Site-specific epitope tagging of G protein-coupled receptors by bioorthogonal modification of a genetically encoded unnatural amino acid.

Abstract:

:We developed a general strategy for labeling expressed membrane proteins with a peptide epitope tag and detecting the tagged proteins in native cellular membranes. First, we genetically encoded the unnatural amino acid p-azido-L-phenylalanine (azF) at various specific sites in a G protein-coupled receptor (GPCR), C-C chemokine receptor 5 (CCR5). The reactive azido moiety facilitates Staudinger ligation to a triarylphosphine-conjugated FLAG peptide. We then developed a whole-cell-based enzyme-linked immunosorbent assay approach to detect the modified azF-CCR5 using anti-FLAG mAb. We optimized conditions to achieve labeling and detection of low-abundance GPCRs in live cells. We also performed an accessibility screen to identify azF positions on CCR5 amenable to labeling. Finally, we demonstrate a preparative strategy for obtaining pure bioorthogonally modified GPCRs suitable for single-molecule detection fluorescence experiments. This peptide epitope tagging strategy, which employs genetic encoding and bioorthogonal labeling of azF in live cells, should be useful for studying biogenesis of polytopic membrane proteins and GPCR signaling mechanisms.

journal_name

Biochemistry

journal_title

Biochemistry

authors

Naganathan S,Ye S,Sakmar TP,Huber T

doi

10.1021/bi301292h

subject

Has Abstract

pub_date

2013-02-12 00:00:00

pages

1028-36

issue

6

eissn

0006-2960

issn

1520-4995

journal_volume

52

pub_type

杂志文章
  • Lycopene-Family Carotenoids Confer Thermostability on Photocomplexes from a New Thermophilic Purple Bacterium.

    abstract::Blastochloris tepida is a newly described thermophilic purple bacterium containing bacteriochlorophyll b. Using purified light-harvesting 1 reaction center (LH1-RC) core complexes from Blc. tepida, we compared the biochemical, spectroscopic, and thermal denaturation properties of these complexes with those of its meso...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/acs.biochem.0c00192

    authors: Seto R,Takaichi S,Kurihara T,Kishi R,Honda M,Takenaka S,Tsukatani Y,Madigan MT,Wang-Otomo ZY,Kimura Y

    更新日期:2020-06-30 00:00:00

  • Adaptation to altered growth temperatures in Acholeplasma laidlawii B: Fourier transform infrared studies of acyl chain conformational order in live cells.

    abstract::Acholeplasma laidlawii B cells, highly enriched in saturated C14 and C15 fatty acids, have been grown at several temperatures. Conformational order in the acyl chains of live cell membranes has been monitored with Fourier-transform infrared (FT-IR) spectroscopy. Coupled CH2 wagging progressions, characteristic of the ...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00179a037

    authors: Moore DJ,Mendelsohn R

    更新日期:1994-04-05 00:00:00

  • Enzymic and physicochemical properties of Streptomyces griseus trypsin.

    abstract::Streptomyces griseus trypsin has been isolated from Pronase by ion-exchange chromatography on CM-Sephadex and SE-Sephadex. The isolated enzyme was homogeneous by the criteria tested except for a low degree of contamination by an enzyme with nontryptic activity. The latter could be partially resolved by chromatography ...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00677a010

    authors: Olafson RW,Smillie LB

    更新日期:1975-03-25 00:00:00

  • Synergistic activation of protein kinase Calpha, -betaI, and -gamma isoforms induced by diacylglycerol and phorbol ester: roles of membrane association and activating conformational changes.

    abstract::Protein kinase Calpha (PKCalpha) has been shown to contain two discrete activator sites with differing binding affinities for phorbol esters and diacylglycerols. The interaction of diacylglycerol with a low-affinity phorbol ester binding site leads to enhanced high-affinity phorbol ester binding and to a potentiated l...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi982778r

    authors: Slater SJ,Milano SK,Stagliano BA,Gergich KJ,Ho C,Mazurek A,Taddeo FJ,Kelly MB,Yeager MD,Stubbs CD

    更新日期:1999-03-23 00:00:00

  • Structural basis for the retroreduction of inactivated peroxiredoxins by human sulfiredoxin.

    abstract::Sufiredoxins (Srx) repair the inactivated forms of typical two-Cys peroxiredoxins (Prx) implicated in hydrogen peroxide-mediated cell signaling. The reduction of the cysteine sulfinic acid moiety within the active site of the Prx by Srx involves novel sulfur chemistry and the use of ATP and Mg(2+). The 1.65 A crystal ...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi050131i

    authors: Jönsson TJ,Murray MS,Johnson LC,Poole LB,Lowther WT

    更新日期:2005-06-21 00:00:00

  • Study of hydrogen exchange in hemoglobin as a function of fractional ligand saturation.

    abstract::We have studied the hydrogen-exchange kinetics of hemoglobin A0 as a function of ligand, CO, saturation. In the noncooperative system, azide binding to methemoglobin, the alterations in exchange kinetics are proportional to the average degree of ligation. However, in the case of CO binding to deoxyhemoglobin the chang...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00297a015

    authors: Hallaway PE,Hallaway BE,Rosenberg A

    更新日期:1984-01-17 00:00:00

  • Effects of Olive Metabolites on DNA Cleavage Mediated by Human Type II Topoisomerases.

    abstract::Several naturally occurring dietary polyphenols with chemopreventive or anticancer properties are topoisomerase II poisons. To identify additional phytochemicals that enhance topoisomerase II-mediated DNA cleavage, a library of 341 Mediterranean plant extracts was screened for activity against human topoisomerase IIα....

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/acs.biochem.5b00162

    authors: Vann KR,Sedgeman CA,Gopas J,Golan-Goldhirsh A,Osheroff N

    更新日期:2015-07-28 00:00:00

  • Human transforming growth factor type beta 2: production by a prostatic adenocarcinoma cell line, purification, and initial characterization.

    abstract::Human type beta 2 transforming growth factor (hTGF-beta 2) was purified from tamoxifen-supplemented, serum-free medium conditioned by the human prostatic adenocarcinoma cell line PC-3. The purification of hTGF-beta 2 was monitored in a growth inhibition assay and was achieved by batch purification on methylsilyl-contr...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00383a002

    authors: Ikeda T,Lioubin MN,Marquardt H

    更新日期:1987-05-05 00:00:00

  • Ultrafast water dynamics at the interface of the polymerase-DNA binding complex.

    abstract::DNA polymerases slide on DNA during replication, and the interface must be mobile for various conformational changes. The role of lubricant interfacial water is not understood. In this report, we systematically characterize the water dynamics at the interface and in the active site of a tight binding polymerase (pol β...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi500810a

    authors: Yang Y,Qin Y,Ding Q,Bakhtina M,Wang L,Tsai MD,Zhong D

    更新日期:2014-08-26 00:00:00

  • Modification of ribonuclease T1 specificity by random mutagenesis of the substrate binding segment.

    abstract::Attempts to modify the guanine specificity of ribonuclease T1 (RNase T1) by rationally designed amino acid substitutions failed so far. Therefore, we applied a semirational approach by randomizing the guanine binding site. A combinatorial library of approximately 1.6 million RNase T1 variants containing permutations o...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi9817515

    authors: Hubner B,Haensler M,Hahn U

    更新日期:1999-01-26 00:00:00

  • 1H NMR assignment and global fold of napin BnIb, a representative 2S albumin seed protein.

    abstract::Napin BnIb is a representative member of the 2S albumin seed proteins, which consists of two polypeptide chains of 3.8 and 8.4 kDa linked by two disulfide bridges. In this work, a complete assignment of the 1H spectra of napin BnIb has been carried out by two-dimensional NMR sequence-specific methods and its secondary...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi961748q

    authors: Rico M,Bruix M,González C,Monsalve RI,Rodríguez R

    更新日期:1996-12-10 00:00:00

  • Unique structural modulation of a non-native substrate by cochaperone DnaJ.

    abstract::The role of bacterial DnaJ protein as a cochaperone of DnaK is strongly appreciated. Although DnaJ unaccompanied by DnaK can bind unfolded as well as native substrate proteins, its role as an individual chaperone remains elusive. In this study, we demonstrate that DnaJ binds a model non-native substrate with a low nan...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi301543g

    authors: Tiwari S,Kumar V,Jayaraj GG,Maiti S,Mapa K

    更新日期:2013-02-12 00:00:00

  • The hemophore HasA from Yersinia pestis (HasAyp) coordinates hemin with a single residue, Tyr75, and with minimal conformational change.

    abstract::Hemophores from Serratia marcescens (HasA(sm)) and Pseudomonas aeruginosa (HasA(p)) bind hemin between two loops, which harbor the axial ligands H32 and Y75. Hemin binding to the Y75 loop triggers closing of the H32 loop and enables binding of H32. Because Yersinia pestis HasA (HasA(yp)) presents a Gln at position 32,...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi400280z

    authors: Kumar R,Lovell S,Matsumura H,Battaile KP,Moënne-Loccoz P,Rivera M

    更新日期:2013-04-23 00:00:00

  • Synthetic Riboswitches: From Plug and Pray toward Plug and Play.

    abstract::In synthetic biology, metabolic engineering, and gene therapy, there is a strong demand for orthogonal or externally controlled regulation of gene expression. Here, RNA-based regulatory devices represent a promising emerging alternative to proteins, allowing a fast and direct control of gene expression, as no synthesi...

    journal_title:Biochemistry

    pub_type: 杂志文章,评审

    doi:10.1021/acs.biochem.6b01218

    authors: Etzel M,Mörl M

    更新日期:2017-03-07 00:00:00

  • A novel CXCR4-selective high-affinity fluorescent probe and its application in competitive binding assays.

    abstract::We recently developed a new, rapid, and specific bioassay system that employs a fluorescent probe fabricated from our discovered CXCR4-specific ligand DV1. This new probe sensitively and selectively blocks the binding of native and synthetic ligands to CXCR4 at nanomolar levels, with a capability comparable to that se...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi500500h

    authors: Yang Y,Zhang Q,Gao M,Yang X,Huang Z,An J

    更新日期:2014-08-05 00:00:00

  • Thermodynamics of transient conformations in the folding pathway of barnase: reorganization of the folding intermediate at low pH.

    abstract::New classes of small proteins have recently been found that refold rapidly with two-state kinetics from a substantially unfolded conformation ("U") and without the accumulation of a folding intermediate. Barnase, on the other hand, is representative of a class of proteins that display multistate kinetics and refold fr...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi950967t

    authors: Oliveberg M,Fersht AR

    更新日期:1996-02-27 00:00:00

  • Molecular architecture of DesI: a key enzyme in the biosynthesis of desosamine.

    abstract::Desosamine is a 3-(dimethylamino)-3,4,6-trideoxyhexose found, for example, in such macrolide antibiotics as erthyromycin, azithromycin, and clarithromycin. The efficacies of these macrolide antibiotics are markedly reduced in the absence of desosamine. In the bacterium Streptomyces venezuelae, six enzymes are required...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi700751d

    authors: Burgie ES,Holden HM

    更新日期:2007-08-07 00:00:00

  • Pinpoint Chemical Modification of the Quinone-Access Channel of Mitochondrial Complex I via a Two-Step Conjugation Reaction.

    abstract::We previously showed that a bulky ring-strained cycloalkyne possessing a rhodamine fluorophore directly reacts (via strain-promoted click chemistry) with the azido group incorporated (via ligand-directed tosyl chemistry) into Asp160 in the 49 kDa subunit of complex I in bovine heart submitochondrial particles [Masuya,...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/acs.biochem.7b00612

    authors: Masuya T,Murai M,Ito T,Aburaya S,Aoki W,Miyoshi H

    更新日期:2017-08-15 00:00:00

  • Mapping of factor XIII solvent accessibility as a function of activation state using chemical modification methods.

    abstract::The transglutaminase Factor XIII (FXIII) catalyzes the formation of covalent cross-links between adjacent noncovalently associated fibrin chains in blood coagulation. The resulting covalently cross-linked hard clot is much more mechanically stable and resistant to proteolytic degradation. FXIII is activated by the ser...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi049260+

    authors: Turner BT Jr,Sabo TM,Wilding D,Maurer MC

    更新日期:2004-08-03 00:00:00

  • Conformation of magainin-2 and related peptides in aqueous solution and membrane environments probed by Fourier transform infrared spectroscopy.

    abstract::The conformational properties of the magainin family of antimicrobial peptides in aqueous solution and in model membranes have been probed by Fourier transform infrared spectroscopy. The magainins were found to be structureless in aqueous solution at neutral pD, confirming other studies by Raman and circular dichroism...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00147a012

    authors: Jackson M,Mantsch HH,Spencer JH

    更新日期:1992-08-18 00:00:00

  • tRNA discrimination at the binding step by a class II aminoacyl-tRNA synthetase.

    abstract::Aminoacyl-tRNA synthetases preserve the fidelity of decoding genetic information by accurately joining amino acids to their cognate transfer RNAs. Here, tRNA discrimination at the level of binding by Escherichia coli histidyl-tRNA synthetase is addressed by filter binding, analytical ultracentrifugation, and iodine fo...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi991182g

    authors: Bovee ML,Yan W,Sproat BS,Francklyn CS

    更新日期:1999-10-12 00:00:00

  • Human class I alcohol dehydrogenases catalyze the interconversion of alcohols and aldehydes in the metabolism of dopamine.

    abstract::The class I human liver alcohol dehydrogenases (ADHs) catalyze the interconversion of the intermediary alcohols and aldehydes of dopamine metabolism in vitro, whereas those of the class II and class III do not. The individual, homogeneous class I isozymes oxidize (3,4-dihydroxyphenyl)ethanol and (4-hydroxy-3-methoxyph...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00371a005

    authors: Mårdh G,Vallee BL

    更新日期:1986-11-18 00:00:00

  • Structure of Ralstonia eutropha flavohemoglobin in complex with three antibiotic azole compounds.

    abstract::Flavohemoglobins (flavoHbs) are enzymes that operate primarily as nitric oxide dioxygenases and shuttle thereby electrons among NAD(P)H, FAD, heme, and a ligated redox-active substrate such as O(2). They function in the bacterial defense against nitrosative stress and are therefore considered as targets for new antibi...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi101650q

    authors: El Hammi E,Warkentin E,Demmer U,Limam F,Marzouki NM,Ermler U,Baciou L

    更新日期:2011-02-22 00:00:00

  • Distal pocket polarity in ligand binding to myoglobin: deoxy and carbonmonoxy forms of a threonine68(E11) mutant investigated by X-ray crystallography and infrared spectroscopy.

    abstract::The crystal structures of the deoxy and carbonmonoxy forms of a distal pocket myoglobin mutant in which valine68(E11) is replaced by threonine have been solved to 2.1- and 2.2-A resolution, respectively. This substitution has been shown previously to cause large decreases in the rate of oxygen binding and to lower the...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00211a016

    authors: Cameron AD,Smerdon SJ,Wilkinson AJ,Habash J,Helliwell JR,Li T,Olson JS

    更新日期:1993-12-07 00:00:00

  • Proximal influences in two-on-two globins: effect of the Ala69Ser replacement on Synechocystis sp. PCC 6803 hemoglobin.

    abstract::The cyanobacterium Synechocystis sp. PCC 6803 (S6803) expresses a two-on-two globin in which His46 (distal side) and His70 (proximal) function as heme iron axial ligands. His46 can be displaced by O2, CO, and CN-, among others, whereas His70 is not labile under native conditions. The residue preceding the proximal his...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi060691x

    authors: Knappenberger JA,Kuriakose SA,Vu BC,Nothnagel HJ,Vuletich DA,Lecomte JT

    更新日期:2006-09-26 00:00:00

  • Distribution of shape-changing compounds across the red cell membrane.

    abstract::The effects of two oppositely charged pyrene derivatives, 1-pyrenebutyrylcholine (PBC) and 1-pyrenebutyric acid (PBA), on red blood cell shape have been examined. Both compounds convert normal biconcave erythrocytes into echinocytes. However, with extended incubation time at elevated temperature, the morphology of PBC...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi00556a002

    authors: Matayoshi ED

    更新日期:1980-07-22 00:00:00

  • Impact of actin glutathionylation on the actomyosin-S1 ATPase.

    abstract::Glutathionylation of intracellular proteins is an established physiological regulator of protein function. In multiple models, including ischemia-reperfusion of the heart, increased oxidative stress results in the glutathionylation of sarcomeric actin. We hypothesized that actin glutathionylation may play a role in th...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi900669m

    authors: Pizarro GO,Ogut O

    更新日期:2009-08-11 00:00:00

  • Interruption of the internal water chain of cytochrome f impairs photosynthetic function.

    abstract::The structure of cytochrome f includes an internal chain of five water molecules and six hydrogen-bonding side chains, which are conserved throughout the phylogenetic range of photosynthetic organisms from higher plants, algae, and cyanobacteria. The in vivo electron transfer capability of Chlamydomonas reinhardtii cy...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi0004596

    authors: Sainz G,Carrell CJ,Ponamarev MV,Soriano GM,Cramer WA,Smith JL

    更新日期:2000-08-08 00:00:00

  • Role of guanine nucleotides in the vinblastine-induced self-association of tubulin: effects of guanosine alpha,beta-methylenetriphosphate and guanosine alpha,beta-methylenediphosphate.

    abstract::It is now well established that guanine nucleotides are allosteric effectors of the vinca alkaloid-induced self-association of tubulin. GDP enhances self-association for vinblastine-, vincristine- and vinorelbine-induced spiral assembly relative to GTP by 0.90 +/- 0.17 kcal/mol [Lobert et al. (1996) Biochemistry 35, 6...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi971120v

    authors: Vulevic B,Lobert S,Correia JJ

    更新日期:1997-10-21 00:00:00

  • Directed evolution of the suicide protein O⁶-alkylguanine-DNA alkyltransferase for increased reactivity results in an alkylated protein with exceptional stability.

    abstract::Here we present a biophysical, structural, and computational analysis of the directed evolution of the human DNA repair protein O(6)-alkylguanine-DNA alkyltransferase (hAGT) into SNAP-tag, a self-labeling protein tag. Evolution of hAGT led not only to increased protein activity but also to higher stability, especially...

    journal_title:Biochemistry

    pub_type: 杂志文章

    doi:10.1021/bi2016537

    authors: Mollwitz B,Brunk E,Schmitt S,Pojer F,Bannwarth M,Schiltz M,Rothlisberger U,Johnsson K

    更新日期:2012-02-07 00:00:00