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PubMed2003

Hypobaric biology: Arabidopsis gene expression at low atmospheric pressure.

As a step in developing an understanding of plant adaptation to low atmospheric pressures, we have identified genes central to the initial response of Arabidopsis to hypobaria. Exposure of plants to an atmosphere of 10 kPa compared with the sea-level pressure of 101 kPa resulted in the significant differential expression of more than 200 genes between the two treatments. Less than one-half of the genes induced by hypobaria are similarly affected by hypoxia, suggesting that response to hypobaria is unique and is more complex than an adaptation to the reduced partial pressure of oxygen inherent to hypobaric environments. In addition, the suites of genes induced by hypobaria confirm that water movement is a paramount issue at low atmospheric pressures, because many of gene products intersect abscisic acid-related, drought-induced pathways. A motivational constituent of these experiments is the need to address the National Aeronautics and Space Administration's plans to include plants as integral components of advanced life support systems. The design of bioregenerative life support systems seeks to maximize productivity within structures engineered to minimize mass and resource consumption. Currently, there are severe limitations to producing Earth-orbital, lunar, or Martian plant growth facilities that contain Earth-normal atmospheric pressures within light, transparent structures. However, some engineering limitations can be offset by growing plants in reduced atmospheric pressures. Characterization of the hypobaric response can therefore provide data to guide systems engineering development for bioregenerative life support, as well as lead to fundamental insights into aspects of desiccation metabolism and the means by which plants monitor water relations.

Adaptation, Physiological↗
PubMed2005

Methionine aminopeptidase 2 inhibition is an effective treatment strategy for neuroblastoma in preclinical models.

Tumor vascularity is correlated with an aggressive disease phenotype in neuroblastoma, suggesting that angiogenesis inhibitors may be a useful addition to current therapeutic strategies. We previously showed that the antiangiogenic compound TNP-470, an irreversible methionine aminopeptidase 2 (MetAP2) inhibitor, suppressed local and disseminated human neuroblastoma growth rates in murine models but had significant associated toxicity at the effective dose. We have recently shown that a novel, reversible MetAP2 inhibitor, A-357300, significantly inhibits CHP-134-derived neuroblastoma s.c. xenograft growth rate with a treatment-to-control (T/C) ratio at day 24 of 0.19 (P < 0.001) without toxicity. We now show that the combination of A-357300 with cyclophosphamide at the maximal tolerated dose sustained tumor regression with a T/C at day 48 of 0.16 (P < 0.001) in the CHP-134 xenograft model. A-357300 also significantly inhibited establishment and growth rate of hematogenous metastatic deposits following tail vein inoculation of CHP-134 cells and increased overall survival (P = 0.021). Lastly, A-357300 caused regression of established tumors in a genetically engineered murine model with progression-free survival in five of eight mice (P < 0.0001). There was no evidence of toxicity. These data show that MetAP2 may be an important molecular target for high-risk human neuroblastomas. We speculate that the growth inhibition may be through both tumor cell intrinsic and extrinsic (antiangiogenic) mechanisms. The potential for a wide therapeutic index may allow for treatment strategies that integrate MetAP2 inhibition with conventional cytotoxic compounds.

Aminopeptidases↗
PubMed2005

Characterization and gene expression profiling of a stable cell line expressing a cell cycle GFP sensor.

The use of stable cell lines expressing fusions with green fluorescent protein (GFP) has increased significantly in recent years. In this study we have used a range of complimentary analytical techniques to examine the characteristics of a cell line stably expressing a EGFP cell cycle sensor relative to parental U2OS cells. Analysis of cell cycle duration and cell cycle phase distribution by cell growth assays and flow cytometry revealed that the two cell lines had identical doubling times and cell cycle distributions. Measurement of EGFP fusion protein mRNA by quantitative RT-PCR indicated a EGFP sensor expression level equivalent to endogenous Cyclin B1 (7000 copies/cell in G2). Microarray analysis showed a 0.9% (>2 fold at p<0.001 across 20,000 genes) difference in global gene expression levels between parental and EGFP expressing U2OS cells, with no significant differences in expression of A, B, C, D, E, F, G, H, I, K, L, M or T type Cyclins between the two cell types. These results confirm that engineering a stable cell line for low expression of a EGFP cell cycle sensor is minimally perturbing to the cell cycle and cellular gene expression.

Cell Cycle↗
PubMed2005

Eye problem following foot surgery--abducens palsy as a complication of spinal anesthesia.

BACKGROUND: Paralysis of abducens nerve is a very rare complication of lumbar puncture, which is a common procedure most often used for diagnostic and anesthetic purposes. CASE REPORT: A 38-year-old man underwent surgery for a left hallux valgus while he was under spinal anesthesia. On the first postoperative day, the patient experienced a severe headache that did not respond to standard nonsteroidal anti-inflammatory medication and hydration. During the second postoperative day, nausea and vomiting occurred. On the fourth postoperative day, nausea ceased completely but the patient complained of diplopia. Examination revealed bilateral strabismus with bilateral abducens nerve palsy. His diplopia resolved completely after 9 weeks and strabismus after 6 months. CONCLUSION: Abducens palsy following spinal anesthesia is a rare and reversible complication. Spinal anesthesia is still a feasible procedure for both the orthopaedic surgeon and the patient. Other types of anesthesia or performing spinal anesthesia with smaller diameter or atraumatic spinal needles may help decrease the incidence of abducens palsy. Informing the patient about the reversibility of the complication is essential during the follow-up because the palsy may last for as long as 6 months. Special attention must be paid to patient positioning following the operation. Recumbency and lying flat should be accomplished as soon as possible to prevent cerebrospinal fluid leakage and resultant intracranial hypotension. This becomes much more important if the patient has postdural puncture headache.

Abducens Nerve Diseases↗
PubMedDate not supplied

Osteoblastic differentiation of rabbit mesenchymal stem cells loaded in A carrier system of Pluronic F127 and Interpore.

BACKGROUND: Ideally, bone tissue engineering products should have the ability of osteoconduction and osteoinduction. According to the tissue engineering principle, mesenchymal stem cells (MSCs) combined with an appropriate scaffold can be used as a bone substitute for bone defects. Here we used Interpore as a scaffold loaded with MSCs mixed in hydrogel (Pluronic F127). In order to demonstrate the osteogenic ability of MSCs in the hydrogel, cell/hydrogel scaffold constructs were cultured in an induction medium to elicit an osteoblastic response. METHODS: MSCs aspirated from rabbit bone marrow were cultured in induction medium. MSCs were then loaded into scaffold Interpore with the aid of hydrogel (Pluronic F127). After culture for 7 and 14 days, osteoblastic differentiation ability was tested using Alizarin Red S stain, reverse transcription polymerase chain reaction (RT-PCR), measurement of calcium and alkaline phosphatase levels, and scanning electron microscopy (SEM). RESULTS: Calcium and alkaline phosphatase levels both increased after 7 and 14 days incubation. Alizarin Red S staining revealed MSCs could survive and differentiate to osteoblasts in the cell/hydrogel scaffold. RT-PCR showed mRNA expression of osteopontin and Core binding factor alpha 1 (Cbfa1). SEM revealed growth of osteoblast-like cells on ceramic pores. CONCLUSIONS: Osteoconductive bone substitute (Interpore) has been used clinically for a long time. This study showed that MSCs could be held on Interpore with the aid of hydrogel (Pluronic F127) and that they could differentiate to osteoblasts.

Alkaline Phosphatase↗
PubMed2002

Complete change of the protein folding transition state upon circular permutation.

Reversing the loop lengths of the small protein S6 by circular permutation has a dramatic effect on the transition state structure: it changes from globally diffuse to locally condensed. The phenomenon arises from a biased dispersion of the contact energies. Stability data derived from point mutations throughout the S6 structure show that interactions between residues that are far apart in sequence are stronger than those that are close. This entropy compensation drives all parts of the protein to fold simultaneously and produces the diffuse transition-state structure typical for two-state proteins. In the circular permutant, where strong contacts and short sequence separations are engineered to concur, the transition state becomes atypically condensed and polarized. Taken together with earlier findings that S6 may also fold by a 'collapsed' trajectory with an intermediate, the results suggest that this protein may fold by a multiplicity of mechanisms. The observations indicate that the diffuse transition state of S6 is not required for folding but could be an evolutionary development to optimize cooperativity.

Kinetics↗
PubMed2004

The alpha 1 isoform of Na,K-ATPase regulates cardiac contractility and functionally interacts and co-localizes with the Na/Ca exchanger in heart.

The primary objective of this study was to examine the functional role of the Na,K-ATPase alpha 1 isoform in the regulation of cardiac contractility. Previous studies using knock-out mice showed that the hearts of animals lacking one copy of the alpha 1 or alpha 2 isoform gene exhibit opposite phenotypes. Hearts from alpha 2(+/-) animals are hypercontractile, whereas those of the alpha 1(+/-) animals are hypocontractile. The cardiac phenotype of the alpha 1(+/-) animals was unexpected as other studies suggest that inhibition of either isoform increases contraction. To help resolve this difference, we have used genetically engineered knock-in mice expressing a ouabain-sensitive alpha 1 isoform and a ouabain-resistant alpha 2 isoform of the Na,K-ATPase, and we analyzed cardiac contractility following selective inhibition of the alpha1 isoform by ouabain. Administration of ouabain to these animals and to isolated heart preparations selectively inhibits only the activity of the alpha 1 isoform without affecting the activity of the alpha 2 isoform. Low concentrations of ouabain resulted in positive cardiac inotropy in both isolated hearts and intact animals expressing the modified alpha 1 and alpha 2 isoforms. Pretreatment with 10 microm KB-R7943, which inhibits the reverse mode of the Na/Ca exchanger, abolished the cardiotonic effects of ouabain in isolated wild type and knock-in hearts. Immunoprecipitation analysis demonstrated co-localization of the alpha1 isoform and the Na/Ca exchanger in cardiac sarcolemma. The alpha 1 isoform co-immunoprecipitated with the Na/Ca exchanger and vice versa. These results demonstrate that the alpha 1 isoform regulates cardiac contractility, and that both the alpha 1 and alpha 2 isoforms are functionally and physically coupled with the Na/Ca exchanger in heart.

Animals↗
PubMed2005

Gene therapy progress and prospects: novel gene therapy approaches for AIDS.

Acquired immunodeficiency syndrome (AIDS), caused by human immunodeficiency virus (HIV), kills millions worldwide every year. Vaccines against HIV still seem a distant promise. Pharmaceutical treatments exist, but these are not always effective, and there is increasing prevalence of viral strains with multidrug resistance. Highly active antiretroviral therapy (HAART) consists of inhibitors of viral enzymes (reverse transcriptase (RT) and protease). Gene therapy, first introduced as intracellular immunization, may offer hopes for new treatments to be used alone, or in conjunction with, conventional small molecule drugs. Gene therapy approaches against HIV-1, including suicide genes, RNA-based technology, dominant negative viral proteins, intracellular antibodies, intrakines, and peptides, are the subject of this review.

Acquired Immunodeficiency Syndrome↗
PubMed2004

Liver regeneration using a hybrid artificial liver support system.

We have developed two types of hybrid artificial liver support system (HALSS) that use hepatocyte organoid culture: (1) a PUF-HALSS comprising an artificial liver module using polyurethane foam (PUF), in which hepatocytes form spheroids in its pores, and maintained liver-specific functions for at least ten days in vitro; (2) an LLS-HALSS that uses a liver lobule-like structure (LLS) module containing hollow fibers with a microregular arrangement in which hepatocytes in the extra-fiber space of the module form the organoids by centrifugation that maintain liver-specific functions for at least two months in vitro. In preclinical experiments, a PUF-HALSS was applied to a pig having liver failure. To evaluate the effect of liver regeneration, a PUF- and an LLS-HALSS were applied to a rat having reversible hepatic failure. Each HALSS was effective in supporting liver function, stabilization of general conditions and recovery from liver failure state. These results indicate that these HALSS may be useful to treat liver failure patients until liver transplantation or until regeneration of the native liver.

Animals↗
PubMed2002

Cellular specificity of proexendin-4 processing in mammalian cells in vitro and in vivo.

Glucagon-like peptide-1 (GLP-1) is a potent stimulator of glucose-dependent insulin secretion. Exendin-4(1-39) (Ex-4), isolated from Gila monster venom, is a highly specific GLP-1 receptor agonist that exhibits a prolonged duration of action in vivo. Although the processing mechanisms underlying liberation of GLP-1 from its prohormone have been elucidated, those for Ex-4 remain unknown. To examine the requirements for proEx-4 processing in mammalian cells, BHK fibroblasts, InR1-G9 islet A cells, and AtT-20 corticotropes, which express different prohormone convertases (furin, prohormone convertase 2, and prohormone convertase 1, respectively) were transfected with full-length lizard proEx-4, and the processing of proexendin was examined by HPLC and RIA (n = 3). All of the transfected cell lines exhibited Ex-4-like immunoreactivity in the media, and Ex-4-like immunoreactivity was detected in extracts of InR1-G9 and AtT-20 cells. However, only media and extracts from AtT-20 cells (not InR1-G9 and BHK cells) contained a single peak by HPLC corresponding to synthetic Ex-4. To establish whether proEx-4 can be processed to Ex-4 in nonimmortalized mammalian cells in vivo, the molecular forms of exendin-4 were examined in mice expressing a metallothionein-proEx-4 transgene (n = 3-6 for both males and females). ProEx4 mRNA transcripts were detected by RT-PCR in a broad range of both endocrine and nonendocrine tissues. Ex-4-like immunoreactivity was detected in pituitary, fat, adrenals, and testes; however HPLC analyses demonstrated that processed Ex-4 was found only in adrenals and testes. These results indicate that lizard proEx-4 is processed to mature bioactive Ex-4 in both rodent endocrine and nonendocrine mammalian cell types in vitro and in murine tissues in vivo. These findings may be useful for engineering cells that express a lizard pro-Ex4 transgene for the treatment of type 2 diabetes.

Adrenocorticotropic Hormone↗
PubMed2003

Domain engineering of the reductase component of soluble methane monooxygenase from Methylococcus capsulatus (Bath).

Soluble methane monooxygenase (sMMO) from Methylococcus capsulatus (Bath) is a three-component enzyme system that catalyzes the conversion of methane to methanol. A reductase (MMOR), which contains [2Fe-2S] and FAD cofactors, facilitates electron transfer from NADH to the hydroxylase diiron active sites where dioxygen activation and substrate hydroxylation take place. By separately expressing the ferredoxin (MMORFd, MMOR residues 1-98) and FAD/NADH (MMOR-FAD, MMOR residues 99-348) domains of the reductase, nearly all biochemical properties of full-length MMOR are retained, except for interdomain electron transfer rates. To investigate the extent to which rapid electron transfer between domains might be restored and further to explore the modularity of MMOR, MMOR-Fd and MMOR-FAD were connected in a non-native fashion. Four different linker sequences were employed to create MMOR reversed-domain (MMOR-RD) constructs, MMOR(99-342)-linker-MMOR(2-98), with a domain connectivity observed in other homologous oxidoreductases. The optical, redox, and electron transfer properties of the four MMOR-RD proteins were characterized and compared with those of wild-type MMOR. The linker sequence plays a key role in controlling solvent accessibility to the FAD cofactor, as evidenced by perturbed flavin optical spectra, decreased FADox/FADsq redox potentials, and increased steady-state oxidase activities in three of the constructs. Stopped-flow optical spectroscopy revealed slow interdomain electron transfer (k < 0.04 s(-1) at 4 degrees C, compared with 90 s(-1) for wild-type MMOR) for all three MMOR-RD proteins with 7-residue linkers. A long (14-residue), flexible linker afforded much faster electron transfer between the FAD and [2Fe-2S] cofactors (k = 0.9 s(-1) at 4 degrees C).

Catalysis↗
PubMedDate not supplied

Analysis of nucleic acids by on-line liquid chromatography-mass spectrometry.

The numerous problems posed by modern biochemistry, biology, and medicine, as well as the growing significance of genetic engineering require the application of fast and reliable methods of utmost sensitivity and selectivity for the analysis of nucleic acids. High-performance liquid chromatography (HPLC) and mass spectrometry (MS) represent established analytical techniques for the characterization and structural elucidation of single- and double-stranded nucleic acids, ranging in size from a few nucleotides to several thousand base pairs. Although both techniques are independently applicable for nucleic acid analysis, the on-line hyphenation significantly enhances their potential for the robust and fully automable routine analysis of minute amounts of biological samples. Among the various chromatographic and mass spectrometric modes available in principle, ion-pair reversed-phase HPLC and electrospray ionization mass spectrometry (ESI-MS) have been shown to be the most suitable for the direct interfacing of liquid chromatography (LC) and MS. Instrumental setup, as well as chromatographic and mass spectrometric experimental conditions, need to be carefully selected in order to maximize the performance of the hyphenated analytical system. Applications of HPLC-ESI-MS include the characterization of oligodeoxynucleotides synthesized by solid-phase synthesis, the analysis of antisense oligodeoxynucleotides, oligonucleotide metabolites, and DNA adducts, the analysis of genomic segments specifically amplified by the polymerase chain reaction (PCR), the characterization of ribonucleic acids, the sizing of double-stranded DNA restriction fragments, the genotyping of short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs), the detection of mutations in nucleic acid sequences, and the sequencing of nucleic acids.

Animals↗
PubMed2004

A mobile loop order-disorder transition modulates the speed of chaperonin cycling.

Molecular machines order and disorder polypeptides as they form and dissolve large intermolecular interfaces, but the biological significance of coupled ordering and binding has been established in few, if any, macromolecular systems. The ordering and binding of GroES co-chaperonin mobile loops accompany an ATP-dependent conformational change in the GroEL chaperonin that promotes client protein folding. Following ATP hydrolysis, disordering of the mobile loops accompanies co-chaperonin dissociation, reversal of the GroEL conformational change, and release of the client protein. "High-affinity" GroEL mutants were identified by their compatibility with "low-affinity" co-chaperonin mutants and incompatibility with high-affinity co-chaperonin mutants. Analysis of binding kinetics using the intrinsic fluorescence of tryptophan-containing co-chaperonin variants revealed that excessive affinity causes the chaperonin to stall in a conformation that forms in the presence of ATP. Destabilizing the beta-hairpins formed by the mobile loops restores the normal rate of dissociation. Thus, the free energy of mobile-loop ordering and disordering acts like the inertia of an engine's flywheel by modulating the speed of chaperonin conformational changes.

Adenosine Triphosphate↗
PubMed1997

A novel Met-to-Thr mutation in the YMDD motif of reverse transcriptase from feline immunodeficiency virus confers resistance to oxathiolane nucleosides.

Variants of feline immunodeficiency virus (FIV) that possess a unique methionine-to-threonine mutation within the YMDD motif of reverse transcriptase (RT) were selected by culturing virus in the presence of inhibitory concentrations of (-)-beta-L-2',3'-dideoxy-5-fluoro-3'-thiacytidine [(-)-FTC]. The mutants were resistant to (-)-FTC and (-)-beta-L-2',3'-dideoxy-3'-thiacytidine (3TC) and additionally exhibited low-level resistance to 2',3'-dideoxycytidine (ddC). DNA sequence analysis of the RT-encoding region of the pol gene amplified from resistant viruses consistently identified a Met-to-Thr mutation in the YMDD motif. Purified RT from the mutants was also resistant to the 5'-triphosphate forms of 3TC, (-)-FTC, and ddC. Site-directed mutants of FIV were engineered which contain either the novel Met-to-Thr mutation or the Met-to-Val mutation seen in oxathiolane nucleoside-resistant HIV-1. Both site-directed mutants displayed resistance to 3TC, thus confirming the role of these mutations in the resistance of FIV to beta-L-3'-thianucleosides.

Animals↗
PubMed2000

Design and intracellular activity of a human single-chain antibody to human immunodeficiency virus type 1 conserved gp41 epitope.

A human lymphoid cell line (F172-D8) excreting a human immunodeficiency virus type 1 (HIV-1) anti-gp41 monoclonal antibody was used to construct a plasmid containing the cDNA of the single-chain variable fragment (scFvD8) corresponding to this antibody. A stable human osteosarcoma cell line was obtained which expressed the scFvD8 protein in the cytoplasm. Whereas a cell line transfected with a control construct (pCI-neo) was readily and productively infected with laboratory (Ba-L) or primary HIV-1 isolates, the scFvD8 cell line did not support productive infection. Binding of the virus, internalization, and reverse transcription were not altered by scFvD8 expression, but gp160 expression was dramatically reduced. These data suggest that cytoplasmic expression of this artificial single-chain antibody can interfere with gp160 expression, thereby reducing the production of mature viral envelope proteins.

Cell Line↗
PubMed2006

[Use of Ssp dnaB mini-intein as a fusion partner for preparation of recombinant human brain natriuretic peptide].

Human brain natriuretic peptide (hBNP) was used clinically for the treatment of acute decompensated congestive heart failure. In this paper, hBNP was expressed as a fusion protein with a histidine tag and Ssp dnaB mini-intein which was capable of self-cleavage. After affinity chromatography with Ni-Sepharose and renaturation, the fusion protein was enriched with CM-cellulose. Ssp dnaB mini-intein mediated peptide-bond hydrolysis was triggered by shifting the pH and temperature in the CM-cellulose column, which let to the release of hBNP from the fusion protein and the separation of hBNP from His-DnaB. The hBNP sample was further purified by C4 reverse phase HPLC, and 2.8mg of the peptide with homogeneity of 97% was obtained from one liter of culture medium. The biological activity was assayed in vitro, which indicated that hBNP had a potent vasodilatory effect on rabbit aortic strips with an EC50 of 1.94 x 10(-6) mg/mL.

Animals↗
PubMed1990

NIH conference. Antiretroviral therapy in AIDS.

OBJECTIVE: To review recent developments of antiretroviral therapy for the acquired immunodeficiency syndrome (AIDS) and related disorders. DATA SOURCES, STUDY SELECTION, AND DATA EXTRACTION: An edited and updated summary of a Clinical Staff Conference held 26 October 1988 at the National Institutes of Health. The speakers discussed their own work as well as related work from other groups. DATA SYNTHESIS: The discovery that human immunodeficiency virus (HIV) causes AIDS has permitted the development of rational antiviral therapy for this disease. Various steps in the replicative cycle of HIV can be targeted for intervention. More basic research into the life cycle of HIV is therefore likely to yield new therapeutic approaches. In 1985, nucleoside analogues called dideoxynucleosides were discovered to be potent inhibitors of HIV replication in vitro. Dideoxynucleosides selectively inhibit HIV reverse transcriptase after they are phosphorylated intracellularly to 5'-triphosphates. One dideoxynucleoside, 3'-azido-2',3'-dideoxythymidine (AZT or zidovudine) has been found to prolong the life of patients with AIDS. This drug can partially reverse HIV dementia and decrease short-term progression to AIDS; it has been approved for treating HIV-infected patients with fewer than 500 CD4+ cells/mm3. AZT is only a first step in developing new therapy for AIDS. Its use is associated with toxicities, particularly bone marrow suppression. Several groups have reported the development of AZT-resistant strains of HIV. Other dideoxynucleosides whose toxicity profiles differ from that of AZT have also shown activity against HIV in early clinical studies. Large-scale, randomized trials of these drugs are now under way. Studies have shown that the binding of HIV to CD4 may be blocked by genetically engineered forms of CD4 and that HIV protease may be inhibited by substrate analogues. Protease inhibitors are an excellent area for further study in patients. Antisense oligonucleotide therapy may target the regulatory genes of HIV and is also being considered. CONCLUSION: With these advances, AIDS is gradually changing from an imminently fatal disease to one that can be managed with the judicious use of drugs and biologics. Progress against AIDS will continue, provided that researchers adhere to the principles of controlled trials.

Acquired Immunodeficiency Syndrome↗
PubMed1997

Introduction of novel substrate oxidation into cytochrome c peroxidase by cavity complementation: oxidation of 2-aminothiazole and covalent modification of the enzyme.

The binding and oxidation of an artificial substrate, 2-aminothiazole, by an engineered cavity of cytochrome c peroxidase is described. The W191G mutant has been shown to create a buried cavity into which a number of small heterocyclic compounds will bind [Fitzgerald, M. M., Churchill, M. J., McRee, D. E., & Goodin, D. B. (1994) Biochemistry 33, 3807-3818], providing a specific site near the heme from which substrates might be oxidized. In this study, we show by titration calorimetry that 2-aminothiazole binds to W191G with a Kd of 0.028 mM at pH 6. A crystal structure at 2.3 A resolution of W191G in the presence of 2-aminothiazole reveals the occupation of this compound in the cavity, and indicates that it is in van der Waals contact with the heme. The WT enzyme reacts with H2O2 to form Compound ES, in which both the iron center and the Trp-191 side chain are reversibly oxidized. For the W191F (and perhaps the W191G) mutants, the iron is still oxidized, but the second equivalent exists transiently as a radical on the porphyrin before migrating to an alternate protein radical site [Erman, J. E., Vitello, L. B., Mauro, J. M., & Kraut, J. (1989) Biochemistry 28, 7992-7995]. Two separate reactions are observed between 2-aminothiazole and the oxidized centers of W191G. In the one reaction, optical and EPR spectra of the heme are used to show that 2-aminothiazole acts as an electron donor to the ferryl (Fe4+&dbd;O) center of W191G to reduce it to the ferric oxidation state. This reaction occurs from within the cavity, as it is not observed for variants that lack this artificial binding site. A second reaction between 2-aminothiazole and peroxide-oxidized W191G, which is much less efficient, results in the specific covalent modification of Tyr-236. Electrospray mass spectra of the W191G after incubation in 2-aminothiazole and H2O2 show a modification of the protein indicative of covalent binding of 2-aminothiazole. The site of modification was determined to be Tyr-236 by CNBr peptide mapping and automated peptide sequencing. The covalent modification is only observed for W191G and W191F which form the alternate radical center. This observation provides an unanticipated assignment of this free radical species to Tyr-236, which is consistent with previous proposals that it is a tyrosine. The oxidation of 2-aminothiazole by W191G represents an example of how the oxidative capacity inherent in the heme prosthetic group and the specific binding behavior of artificial protein cavities can be harnessed and redirected toward the oxidation of organic substrates.

Binding Sites↗