Burlingame AL

References (7)

Title : Nicotinic agonist binding site mapped by methionine- and tyrosine-scanning coupled with azidochloropyridinyl photoaffinity labeling - Tomizawa_2009_J.Med.Chem_52_3735
Author(s) : Tomizawa M , Talley TT , Park JF , Maltby D , Medzihradszky KF , Durkin KA , Cornejo-Bravo JM , Burlingame AL , Casida JE , Taylor P
Ref : Journal of Medicinal Chemistry , 52 :3735 , 2009
Abstract : Agonists activating nicotinic acetylcholine receptors (nAChR) include potential therapeutic agents and also toxicants such as epibatidine and neonicotinoid insecticides with a chloropyridinyl substituent. Nicotinic agonist interactions with mollusk (Aplysia californica) acetylcholine binding protein, a soluble surrogate of the nAChR extracellular domain, are precisely defined by scanning with 17 methionine and tyrosine mutants within the binding site by photoaffinity labeling with 5-azido-6-chloropyridin-3-yl probes that have similar affinities to their nonazido counterparts. Methionine and tyrosine are the only residues found derivatized, and their reactivity exquisitely depends on the direction of the azido moiety and its apposition to the reactive amino acid side chains.
ESTHER : Tomizawa_2009_J.Med.Chem_52_3735
PubMedSearch : Tomizawa_2009_J.Med.Chem_52_3735
PubMedID: 19459645

Title : Defining nicotinic agonist binding surfaces through photoaffinity labeling - Tomizawa_2007_Biochemistry_46_8798
Author(s) : Tomizawa M , Maltby D , Medzihradszky KF , Zhang N , Durkin KA , Presley J , Talley TT , Taylor P , Burlingame AL , Casida JE
Ref : Biochemistry , 46 :8798 , 2007
Abstract : Nicotinic acetylcholine (ACh) receptor (nAChR) agonists are potential therapeutic agents for neurological dysfunction. In the present study, the homopentameric mollusk ACh binding protein (AChBP), used as a surrogate for the extracellular ligand-binding domain of the nAChR, was specifically derivatized by the highly potent agonist azidoepibatidine (AzEPI) prepared as a photoaffinity probe and radioligand. One EPI-nitrene photoactivated molecule was incorporated in each subunit interface binding site based on analysis of the intact derivatized protein. Tryptic fragments of the modified AChBP were analyzed by collision-induced dissociation and Edman sequencing of radiolabeled peptides. Each specific EPI-nitrene-modified site involved either Tyr195 of loop C on the principal or (+)-face or Met116 of loop E on the complementary or (-)-face. The two derivatization sites were observed in similar frequency, providing evidence of the reactivity of the azido/nitrene probe substituent and close proximity to both residues. [3H]AzEPI binds to the alpha4beta2 nAChR at a single high-affinity site and photoaffinity-labels only the alpha4 subunit, presumably modifying Tyr225 spatially corresponding to Tyr195 of AChBP. Phe137 of the beta2 nAChR subunit, equivalent to Met116 of AChBP, conceivably lacks sufficient reactivity with the nitrene generated from the probe. The present photoaffinity labeling in a physiologically relevant condition combined with the crystal structure of AChBP allows development of precise structural models for the AzEPI interactions with AChBP and alpha4beta2 nAChR. These findings enabled us to use AChBP as a structural surrogate to define the nAChR agonist site.
ESTHER : Tomizawa_2007_Biochemistry_46_8798
PubMedSearch : Tomizawa_2007_Biochemistry_46_8798
PubMedID: 17614369

Title : Mapping the elusive neonicotinoid binding site - Tomizawa_2007_Proc.Natl.Acad.Sci.U.S.A_104_9075
Author(s) : Tomizawa M , Talley TT , Maltby D , Durkin KA , Medzihradszky KF , Burlingame AL , Taylor P , Casida JE
Ref : Proc Natl Acad Sci U S A , 104 :9075 , 2007
Abstract : Two types of structurally similar nicotinic agonists have very different biological and physicochemical properties. Neonicotinoids, important insecticides including imidacloprid and thiacloprid, are nonprotonated and selective for insects and their nicotinic receptors, whereas nicotinoids such as nicotine and epibatidine are cationic and selective for mammalian systems. We discovered that a mollusk acetylcholine binding protein (AChBP), as a structural surrogate for the extracellular ligand-binding domain of the nicotinic receptor, is similarly sensitive to neonicotinoids and nicotinoids. It therefore seemed possible that the proposed very different interactions of the neonicotinoids and nicotinoids might be examined with a single AChBP by using optimized azidochloropyridinyl photoaffinity probes. Two azidoneonicotinoids with a nitro or cyano group were compared with the corresponding desnitro or descyano azidonicotinoids. The four photoactivated nitrene probes modified AChBP with up to one agonist for each subunit based on analysis of the intact derivatized protein. Identical modification sites were observed by collision-induced dissociation analysis for the neonicotinoids and nicotinoids with similar labeling frequency of Tyr-195 of loop C and Met-116 of loop E at the subunit interface. The nitro- or cyano-substituted guanidine/amidine planes of the neonicotinoids provide a unique electronic conjugation system to interact with loop C Tyr-188. The neonicotinoid nitro oxygen and cyano nitrogen contact loop C Cys-190/Ser-189, whereas the cationic head of the corresponding nicotinoids is inverted for hydrogen-bonding and cation-pi contact with Trp-147 and Tyr-93. These structural models based on AChBP directly map the elusive neonicotinoid binding site and further describe the molecular determinants of agonists on nicotinic receptors.
ESTHER : Tomizawa_2007_Proc.Natl.Acad.Sci.U.S.A_104_9075
PubMedSearch : Tomizawa_2007_Proc.Natl.Acad.Sci.U.S.A_104_9075
PubMedID: 17485662

Title : Comprehensive identification of phosphorylation sites in postsynaptic density preparations - Trinidad_2006_Mol.Cell.Proteomics_5_914
Author(s) : Trinidad JC , Specht CG , Thalhammer A , Schoepfer R , Burlingame AL
Ref : Mol Cell Proteomics , 5 :914 , 2006
Abstract : In the mammalian central nervous system, the structure known as the postsynaptic density (PSD) is a dense complex of proteins whose function is to detect and respond to neurotransmitter released from presynaptic axon terminals. Regulation of protein phosphorylation in this molecular machinery is critical to the activity of its components, which include neurotransmitter receptors, kinases/phosphatases, scaffolding molecules, and proteins regulating cytoskeletal structure. To characterize the phosphorylation state of proteins in PSD samples, we combined strong cation exchange (SCX) chromatography with IMAC. Initially, tryptic peptides were separated by cation exchange and analyzed by reverse phase chromatography coupled to tandem mass spectrometry, which led to the identification of phosphopeptides in most SCX fractions. Because each of these individual fractions was too complex to characterize completely in single LC-MS/MS runs, we enriched for phosphopeptides by performing IMAC on each SCX fraction, yielding at least a 3-fold increase in identified phosphopeptides relative to either approach alone (SCX or IMAC). This enabled us to identify at least one site of phosphorylation on 23% (287 of 1,264) of all proteins found to be present in the postsynaptic density preparation. In total, we identified 998 unique phosphorylated peptides, mapping to 723 unique sites of phosphorylation. At least one exact site of phosphorylation was determined on 62% (621 of 998) of all phosphopeptides, and approximately 80% of identified phosphorylation sites are novel.
ESTHER : Trinidad_2006_Mol.Cell.Proteomics_5_914
PubMedSearch : Trinidad_2006_Mol.Cell.Proteomics_5_914
PubMedID: 16452087
Gene_locus related to this paper: mouse-q6wqj1

Title : Site-specific detection and structural characterization of the glycosylation of human plasma proteins lecithin:cholesterol acyltransferase and apolipoprotein D using HPLC\/electrospray mass spectrometry and sequential glycosidase digestion - Schindler_1995_Protein.Sci_4_791
Author(s) : Schindler PA , Settineri CA , Collet X , Fielding CJ , Burlingame AL
Ref : Protein Science , 4 :791 , 1995
Abstract : Site-specific structural characterization of the glycosylation of human lecithin:cholesterol acyltransferase (LCAT) was carried out using microbore reversed-phase high performance liquid chromatography coupled with electrospray ionization mass spectrometry (HPLC/ESIMS). A recently described mass spectrometric technique involving monitoring of carbohydrate-specific fragment ions during HPLC/ESIMS was employed to locate eight different groups of glycopeptides in a digest of a human LCAT protein preparation. In addition to the four expected N-linked glycopeptides of LCAT, a di-O-linked glycopeptide was detected, as well as three additional glycopeptides. Structural information on the oligosaccharides from all eight glycopeptides was obtained by sequential glycosidase digestion of the glycopeptides followed by HPLC/ESIMS. All four potential N-linked glycosylation sites (Asn20, Asn84, Asn272, and Asn384) of LCAT were determined to contain sialylated triantennary and/or biantennary complex structures. Two unanticipated O-linked glycosylation sites were identified at Thr407 and Ser409 of the LCAT O-linked glycopeptide, each of which contain sialylated galactose beta 1-->3N-acetylgalactosamine structures. The three additional glycopeptides were determined to be from a copurifying protein, apolipoprotein D, which contains potential N-linked glycosylation sites at Asn45 and Asn78. These glycopeptides were determined to bear sialylated triantennary oligosaccharides or fucosylated sialylated biantennary oligosaccharides. Previous studies of LCAT indicated that removal of the glycosylation site at Asn272 converts this protein to a phospholipase (Francone OL, Evangelista L, Fielding CJ, 1993, Biochim Biophys Acta 1166:301-304). Our results indicate that the carbohydrate structures themselves are not the source of this functional discrimination; rather, it must be mediated by the structural environment around Asn272.
ESTHER : Schindler_1995_Protein.Sci_4_791
PubMedSearch : Schindler_1995_Protein.Sci_4_791
PubMedID: 7613477
Gene_locus related to this paper: human-LCAT

Title : Structure, oligosaccharide structures, and posttranslationally modified sites of the nicotinic acetylcholine receptor - Poulter_1989_Proc.Natl.Acad.Sci.U.S.A_86_6645
Author(s) : Poulter L , Earnest JP , Stroud RM , Burlingame AL
Ref : Proc Natl Acad Sci U S A , 86 :6645 , 1989
Abstract : Using mass spectrometry, we have examined the transmembrane topography of the nicotinic acetylcholine receptor, a five-subunit glycosylated protein complex that forms a gated ion channel in the neuromuscular junction. The primary sequences of the four polypeptide chains making up the acetylcholine receptor from Torpedo californica contain many possible sites for glycosylation or phosphorylation. We have used liquid secondary ion mass spectrometry to identify posttranslationally modified residues and to determine the intact oligosaccharide structures of the carbohydrate present on the acetylcholine receptor. Asparagine-143 of the alpha subunit (in consensus numbering) is shown to be glycosylated with high-mannose oligosaccharide. Asparagine-453 of the gamma subunit is not glycosylated, a fact that bears on the question of the orientations of putative transmembranous helices M3, MA, and M4. The structures of the six major acetylcholine receptor oligosaccharides are determined: the major components (70%) are of the high-mannose type, with bi-, tri-, and tetraantennary complex oligosaccharides making up approximately equal to 22 mol% of the total carbohydrate. This application of a multichannel array detector mass spectrometer provided a breakthrough in sensitivity that allowed us to identify the site of attachment of, and the sequence of, oligosaccharides on a 300-kDa membrane protein from only 5 pmol of the isolated oligosaccharide.
ESTHER : Poulter_1989_Proc.Natl.Acad.Sci.U.S.A_86_6645
PubMedSearch : Poulter_1989_Proc.Natl.Acad.Sci.U.S.A_86_6645
PubMedID: 2771948

Title : Cesium ion liquid secondary ion mass spectrometry of membrane-bound glycoproteins: structural and topological considerations of acetylcholine receptor from Torpedo californica - Poulter_1988_Biomed.Environ.Mass.Spectrom_16_25
Author(s) : Poulter L , Earnest JP , Stroud RM , Burlingame AL
Ref : Biomed Environ Mass Spectrom , 16 :25 , 1988
Abstract : We report mass mapping of a large (270 kD) multisubunit membrane bound glycoprotein, nicotinic acetylcholine receptor from Torpedo californica, using enzymic digests of the affinity purified whole receptor and cesium ion liquid secondary ion mass spectrometry. Peptides, glycopeptides and derivatized N-linked oligosaccharides were isolated by HPLC and identified by LSIMS. We have shown that mass spectrometric sensitivity is improved a hundred-fold through use of computer-controlled mass window stepping of an electro-optical multichannel array detection system on a LSIMS double focusing mass spectrometer. This new method permitted determination of the complete fragmentation pattern of Man8N2-ABEE using only 5 picomoles of sample.
ESTHER : Poulter_1988_Biomed.Environ.Mass.Spectrom_16_25
PubMedSearch : Poulter_1988_Biomed.Environ.Mass.Spectrom_16_25
PubMedID: 3242677