Alice Y. Ting

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Name: Ting, Alice

Organization: Massachusetts Institute of Technology , USA

Department: Department of Chemistry

Title: Professor(PhD)

TOPICS

Organic Chemistry

Click chemistry

Chemicals
References

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

Co-reporter:Peter J. Goldman;Douglas Richardson;Daniel S. Liu;Anne Z. Ye;William S. Phipps;Catherine L. Drennan;David Baker;Jennifer Z. Yao;Lucas G. Nivón;Florian Richter;Thomas J. Deerinck;Mark H. Ellisman

PNAS 2014 Volume 111 (Issue 43 ) pp:E4551-E4559

Publication Date(Web):2014-10-28

DOI:10.1073/pnas.1404736111

Chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.

Proteomic Mapping of Mitochondria in Living Cells via Spatially Restricted Enzymatic Tagging

Co-reporter:Vamsi K. Mootha;Jeffrey D. Martell;Hyun-Woo Rhee;Namrata D. Udeshi;Peng Zou;Steven A. Carr

Science 2013 Volume 339(Issue 6125) pp:1328-1331

Publication Date(Web):15 Mar 2013

DOI:10.1126/science.1230593

Mitochondrial Makeup Mapped

Because mass spectrometry (MS) cannot be performed on living cells, biologists currently recover spatial information indirectly, by purifying organelles or protein complexes prior to MS analysis. These purifications often yield false positives because of sample contamination and false negatives because of material loss. Rhee et al. (p. 1328, published online 31 January) present an approach that bridges microscopy and proteomics to produce a spatially and temporally resolved proteomic map of mitochondria from living cells. A nonspecific labeling enzyme (peroxidase) was genetically targeted to the mitochondria within live cells, where it tagged endogenous proteins in a spatially restricted manner within a 1-minute window, for subsequent identification and analysis by MS. This rapid and straightforward technology provides the ability to access otherwise inaccessible cellular regions and requires a very small amount of starting material.

Fluorophore Targeting to Cellular Proteins via Enzyme-Mediated Azide Ligation and Strain-Promoted Cycloaddition

Co-reporter:Jennifer Z. Yao ; Chayasith Uttamapinant ; Andrei Poloukhtine ; Jeremy M. Baskin ; Julian A. Codelli ; Ellen M. Sletten ; Carolyn R. Bertozzi ; Vladimir V. Popik

Journal of the American Chemical Society 2012 Volume 134(Issue 8) pp:3720-3728

Publication Date(Web):January 4, 2012

DOI:10.1021/ja208090p

Methods for targeting of small molecules to cellular proteins can allow imaging with fluorophores that are smaller, brighter, and more photostable than fluorescent proteins. Previously, we reported targeting of the blue fluorophore coumarin to cellular proteins fused to a 13-amino acid recognition sequence (LAP), catalyzed by a mutant of the Escherichia coli enzyme lipoic acid ligase (LplA). Here, we extend LplA-based labeling to green- and red-emitting fluorophores by employing a two-step targeting scheme. First, we found that the W37I mutant of LplA catalyzes site-specific ligation of 10-azidodecanoic acid to LAP in cells, in nearly quantitative yield after 30 min. Second, we evaluated a panel of five different cyclooctyne structures and found that fluorophore conjugates to aza-dibenzocyclooctyne (ADIBO) gave the highest and most specific derivatization of azide-conjugated LAP in cells. However, for targeting of hydrophobic fluorophores such as ATTO 647N, the hydrophobicity of ADIBO was detrimental, and superior targeting was achieved by conjugation to the less hydrophobic monofluorinated cyclooctyne (MOFO). Our optimized two-step enzymatic/chemical labeling scheme was used to tag and image a variety of LAP fusion proteins in multiple mammalian cell lines with diverse fluorophores including fluorescein, rhodamine, Alexa Fluor 568, ATTO 647N, and ATTO 655.

Fast, Cell-Compatible Click Chemistry with Copper-Chelating Azides for Biomolecular Labeling

Co-reporter:Chayasith Uttamapinant;Anupong Tangpeerachaikul;Dr. Scott Grecian;Dr. Scott Clarke;Dr. Upinder Singh;Dr. Peter Slade;Dr. Kyle R. Gee; Alice Y. Ting

Angewandte Chemie International Edition 2012 Volume 51( Issue 24) pp:5852-5856

Publication Date(Web):

DOI:10.1002/anie.201108181

Site-Specific Protein Modification Using Lipoic Acid Ligase and Bis-Aryl Hydrazone Formation

Co-reporter:Dr. Justin D. Cohen;Peng Zou ; Alice Y. Ting

ChemBioChem 2012 Volume 13( Issue 6) pp:888-894

Publication Date(Web):

DOI:10.1002/cbic.201100764

Abstract

A screen of Trp37 mutants of Escherichia coli lipoic acid ligase (LplA) revealed enzymes capable of ligating an aryl-aldehyde or aryl-hydrazine substrate to LplA's 13-residue acceptor peptide. Once site-specifically attached to recombinant proteins fused to this peptide, aryl-aldehydes could be chemoselectively derivatized with hydrazine-probe conjugates, and aryl-hydrazines could be derivatized in an analogous manner with aldehyde-probe conjugates. Such two-step labeling was demonstrated for AlexaFluor568 targeting to monovalent streptavidin in vitro, and to neurexin-1β on the surface of living mammalian cells. To further highlight this technique, we labeled the low-density lipoprotein receptor on the surface of live cells with fluorescent phycoerythrin protein to allow single-molecule imaging and tracking over time.

Quantum Dot Targeting with Lipoic Acid Ligase and HaloTag for Single-Molecule Imaging on Living Cells

Co-reporter:Daniel S. Liu, William S. Phipps, Ken H. Loh, Mark Howarth, and Alice Y. Ting

ACS Nano 2012 Volume 6(Issue 12) pp:11080

Publication Date(Web):November 26, 2012

DOI:10.1021/nn304793z

We present a methodology for targeting quantum dots to specific proteins on living cells in two steps. In the first step, Escherichia coli lipoic acid ligase (LplA) site-specifically attaches 10-bromodecanoic acid onto a 13 amino acid recognition sequence that is genetically fused to a protein of interest. In the second step, quantum dots derivatized with HaloTag, a modified haloalkane dehalogenase, react with the ligated bromodecanoic acid to form a covalent adduct. We found this targeting method to be specific, fast, and fully orthogonal to a previously reported and analogous quantum dot targeting method using E. coli biotin ligase and streptavidin. We used these two methods in combination for two-color quantum dot visualization of different proteins expressed on the same cell or on neighboring cells. Both methods were also used to track single molecules of neurexin, a synaptic adhesion protein, to measure its lateral diffusion in the presence of neuroligin, its trans-synaptic adhesion partner.Keywords: fluorescence microscopy; HaloTag; lipoic acid ligase; quantum dot targeting; single-molecule imaging

Fast, Cell-Compatible Click Chemistry with Copper-Chelating Azides for Biomolecular Labeling

Co-reporter:Chayasith Uttamapinant;Anupong Tangpeerachaikul;Dr. Scott Grecian;Dr. Scott Clarke;Dr. Upinder Singh;Dr. Peter Slade;Dr. Kyle R. Gee; Alice Y. Ting

Angewandte Chemie 2012 Volume 124( Issue 24) pp:5954-5958

Publication Date(Web):

DOI:10.1002/ange.201108181

Diels–Alder Cycloaddition for Fluorophore Targeting to Specific Proteins inside Living Cells

Co-reporter:Daniel S. Liu ; Anupong Tangpeerachaikul ; Ramajeyam Selvaraj ; Michael T. Taylor ; Joseph M. Fox

Journal of the American Chemical Society 2011 Volume 134(Issue 2) pp:792-795

Publication Date(Web):December 16, 2011

DOI:10.1021/ja209325n

The inverse-electron-demand Diels–Alder cycloaddition between trans-cyclooctenes and tetrazines is biocompatible and exceptionally fast. We utilized this chemistry for site-specific fluorescence labeling of proteins on the cell surface and inside living mammalian cells by a two-step protocol. Escherichia coli lipoic acid ligase site-specifically ligates a trans-cyclooctene derivative onto a protein of interest in the first step, followed by chemoselective derivatization with a tetrazine–fluorophore conjugate in the second step. On the cell surface, this labeling was fluorogenic and highly sensitive. Inside the cell, we achieved specific labeling of cytoskeletal proteins with green and red fluorophores. By incorporating the Diels–Alder cycloaddition, we have broadened the panel of fluorophores that can be targeted by lipoic acid ligase.

Imaging Protein–Protein Interactions inside Living Cells via Interaction-Dependent Fluorophore Ligation

Co-reporter:Sarah A. Slavoff ; Daniel S. Liu ; Justin D. Cohen

Journal of the American Chemical Society 2011 Volume 133(Issue 49) pp:19769-19776

Publication Date(Web):November 18, 2011

DOI:10.1021/ja206435e

We report a new method, Interaction-Dependent PRobe Incorporation Mediated by Enzymes, or ID-PRIME, for imaging protein–protein interactions (PPIs) inside living cells. ID-PRIME utilizes a mutant of Escherichia coli lipoic acid ligase, LplAW37V, which can catalyze the covalent ligation of a coumarin fluorophore onto a peptide recognition sequence called LAP1. The affinity between the ligase and LAP1 is tuned such that, when each is fused to a protein partner of interest, LplAW37V labels LAP1 with coumarin only when the protein partners to which they are fused bring them together. Coumarin labeling in the absence of such interaction is low or undetectable. Characterization of ID-PRIME in living mammalian cells shows that multiple protein–protein interactions can be imaged (FRB–FKBP, Fos–Jun, and neuroligin–PSD-95), with as little as 10 min of coumarin treatment. The signal intensity and detection sensitivity are similar to those of the widely used fluorescent protein complementation technique (BiFC) for PPI detection, without the disadvantage of irreversible complex trapping. ID-PRIME provides a powerful and complementary approach to existing methods for visualization of PPIs in living cells with spatial and temporal resolution.

Imaging LDL Receptor Oligomerization during Endocytosis Using a Co-internalization Assay

Co-reporter:Peng Zou and Alice Y. Ting

ACS Chemical Biology 2011 Volume 6(Issue 4) pp:308

Publication Date(Web):December 31, 2010

DOI:10.1021/cb100361k

Methods to probe receptor oligomerization are useful to understand the molecular mechanisms of receptor signaling. Here we report a fluorescence imaging method to determine receptor oligomerization state in living cells during endocytic internalization. The wild-type receptor is co-expressed with an internalization-defective mutant, and the internalization kinetics of each are independently monitored. If the receptor internalizes as an oligomer, then the wild-type and mutant isoforms will mutually influence each others’ trafficking properties, causing co-internalization of the mutant or co-retention of the wild-type at the cell surface. Using this approach, we found that the low density lipoprotein (LDL) receptor internalizes as an oligomer into cells, both in the presence and absence of LDL ligand. The internalization kinetics of the wild-type receptor are not changed by LDL binding. We also found that the oligomerization domain of the LDL receptor is located in its cytoplasmic tail.

Structure-Guided Engineering of a Pacific Blue Fluorophore Ligase for Specific Protein Imaging in Living Cells

Co-reporter:Justin D. Cohen, Samuel Thompson, and Alice Y. Ting

Biochemistry 2011 Volume 50(Issue 38) pp:

Publication Date(Web):August 23, 2011

DOI:10.1021/bi201037r

Mutation of a gatekeeper residue, tryptophan 37, in E. coli lipoic acid ligase (LplA), expands substrate specificity such that unnatural probes much larger than lipoic acid can be recognized. This approach, however, has not been successful for anionic substrates. An example is the blue fluorophore Pacific Blue, which is isosteric to 7-hydroxycoumarin and yet not recognized by the latter’s ligase (W37VLplA) or any tryptophan 37 point mutant. Here we report the results of a structure-guided, two-residue screening matrix to discover an LplA double mutant, E20G/W37TLplA, that ligates Pacific Blue as efficiently as W37VLplA ligates 7-hydroxycoumarin. The utility of this Pacific Blue ligase for specific labeling of recombinant proteins inside living cells, on the cell surface, and inside acidic endosomes is demonstrated.

Synthesis of 7-Aminocoumarin by BuchwaldHartwig Cross Coupling for Specific Protein Labeling in Living Cells

Co-reporter:Xin Jin;Chayasith Uttamapinant; Dr. Alice Y. Ting

ChemBioChem 2011 Volume 12( Issue 1) pp:65-70

Publication Date(Web):

DOI:10.1002/cbic.201000414

Pyrenebutyrate Leads to Cellular Binding, Not Intracellular Delivery, of Polyarginine Quantum Dots

Co-reporter:Amy E. Jablonski, Takashi Kawakami, Alice Y. Ting and Christine K. Payne

The Journal of Physical Chemistry Letters 2010 Volume 1(Issue 9) pp:1312-1315

Publication Date(Web):April 6, 2010

DOI:10.1021/jz100248c

The intracellular, cytosolic, delivery of quantum dots is an important goal for cellular imaging. Recently, a hydrophobic anion, pyrenebutyrate, has been proposed to serve as a delivery agent for cationic quantum dots as characterized by confocal microscopy. Using an extracellular quantum dot quencher, QSY-21, as an alternative to confocal microscopy, we demonstrate that quantum dots remain on the cell surface and do not cross the plasma membrane following pyrenebutyrate treatment, a result that is confirmed with transmission electron microscopy. Pyrenebutyrate leads to increased cellular binding of quantum dots rather than intracellular delivery. These results characterize the use of QSY-21 as a quantum dot quencher and highlight the importance of the use of complementary techniques when using confocal microscopy.Keywords (keywords): biophysical chemistry; fluorescence microscopy; live cell imaging; pyrenebutyrate; quantum dots;

A fluorophore ligase for site-specific protein labeling inside living cells

Co-reporter:Marta Fernández-Suárez;Samuel Thompson;Hemanta Baruah;Chayasith Uttamapinant;Katharine A. White;Sujiet Puthenveetil

PNAS 2010 Volume 107 (Issue 24 ) pp:10914-10919

Publication Date(Web):2010-06-15

DOI:10.1073/pnas.0914067107

Biological microscopy would benefit from smaller alternatives to green fluorescent protein for imaging specific proteins in living cells. Here we introduce PRIME (PRobe Incorporation Mediated by Enzymes), a method for fluorescent labeling of peptide-fused recombinant proteins in living cells with high specificity. PRIME uses an engineered fluorophore ligase, which is derived from the natural Escherichia coli enzyme lipoic acid ligase (LplA). Through structure-guided mutagenesis, we created a mutant ligase capable of recognizing a 7-hydroxycoumarin substrate and catalyzing its covalent conjugation to a transposable 13-amino acid peptide called LAP (LplA Acceptor Peptide). We showed that this fluorophore ligation occurs in cells in 10 min and that it is highly specific for LAP fusion proteins over all endogenous mammalian proteins. By genetically targeting the PRIME ligase to specific subcellular compartments, we were able to selectively label spatially distinct subsets of proteins, such as the surface pool of neurexin and the nuclear pool of actin.

Yeast Display Evolution of a Kinetically Efficient 13-Amino Acid Substrate for Lipoic Acid Ligase

Co-reporter:Sujiet Puthenveetil ; Daniel S. Liu ; Katharine A. White ; Samuel Thompson

Journal of the American Chemical Society 2009 Volume 131(Issue 45) pp:16430-16438

Publication Date(Web):October 28, 2009

DOI:10.1021/ja904596f

Escherichia coli lipoic acid ligase (LplA) catalyzes ATP-dependent covalent ligation of lipoic acid onto specific lysine side chains of three acceptor proteins involved in oxidative metabolism. Our lab has shown that LplA and engineered mutants can ligate useful small-molecule probes such as alkyl azides ( Nat. Biotechnol. 2007, 25, 1483−1487) and photo-cross-linkers ( Angew. Chem., Int. Ed. 2008, 47, 7018−7021) in place of lipoic acid, facilitating imaging and proteomic studies. Both to further our understanding of lipoic acid metabolism, and to improve LplA’s utility as a biotechnological platform, we have engineered a novel 13-amino acid peptide substrate for LplA. LplA’s natural protein substrates have a conserved β-hairpin structure, a conformation that is difficult to recapitulate in a peptide, and thus we performed in vitro evolution to engineer the LplA peptide substrate, called “LplA Acceptor Peptide” (LAP). A ∼107 library of LAP variants was displayed on the surface of yeast cells, labeled by LplA with either lipoic acid or bromoalkanoic acid, and the most efficiently labeled LAP clones were isolated by fluorescence activated cell sorting. Four rounds of evolution followed by additional rational mutagenesis produced a “LAP2” sequence with a kcat/Km of 0.99 μM−1 min−1, >70-fold better than our previous rationally designed 22-amino acid LAP1 sequence (Nat. Biotechnol. 2007, 25, 1483−1487), and only 8-fold worse than the kcat/Km values of natural lipoate and biotin acceptor proteins. The kinetic improvement over LAP1 allowed us to rapidly label cell surface peptide-fused receptors with quantum dots.

An Engineered Aryl Azide Ligase for Site-Specific Mapping of ProteinProtein Interactions through Photo-Cross-Linking

Co-reporter:Hemanta Baruah Dr.;Sujiet Puthenveetil Dr.;Yoon-Aa Choi;Samit Shah Dr. ;AliceY. Ting

Angewandte Chemie 2008 Volume 120( Issue 37) pp:7126-7129

Publication Date(Web):

DOI:10.1002/ange.200802088

An Engineered Aryl Azide Ligase for Site-Specific Mapping of ProteinProtein Interactions through Photo-Cross-Linking

Co-reporter:Hemanta Baruah Dr.;Sujiet Puthenveetil Dr.;Yoon-Aa Choi;Samit Shah Dr. ;AliceY. Ting

Angewandte Chemie International Edition 2008 Volume 47( Issue 37) pp:7018-7021

Publication Date(Web):

DOI:10.1002/anie.200802088

Redirecting lipoic acid ligase for cell surface protein labeling with small-molecule probes

Co-reporter:Laura Martínez-Hernández;Jeremy M Baskin;Marta Fernández-Suárez;Hemanta Baruah;Carolyn R Bertozzi;Kathleen T Xie;Alice Y Ting;Hemanta Baruah;Carolyn R Bertozzi;Jeremy M Baskin;Alice Y Ting;Laura Martínez-Hernández;Kathleen T Xie;Marta Fernández-Suárez

Nature Biotechnology 2007 Volume 25(Issue 12) pp:1483-1487

Publication Date(Web):2007-12-02

DOI:10.1038/nbt1355

Live cell imaging is a powerful method to study protein dynamics at the cell surface, but conventional imaging probes are bulky, or interfere with protein function1, 2, or dissociate from proteins after internalization3, 4. Here, we report technology for covalent, specific tagging of cellular proteins with chemical probes. Through rational design, we redirected a microbial lipoic acid ligase (LplA)5 to specifically attach an alkyl azide onto an engineered LplA acceptor peptide (LAP). The alkyl azide was then selectively derivatized with cyclo-octyne6 conjugates to various probes. We labeled LAP fusion proteins expressed in living mammalian cells with Cy3, Alexa Fluor 568 and biotin. We also combined LplA labeling with our previous biotin ligase labeling7, 8, to simultaneously image the dynamics of two different receptors, coexpressed in the same cell. Our methodology should provide general access to biochemical and imaging studies of cell surface proteins, using small fluorophores introduced via a short peptide tag.

Targeting quantum dots to surface proteins in living cells with biotin ligase

Co-reporter:Mark Howarth;Keizo Takao;Yasunori Hayashi

PNAS 2005 102 (21 ) pp:7583-7588

Publication Date(Web):2005-05-24

DOI:10.1073/pnas.0503125102

Escherichia coli biotin ligase site-specifically biotinylates a lysine side chain within a 15-amino acid acceptor peptide (AP) sequence. We show that mammalian cell surface proteins tagged with AP can be biotinylated by biotin ligase added to the medium, while endogenous proteins remain unmodified. The biotin group then serves as a handle for targeting streptavidin-conjugated quantum dots (QDs). This labeling method helps to address the two major deficiencies of antibody-based labeling, which is currently the most common method for targeting QDs to cells: the size of the QD conjugate after antibody attachment and the instability of many antibody–antigen interactions. To demonstrate the versatility of our method, we targeted QDs to cell surface cyan fluorescent protein and epidermal growth factor receptor in HeLa cells and to α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors in neurons. Labeling requires only 2 min, is extremely specific for the AP-tagged protein, and is highly sensitive. We performed time-lapse imaging of single QDs bound to AMPA receptors in neurons, and we compared the trafficking of different AMPA receptor subunits by using two-color pulse–chase labeling.

A Genetically Encoded Fluorescent Reporter of Histone Phosphorylation in Living Cells

Co-reporter:Chi-Wang Lin

Angewandte Chemie International Edition 2004 Volume 43(Issue 22) pp:

Publication Date(Web):19 MAY 2004

DOI:10.1002/anie.200353375

An increase in FRET indicates phosphorylation of histone H3 at serine 28. The protein-based reporter (see picture) responds to phosphorylation through intramolecular complexation between a substrate domain derived from histone H3 and a linked phosphoserine-recognition domain. The reporter is also effective inside living mammalian cells. FRET=fluorescence resonance energy transfer.

A Genetically Encoded Fluorescent Reporter of Histone Phosphorylation in Living Cells

Co-reporter:Chi-Wang Lin

Angewandte Chemie 2004 Volume 116(Issue 22) pp:

Publication Date(Web):19 MAY 2004

DOI:10.1002/ange.200353375

Eine Zunahme des FRET zeigt die Phosphorylierung von Histon H3 an Serin 28 an. Der proteinbasierte Rezeptor (siehe Bild) reagiert auf die Phosphorylierung mit intramolekularer Komplexbildung zwischen einer von H3 abgeleiteten Substratdomäne und einer damit verknüpften Phosphoserin-Erkennungsdomäne. Der Rezeptor funktioniert auch in lebenden Säugerzellen. FRET=fluorescence resonance energy transfer.

Proteomic Mapping of the Human Mitochondrial Intermembrane Space in Live Cells via Ratiometric APEX Tagging

Co-reporter:Victoria Hung, Peng Zou, Hyun-Woo Rhee, Namrata D. Udeshi, ... Alice Y. Ting

Molecular Cell (17 July 2014) Volume 55(Issue 2) pp:332-341

Publication Date(Web):17 July 2014

DOI:10.1016/j.molcel.2014.06.003

•Ratiometric tagging with an engineered peroxidase gives nanometer spatial resolution•Mitochondrial intermembrane space proteome mapped with >94% specificity•Nine newly discovered mitochondrial proteins confirmed by imaging and western blotting•Data support MICU1 and MICU2 localization in the mitochondrial intermembrane spaceObtaining complete protein inventories for subcellular regions is a challenge that often limits our understanding of cellular function, especially for regions that are impossible to purify and are therefore inaccessible to traditional proteomic analysis. We recently developed a method to map proteomes in living cells with an engineered peroxidase (APEX) that bypasses the need for organellar purification when applied to membrane-bound compartments; however, it was insufficiently specific when applied to unbounded regions that allow APEX-generated radicals to escape. Here, we combine APEX technology with a SILAC-based ratiometric tagging strategy to substantially reduce unwanted background and achieve nanometer spatial resolution. This is applied to map the proteome of the mitochondrial intermembrane space (IMS), which can freely exchange small molecules with the cytosol. Our IMS proteome of 127 proteins has >94% specificity and includes nine newly discovered mitochondrial proteins. This approach will enable scientists to map proteomes of cellular regions that were previously inaccessible.Download high-res image (251KB)Download full-size image