Structural Studies of Copper Proteins Using X-Ray Absorption Spectroscopy
René Lontie in Copper Proteins and Copper Enzymes, 1984
Hemocyanin22 (Hc) is a copper-containing protein which functions as a dioxygen carrier in molluscs and arthropods. The smallest functional units, with a relative molecular mass of approximately 50,000 for molluscs and 75,000 for arthropods, contain two copper atoms and bind one molecule of dioxygen reversibly. The two copper ions in OxyHc are EPR nondetectable, present as a type-3 binuclear site. Many studies have been aimed at elucidating the structure of the active site. Recent resonance Raman studies23 have established that the dioxygen is bound in the form of peroxide in OxyHc and suggested that imidazole ligands are coordinated to the copper site,24,25 consistent with earlier spectrosopic, acid-base titration, and photooxidation studies. However, these studies fail to establish an acceptable number of coordinating imidazoles. Recent EPR studies26 on several derivatives of Hc have provided a better view of the active-site structure. However, a structural model for the active site is still not completely clear. As a great deal of kinetic data have been collected on Hc over the years,27 a good structural model for the active site could definitely provide insight into the mechanism of oxygen binding and perhaps explain the observed cooperativity of oxygen binding.
Immunocytochemical Detection Systems
Lars-Inge Larsson in Immunocytochemistry: Theory and Practice, 2020
Protein A has been used in many immunocytochemical variants. Its use as a substitute for the link antibody in the PAP and related unlabeled immunoenzyme techniques has already been discussed. Moreover, its use in combination with colloidal gold techniques will be discussed in Section II.E. In addition, FITC-conjugated protein A has been used for indirect immunofluorescence.25,254 Peroxidase conjugates of protein A also have been found useful for immunocytochemistry (cf. References 47 and 77), for screening cDNA expression libraries (cf. Reference 370), and for detecting human autoantibodies.338 However, as not all human IgG subclasses react with protein A, perhaps not all possible autoantibodies are detectable.390 Comparisons and discussions of different methods for conjugating protein A with HRP have been published by Nygren and Hansson and by Pain and Surolia.257,264 Protein A-ferritin conjugates also have been exploited using an erythrocyte model system (cf. Reference 39), and Miller et al.224 have conjugated hemocyanin to protein A for use in ultrastructural immunocytochemistry. An interesting approach was devised by Widder et al.,378 using iron-containing protein A microspheres for immunofluorescent staining. By use of the Prussian blue reaction, the microspheres also could be visualized in the light microscope. Finally, protein A has also been labeled with radioiodine [125I], mainly for use in immunoassays.25,80,184
Reconstituted Membrane Systems for Assaying Membrane Proteins in Controlled Lipid Environments
Qiu-Xing Jiang in New Techniques for Studying Biomembranes, 2020
Voltage-sensitive dyes can measure the change in transmembrane electrostatic potential. They can be called potential-sensitive probes. These dyes change their absorption or emission fluorescence as a function of transmembrane potential. This method was started by Lawrence Cohen in the middle of 1970s when he used squid giant axons to screen available dyes,62–64 and later developed several of them, such as diASP, di-4-ANEPPS,65–67 di-8-ANEPPS,68 and di-4-ANEPPDHQ.69 Due to their chromophores, these dyes are classified as hemocyanin. An array of different potential-sensitive dyes were later developed in order to investigate varying aspects of neuronal functions in real time.
Structural characterization of monoclonal antibodies targeting C-terminal Ser404 region of phosphorylated tau protein
Published in mAbs, 2019
Jessica E. Chukwu, Erin E. Congdon, Einar M. Sigurdsson, Xiang-Peng Kong
MAbs 8B2, 6B2, and 4E6 were generated by GenScript as previously described.11,12 Briefly, wild type BALB/c mice were immunized with a peptide encompassing the pSer396/pSer404 region conjugated to keyhole limpet hemocyanin via a cysteine residue (cTDHGAEIVYK(pS)PVVSGDT(pS)PRHL). Hybridoma fusions were screened by ELISA and 8B2, 6B2, and 4E6 clones were selected based on binding to the phospho-tau immunogen. Although mAbs 4E6 and 6B2 were functionally characterized previously,11,12 mAb 8B2 has never been published before. Peptides used in ELISA and crystallization were synthesized by W.M. Keck Biotechnology Resource Center or GenScript (Table 1). The lyophilized peptides were solubilized in water to a stock concentration of 10 mg/mL before mixing with the Fabs for crystallization.
Histidine residues at the copper-binding site in human tyrosinase are essential for its catalytic activities
Published in Journal of Enzyme Inhibition and Medicinal Chemistry, 2020
Hyangsoon Noh, Sung Jun Lee, Hyun-Joo Jo, Hye Won Choi, Sungguan Hong, Kwang-Hoon Kong
In this study, we sought to demonstrate whether the seven copper-binding histidine residues (H180, H202, H211, H363, H367, H389 and H390) around the two copper binding sites are necessary for the catalytic activities and folding of tyrosinases. Nakamura et al.23 reported that seven histidine residues – H63, H84, H93, H290, H294, H332, and H333 – are essential for the tyrosinase activity of Aspergillus oryzae and demonstrated that replacement of each residue with asparagine abolished the catalytic activities of the mutant enzymes. Moreover, a crystallographic analysis of Palinurus interruptus haemocyanin showed that one of the pairs of copper ions, CuA, is enclosed by residues H196, H200 and H226, while the other, CuB, is surrounded by residues H346, H350 and H38612. Against this background, in the present study, the mutation positions were selected by the amino acid sequence alignment of H. sapiens tyrosinase with those of other tyrosinases from Mus musculus, Oryzias latipes, Streptomyces antibioticus, Streptomyces glaucescens, Streptomyces lavendulae, Streptomyces lincolnensis, Neurospora crassa, A. oryzae and Sinorhizobium meliloti (Figure 1), focussing on the positions of seven histidine residues (H180, H202, H211, H363, H367, H389 and H390). Further, we replaced each histidine residue around the CuA and CuB sites with a non-polar amino acid, alanine, using a site-directed mutagenesis approach. We then compared the hydroxylation and oxidation activities of these mutants. These findings shed light on the essential residues responsible for the catalytic activity of human tyrosinase, which would be of enormous value in predicting the structure and designing new inhibitors.
Utilization of lipopolysaccharide challenge in cynomolgus macaques to assess IL-10 receptor antagonism
Published in Journal of Immunotoxicology, 2019
Cris Kamperschroer, Richard Goldstein, Patricia A. Schneider, Bing Kuang, Michael D. Eisenbraun
The anti-human IL-10R1 mAb, PF-05179147, is a fully human IgG4 that was co-developed by Pfizer and Medarex (Redwood City, CA). PF-05179147 was selected based on its high-affinity binding to human and cynomolgus monkey IL-10R1, lack of cross-reactivity to other related cytokine receptors and proteins, and ability to antagonize IL-10-induced signaling in a dose-dependent manner (see Results and Supplemental Materials for mAb characterization). An isotype-matched mAb specific to keyhole limpet hemocyanin (KLH), i.e. PF-05186829, was produced at Pfizer and used as a negative control in experiments.
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