia coli. Although expressed apo-subunits formed inclusion bodies, fluorescent holo-subunits were constituted after incubation of E. coli cells with phycoerythrobilin. Holo-subunits contained both phycoerythrobilin and urobilin chromophores. Fluorescence and differential interference contrast microscopy showed polar location of holo-subunit inclusion bodies in bacterial cells. Cells containing fluorescent holo-subunits were several times brighter than control cells as found by fluorescence microscopy and flow cytometry. The addition of phycoerythrobilin to cells did not show cytotoxic effects, in contrast to expression of proteins in inclusion bodies. In an attempt to improve solubility, R-phycoerythrin apo-subunits were fused to maltose-binding protein and incubated with phycoerythrobilin both in vitro and in vivo. Highly fluorescent soluble fusion proteins containing phycoerythrobilin as the sole chromophore were formed. Fusion proteins were localized by fluorescence microscopy either throughout E. coli cells or at cell poles. Flow cytometry showed that cells containing fluorescent fusion proteins were up to 10 times brighter than control cells. Results indicate that fluorescent proteins formed by attachment of phycoerythrobilin to expressed apo-subunits of phycobiliproteins can be used as fluorescent probes for analysis of cells by microscopy and flow cytometry. A unique property of these fluorescent reporters is their utility in both properly folded (soluble) subunits and subunits aggregated in inclusion bodies.

Transient expression of apoaequorin in zebrafish embryos: extending the ability to image calcium transients during later stages of development.

When aequorin is microinjected into cleavage-stage zebrafish embryos, it is largely used up by ~24 hours. Thus, it is currently not possible to image Ca(2+) signals from later stages of zebrafish development using this approach. We have, therefore, developed protocols to express apoaequorin, i.e., the protein component of aequorin, transiently in zebrafish embryos and then reconstitute intact aequorin in vivo by loading the coelenterazine co-factor into the embryos separately. Two types of apoaequorin mRNA, aeq-mRNA and aeq::EGFP-mRNA, the latter containing the enhanced green fluorescent protein (EGFP) sequence, were in vitro transcribed and when these were microinjected into embryos, they successfully translated apoaequorin and a fusion protein of apoaequorin and EGFP (apoaequorin-EGFP), respectively. We show that aeq::EGFP -mRNA was more toxic to embryos than equivalent amounts of aeq-mRNA. In addition, in an in vitro reconstitution assay, apoaequorin-EGFP produced less luminescence than apoaequorin, after reconstitution with coelenterazine and with the addition of Ca(2+). Furthermore, when imaging intact coelenterazine-loaded embryos that expressed apoaequorin, Ca(2+ )signals from ~2.5 to 48 hpf were observed, with the spatio-temporal pattern of these signals up to 24 hpf, being comparable to that observed with aequorin. This transient aequorin expression approach using aeq-mRNA provides a valuable tool for monitoring Ca(2+ )signaling during the 2448 hpf period of zebrafish development. Thus, it effectively extends the aequorin-based Ca(2+) imaging window by an additional 24 hours.

Preparation of neocarzinostatin apoprotein mutants and the randomized library on the chromophore-binding cavity.

W39F, F52Y, S98G, S98A, and S98C mutants of the neocarzinostatin apoprotein (apo-NCS) were newly prepared and investigated their physicochemical properties. The circular dichroism (CD) spectra of F78W, F52Y, S98A, S98G, S98C were superimposable with that of wild type 1R49 protein although the minor spectral change seemed to be in the ellipticity of W39F. The results suggest that position 52, 78, and 98 involving natural chromophore binding do not play a major role in the inducing overall structural changes of the protein. Conversely, the position 39 would be affected slightly. Ethidium bromide (EtdBr) binding to mutants was also evaluated by the monitoring of total fluorescence intensity and fluorescence polarization (FP). The observed dissociation constant in the FP study was 4.4 microM for wild type, 2.2 microM for S98A, 1.3 microM for S98G, 9.7 microM for S98C, respectively. When S98G and F52Y, the calculated maximum change of the total fluorescence intensity was increased, suggesting that the EtdBr binding to S98G or F52Y were slightly improved compared with the wild type. Then, a total of 14 amino acids randomly substituted phage displayed library of apo-NCS was successfully prepared, because substitution of the amino acid structured the chromophore-binding cavity were not change the overall structural features. The phages which bound glycyrrhetic acid conjugated bovine serum albumin were enriched from this library using phage display technique as the pilot experiments. Although more precision investigation still needs, it should be possible to select variants that have new functions not found in nature.

Immunohistochemical analysis of MUC5B apomucin expression in breast cancer and non-malignant breast tissues.

A deregulation of several MUC genes (MUC1, MUC2, MUC3, MUC5AC, and MUC6) was previously demonstrated in breast carcinomas. Considering that recently we found the "non-mammary" MUC5B mRNA in primary breast tumors (Berois et al. 2003), we undertook the present study to evaluate the expression profile of MUC5B protein product in breast tissues, using LUM5B-2 antisera raised against sequences within the non-glycosylated regions of this apomucin. Expression of MUC5B by breast cancer cells was confirmed by immunocytochemistry, in situ hybridization, and Western blot on MCF-7 cancer cells. Using an immunohistochemical procedure, MUC5B apomucin was detected in 34/42 (81%) primary breast tumors, in 13/14 (92.8%) samples of non-malignant breast diseases, in 8/19 (42.1%) samples of normal-appearing breast epithelia adjacent to cancer, and in 0/5 normal control breast samples. The staining pattern of MUC5B was very different when comparing breast cancer cells (cytoplasmic) and non-malignant breast cells (predominantly apical and in the secretory material). We analyzed MUC5B mRNA expression using RT-PCR in bone marrow aspirates from 22/42 patients with breast cancer to compare with MUC5B protein expression in the primary tumors. Good correlation was observed because the six MUC5B-positive bone marrow samples also displayed MUC5B expression in the tumor. Our results show, for the first time at the protein level, that MUC5B apomucin is upregulated in breast cancer. Its characterization could provide new insights about the glycobiology of breast cancer cells.

Rational design of a supra C-1027: kinetically stabilized analogue of the antitumor enediyne chromoprotein.

C-1027, an extremely potent antitumor agent, is composed of a highly reactive chromophore and an apoprotein. While the chromophore causes DNA cleavage, the apoprotein functions as its carrier. Despite these ideal properties as an anticancer agent, C-1027 slowly self-decomposes through chromophore-mediated abstraction of hydrogens from the apoprotein. In this paper, we report the design and preparation of an engineered C-1027 apoprotein that decelerates this self-decomposition pathway. Our design is based on the kinetic isotope effect, and deuterium is incorporated instead of protium into the hydrogen-abstraction site. The deuterated supra C-1027 was found to have a 4-fold longer lifetime than the natural C-1027.