Published Studies Using Biotinylated Nucleotide Photoaffinity Analogs
Compromised ATP binding as a mechanism of phosphoinositide modulation of ATP-sensitive K+ channelsCongmiao Wang, Kun Wang, Wenxia Wang, Yijun Cui and Zheng Fan * zfan@physio1.utmem.eduDepartment of Physiology, The University of Tennessee Health Science Center, 894 Union Avenue, Memphis, TN 38163, USAEdited by Maurice Montal FEBS Letters Volume 532, Issue 1-2, pp. 177-182 Received 9 August 2002; accepted 22 October 2002 http://www.elsevier.com/febs/774/19/56/abstract.html Full Text Version: http://www.elsevier.com/febs/774/19/56/article.html
AbstractInhibition of ATP-sensitive K+ (KATP) channels by ATP, a process presumably initiated by binding of ATP to the pore-forming subunit, Kir6.2, is reduced in the presence of phosphoinositides (PPIs). Previous studies led to the hypothesis that PPIs compromise ATP binding. Here, this hypothesis was tested using purified Kir6.2. We show that PPIs bind purified Kir6.2 in an isomer-specific manner, that biotinylated ATP analogs photoaffinity label purified Kir6.2, and that this labeling is weakened in the presence of PPIs. Patch-clamp measurements confirmed that these ATP analogs inhibited Kir6.2 channels, and that PPIs decreased the level of inhibition. These results indicate that interaction of PPIs with Kir6.2 impedes ATP-binding activity. The PPI regulation of ATP binding revealed in this study provides a putative molecular mechanism that is potentially pivotal to the nucleotide sensitivity of KATP channels. Keywords: Inwardly rectifying potassium channel; Purification; Photoaffinity labeling; ATP analog; PhosphoinositideAbbreviations: KATP channels,
ATP-sensitive K+ channels; PPI,
phosphoinositide; 2-N3-ATP-[ *Corresponding author. Fax: (1)-901-448 7126
Viperin (cig5), an IFN-inducible antiviral protein directly induced by human cytomegalovirusKeh-Chuang Chin and Peter Cresswell*Section of Immunobiology, Howard Hughes Medical Institute, Yale University School of Medicine, 310 Cedar Street, New Haven, CT 06520-8011 PNAS | December 18, 2001 | vol. 98 | no. 26 | 15125-15130 Contributed by Peter Cresswell, November 6, 2001 http://www.pnas.org/cgi/content/full/98/13/7431=&author1=cresswell&searchid=1059571761110
Little is known about the mechanism by which IFNs inhibit human cytomegalovirus (HCMV) replication. Indeed, infection of fibroblasts with HCMV initiates the expression of a subset of type I IFN-inducible genes whose role in the infectious process is unclear. We describe here the identification of a cytoplasmic antiviral protein that is induced by IFNs, by HCMV infection, and by the HCMV envelope protein, glycoprotein B (gB). Stable expression of the protein in fibroblasts inhibits productive HCMV infection, down-regulating several HCMV structural proteins (gB, pp28, and pp65) known to be indispensable for viral assembly and maturation. We have named the protein viperin (for virus inhibitory protein, endoplasmic reticulum-associated, interferon-inducible). HCMV infection causes the redistribution of the induced viperin from its normal endoplasmic reticulum association, first to the Golgi apparatus and then to cytoplasmic vacuoles containing gB and pp28. Expression before HCMV infection reduces viperin redistribution from the endoplasmic reticulum to the Golgi apparatus and prevents vacuolar localization, perhaps reflecting the mechanism used by HCMV to evade the antiviral function. * To whom reprint requests should be addressed. E-mail: peter.cresswell@yale.edu. www.pnas.org/cgi/doi/10.1073/pnas.011593298 IMMUNOLOGY:
Novel unconventional binding site in the variable region of immunoglobulins. Rajagopalan K, Pavlinkova G, Levy S, Pokkuluri PR, Schiffer M, Haley BE, Kohler H Division of Medicinal Chemistry and Pharmaceutics, College of Pharmacy, Stanford University School of Medicine, CA 94305, USA. Proc Natl Acad Sci U S A 1996 Jun 11;93(12):6019-24 The variable immunoglobulin (Ig) domains contain hypervariable regions that are involved in the formation of the antigen binding site. Besides the canonical antigen binding site, so-called unconventional sites also reside in the variable region that bind bacterial and viral proteins. Docking to these unconventional sites does not typically interfere with antigen binding, which suggests that these sites may be a part of the biological functions of Igs. Herein, a novel unconventional binding site is described. The site is detected with 8-azidopurine nucleotide photoaffinity probes that label antibodies efficiently and under mild conditions. Tryptic peptides were isolated from photolabeled monoclonal antibodies and aligned with the variable antibody domains of heavy and light chains. The structure of a variable Ig fragment was used to model the binding of the purine nucleotide to invariant residues in a hydrophobic pocket of the Ig molecule at a location distant from the antigen binding site. Monoclonal and polyclonal antibodies were biotinylated with the photoaffinity linker and used in fluorescence-activated cell sorter and ELISA analyses. The data support the utility of this site for tethering diagnostic and therapeutic agents to the variable Ig fragment region without impairing the structural and functional integrity of antibodies. PNAS On-Line: http://www.pnas.org/cgi/content/abstract/93/12/6019 PubMed: http://www.ncbi.nlm.nih.gov:80/entrez/PubMed&list_uids=8650212
Site-specific photobiotinylation of antibodies, light chains, and immunoglobulin fragments. Pavlinkova G, Lou D, Kohler H Immpheron Inc., Lexington, Kentucky, 40509, USA. Methods 2000 Sep;22(1):44-8 The high affinity of biotin for avidin has been exploited for many antibody-based assays. This requires that biotin is covalently conjugated to the antibody molecule. Several chemically reactive biotinylation reagents are commercially available. Except for the attachment via sulfhydryl groups in the immunoglobulin (Ig) molecule, these reagents attach biotin randomly to various amino acid side chains. Although non-site-specific modification of antibodies does not interfere in most immunoassays, specific application and sensitive antibodies would benefit from site-specific biotinylation. Here we describe an affinity biotinylation technique based on a photoreactive biotin reagent. The design of this reaction was possible from the discovery of a conserved binding site in the variable Ig domain for nucleotides and nucleosides. The described photoaffinity biotinylation offers the advantages of ease, convenience, and production of a reproducible and defined biotinylated antibody preparation. Copyright 2000 Academic Press. http://www.ncbi.nlm.nih.gov:80/entrez/PubMed&list_uids=11020316
Site-specific photobiotinylation of immunoglobulins, fragments and light chain dimers. Pavlinkova G, Rajagopalan K, Muller S, Chavan A, Sievert G, Lou D, O'Toole C, Haley B, Kohler H Department of Microbiology and Immunology, University of Kentucky, Lexington 40436, USA. J Immunol Methods 1997 Feb 14;201(1):77-88 Herein we report a new method to rapidly photoinsert biotin into a specific and highly conserved site on the Ig structure using a mild photochemical activation step. This site resides in the Fv fragment and involves invariant residues which provide base stacking interactions to the purine ring of ATP (Rajagopalan et al. (1996) Proc. Natl. Acad. Sci. USA 93, 6019-6024). Biotin was coupled to either the phosphate or the ribose of the 8-azidopurine nucleotide or nucleoside photoaffinity probe and shown to insert into the affinity site efficiently. Several monoclonal and polyclonal antibodies, as well as enzymatic and recombinant antibody fragments and light chain dimers were photoaffinity biotinylated and used in ELISA, FACS and Western blots. The selectivity of this site-specific biotinylation method also allows for biotinylation of antibodies in culture supernatants and immune sera without prior purification. Because the biotinylation takes place under physiological conditions and within a short time period, photobiotinylation would be the preferred method for antibodies which are easily damaged by classical non-site specific random biotinylation chemistry. http://www.ncbi.nlm.nih.gov:80/entrez/PubMed&list_uids=9032411
Enhanced molecular mimicry of CEA using photoaffinity crosslinked C3d peptide. Lou D, Kohler H Immpheron, Inc., Lexington, KY 40509, USA. Nat Biotechnol 1998 May;16(5):458-62 Antigen mimicry of using anti-idiotypic antibodies for use as cancer vaccines has been disappointing due to the weak immunogenicity of immunoglobulin variable domains. To enhance the immunogenicity of an anti-idiotype vaccine we incorporated a molecular adjuvant peptide into the antibody. The peptide is derived from the C3d region known to bind CR2 receptors on B-cells. A photoreactive peptide is synthesized that affinity-labels a single site in the antibody variable domain. The molecular adjuvant peptide is crosslinked to the anti-idiotype mimetic by chemical means without modifying other sites on the antibody. The C3d-conjugated anti-idiotype antibody induces a strong idiotype and antigen-specific response in mice. http://www.ncbi.nlm.nih.gov:80/entrez/PubMed&list_uids=9592395 http://www.nature.com/nbt/wilma/v16n5.894290857.html
For ALT Technical Briefs on Photobiotinylation of Nucleotide Binding Proteins see the following:
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