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Research Focus

Since its founding, the Link lab has been interested in applying the tools of protein engineering and bioconjugate chemistry to engineer peptides and proteins with conformational constraints. Conformational constraints within a polypeptide can lead to improvements in properties such as protease stability, thermostability, and binding affinity. With the development of bioorthogonal chemistry in the early 2000’s, the toolbox for conformational constraints expanded. We have used a combination of protein engineering and azide-alkyne click chemistry to carry out “protein stapling” on small proteins (Abdeljabbar et al. Chem. Comm. 2014). We have also used olefin metathesis chemistry to constrain short engineered peptides that function to turn on apoptosis in cells (Link and Zhang, US patent 9,464,125). We have also looked to nature for inspiration in strategies for conformationally constraining peptides. This has led to our program on lasso peptides, an ever expanding class of natural products defined by their slipknot-like topology (see figure). Our group started out working on the antimicrobial lasso peptide microcin J25 as an interesting substrate for engineering by directed evolution (Pan et al. JACS 2011) and with unnatural amino acids (Piscotta et al. Chem. Comm 2015). We became interested in the variety of different lasso peptides present in nature and developed the first algorithm for large-scale genome mining of lasso peptides (Maksimov et al. PNAS 2012). We continue to look to lasso peptides as a source of new antibiotics, interesting new enzymology, and even as building blocks for molecular machines. Please read below for more details on these specific projects.

Research Areas
Chemical Biology
Honors

Participant in German-American Frontiers of Science Symposium, 2016
Participant in NAE US/EU Frontiers of Engineering, 2014
Alfred P. Sloan Research Fellowship (Chemistry), 2013-2015
DuPont Young Professor, 2011
NSF CAREER Award, 2010
NIH Kirschstein/NRSA Fellowship, 2006
NSF Graduate Research Fellowship, 2000
Tau Beta Pi Fellowship, 2000

Selected Recent Publications

Elashal HE, Cohen RD, Elashal HE, Zong CH, Link AJ, Raj M: Cyclic and Lasso Peptides: Sequence Determination, Topology Analysis, and Rotaxane FormationAngewandte Chemie-International Edition 2018, 57: 6150-6154.

Zong C, Cheung-Lee WL, Elashal HE, Raj M, Link AJ: Albusnodin: an acetylated lasso peptide from Streptomyces albusChemical Communications 2018, 54: 1339-1342.

Zong C, Wu MJ, Qin JZ, Link AJ: Lasso Peptide Benenodin-1 is a Thermally Actuated [1]Rotaxane SwitchJournal of the American Chemical Society 2017, 139: 10403-10409.

Chekan JR, Koos JD, Zong C, Maksimov MO, Link AJ, Nair SK: Structure of the Lasso Peptide Isopeptidase Identifies a Topology for Processing Threaded SubstratesJournal of the American Chemical Society 2016, 138: 16452-16458.

Allen CD, Link AJ: Self-Assembly of Catenanes from Lasso PeptidesJournal of the American Chemical Society 2016, 138: 14214-14217.

Cheung WL, Chen MY, Maksimov MO, Link AJ: Lasso Peptide Biosynthetic Protein LarB1 Binds Both Leader and Core Peptide Regions of the Precursor Protein LarAACS Central Science 2016, 2: 702-709.

Allen CD, Chen MY, Trick AY, Le DT, Ferguson AL, Link AJ: Thermal Unthreading of the Lasso Peptides Astexin-2 and Astexin-3ACS Chemical Biology 2016, 11: 3043-3051.

Arnaudo AM, Link AJ, Garcia BA: Bioorthogonal Chemistry for the Isolation and Study of Newly Synthesized Histones and their ModificationsACS Chemical Biology 2016, 11: 782-791.

Zong C, Maksimov MO, Link AJ: Construction of Lasso Peptide Fusion ProteinsACS Chemical Biology 2016, 11: 61-68.

Maksimov MO, Koos JD, Zong C, Lisko B, Link AJ: Elucidating the Specificity Determinants of the AtxE2 Lasso Peptide IsopeptidaseJournal of Biological Chemistry 2015, 290: 30806-30812.

Link AJ: Leading the way to RiPPsNature Chemical Biology 2015, 11: 551-552.

Futran AS, Kyin S, Shvartsman SY, Link AJ: Mapping the binding interface of ERK and transcriptional repressor Capicua using photocrosslinkingProceedings of the National Academy of Sciences 2015, 112: 8590-8595.

Piscotta FJ, Tharp JM, Liu WR, Link AJ: Expanding the chemical diversity of lasso peptide MccJ25 with genetically encoded noncanonical amino acidsChemical Communications 2015, 51: 409-412.

Abdeljabbar DM, Piscotta FJ, Zhang SY, Link AJ: Protein stapling via azide-alkyne ligationChemical Communications2014, 50: 14900-14903.

Maksimov MO, Link AJ: Prospecting Genomes for Lasso PeptidesJournal of Industrial Microbiology & Biotechnology2014, 41: 333-344.

Futran AS, Link AJ, Seger R, Shvartsman SY: ERK as a Model for Systems Biology of Enzyme Kinetics in CellsCurrent Biology 2013, 23: R972-R979.

Maksimov MO, Link AJ: Discovery and Characterization of an Isopeptidase that Linearizes Lasso PeptidesJournal of the American Chemical Society 2013, 135: 12038-12047.

Arnison PG, Bibb MJ, Bierbaum G, Bowers AA, Bugni TS, Bulaj G, Camarero JA, Campopiano DJ, Challis GL, Clardy J, Cotter PD, Craik DJ, Dawson M, Dittmann E, Donadio S, Dorrestein PC, Entian K-D, Fischbach MA, Garavelli JS, Goransson U, Gruber CW, Haft DH, Hemscheidt TK, Hertweck C, Hill C, Horswill AR, Jaspars M, Kelly WL, Klinman JP, Kuipers OP, Link AJ, Liu W, Marahiel MA, Mitchell DA, Moll GN, Moore BS, Muller R, Nair SK, Nes IF, Norris GE, Olivera BM, Onaka H, Patchett ML, Piel J, Reaney MJT, Rebuffat S, Ross RP, Sahl H-G, Schmidt EW, Selsted ME, Severinov K, Shen B, Sivonen K, Smith L, Stein T, Sussmuth RD, Tagg JR, Tang G-L, Truman AW, Vederas JC, Walsh CT, Walton JD, Wenzel SC, Willey JM, van der Donk WA: Ribosomally synthesized and post-translationally modified peptide natural products: overview and recommendations for a universal nomenclatureNatural Product Reports 2013, 30:108-160.

Maksimov MO, Pelczer I, Link AJ: Precursor-centric genome-mining approach for lasso peptide discoveryProceedings of the National Academy of Sciences 2012, 109:15223-15228.

Maksimov MO, Pan SJ, Link AJ: Lasso peptides: structure, function, biosynthesis, and engineeringNatural Product Reports 2012, 29: 996-1006.

Park EJ, Wagenaar T, Zhang SY, Link AJ, Prud'homme RK, Koberstein JT, Turro NJ: Using Light to Covalently Immobilize and Pattern Nanoparticles onto SurfacesLangmuir 2012, 28: 10934-10941.

Zhang SY, Chan KH, Prud'homme RK, Link AJ: Synthesis and Evaluation of Clickable Block Copolymers for Targeted Nanoparticle Drug DeliveryMolecular Pharmaceutics 2012, 9: 2228-2236.

Pan SJ, Rajniak J, Maksimov MO, Link AJ: The Role of a Conserved Threonine Residue in the Leader Peptide of Lasso Peptide PrecursorsChemical Communications 2012, 48: 1880-1882.

Pan SJ, Rajniak J, Cheung WL, Link AJ: Construction of a Single Polypeptide that Matures and Exports the Lasso Peptide Microcin J25ChemBioChem 2012, 13: 367-370.

Zhang SY, Long A, Link AJ: A comparison of two strategies for affinity maturation of a BH3 peptide toward pro-survival Bcl-2 proteins. ACS Synthetic Biology 2012, 1: 89-98.

Abdeljabbar DM, Song HJ, Link AJ: Trichoderma reesei cellobiohydrolase II is associated with the outer membrane when overexpressed in Escherichia coli. Biotechnology Letters 2012, 34: 91-96.

Zhang SY, Link AJ: Bcl-2 Family Interactome Analysis Using Bacterial Surface DisplayIntegrative Biology 2011, 3: 823-831.

Abdeljabbar DM, Klein TJ, Link AJ: An Engineered Methionyl-tRNA Synthetase Enables Azidonorleucine Incorporation in Methionine Prototrophic BacteriaChemBioChem 2011, 12: 1699-1702.

Zhang SY, Prud’homme RK, Link AJ: Block Copolymer Nanoparticles as Nanobeads for the Polymerase Chain ReactionNano Letters 2011, 11: 1123-1126.

Pan SJ, Link AJ: Sequence Diversity in the Lasso Peptide Framework: Discovery of Functional Microcin J25 Variants with Multiple Amino Acid SubstitutionsJournal of the American Chemical Society 2011, 133: 5016-5023.

Zhang SY, Adamson DH, Prud’homme RK, Link AJ: Photocrosslinking the Polystyrene Core of Block-Copolymer Nanoparticles. Polymer Chemistry 2011, 2: 665-671.

Pan SJ, Cheung WL, Fung HK, Floudas CA, Link AJ: Computational Design of Functional Variants of the Antimicrobial Peptide Microcin J25Protein Engineering Design & Selection 2011, 24: 275-282.

Ferguson AL, Zhang SY, Dikiy I, Panagiotopoulos AZ, Debenedetti PG, Link AJ: An Experimental and Computational Investigation of Spontaneous Lasso Formation in Microcin J25Biophysical Journal 2010, 99: 3056-3065.

Mannoor MS, Zhang SY, Link AJ, McAlpine MC: Electrical detection of pathogenic bacteria via immobilized antimicrobial peptidesProceedings of the National Academy of Sciences 2010, 107: 19207-19212.

Cheung WL, Pan SJ, Link AJ: Much of the Microcin J25 Leader Peptide is DispensableJournal of the American Chemical Society 2010, 132: 2514-2515.

Pan SJ, Cheung WL, Link AJ: Engineered Gene Clusters for the Production of the Antimicrobial Peptide Microcin J25Protein Expression and Purification 2010, 71: 200-206.

Abdeljabbar DM, Klein TJ, Zhang SY, Link AJ: A Single Genomic Copy of an Engineered Methionyl-tRNA Synthetase Enables Robust Incorporation of Azidonorleucine into Recombinant Proteins in E. coliJournal of the American Chemical Society 2009, 131: 17078-17079.

Sun JJ, Abdeljabbar DM, Clarke N, Bellows ML, Floudas CA, Link AJ: Reconstitution and Engineering of Apoptotic Protein Interactions on the Bacterial Cell SurfaceJournal of Molecular Biology 2009, 394: 297-305.

Tang Y, Wang P, Van Deventer JA, Link AJ, Tirrell DA: Introduction of an Aliphatic Ketone into Recombinant Proteins in a Bacterial Strain that Overexpresses an Editing-Impaired Leucyl-tRNA SynthetaseChembiochem 2009, 10: 2188-2190.

Xu LY, Link AJ: Stress responses to heterologous membrane protein expression in Escherichia coliBiotechnology Letters 2009, 31: 1775-1782.

Dieterich DC, Link AJ: Click Chemistry in Protein Engineering, Design, Detection and Profiling. in Click Chemistry in Biotechnology and Materials Science, Wiley, ed. Joerg Lahann.

Link AJ, Skretas G, Chari NS, Strauch EM, Georgiou, G: Efficient production of membrane-integrated and detergent-soluble G protein-coupled receptors in Escherichia coli Protein Science 2008, 17: 1857-1863.

Strable E, Prasuhn DE, Udit AK, Brown S, Link AJ, Ngo JT, Lander G, Quispe J, Potter CS, Carragher B, et al.: Unnatural amino acid incorporation into virus-like particlesBioconjugate Chemistry 2008, 19: 866-875.

Yoo TH, Link AJ, Tirrell DA: Evolution of a fluorinated green fluorescent proteinProceedings of the National Academy of Sciences 2007, 104: 13887-13890.

Link AJ, Jeong KJ, Georgiou, G: Beyond toothpicks: new methods for isolating mutant bacteriaNature Reviews Microbiology 2007, 5: 680-688.

Link AJ, Vink MKS, Tirrell DA: Synthesis of the functionalizable methionine surrogate azidohomoalanine using Boc-homoserine as precursorNature Protocols 2007, 2: 1884-1887.

Link AJ, Vink MKS, Tirrell DA: Preparation of the functionalizable methionine surrogate azidohomoalanine via copper-catalyzed diazo transfer Nature Protocols 2007, 2: 1879-1883.

Link AJ, Georgiou G: Advances and Challenges in Membrane Protein ExpressionAIChE Journal 2007, 53: 752-756.

Dieterich DC, Lee JJ, Link AJ, Graumann J, Tirrell DA, Schuman EM: Labeling, detection and identification of newly synthesized proteomes with bioorthogonal non-canonical amino-acid taggingNature Protocols 2007, 2: 532-540.

Link AJ, Vink MKS, Agard NJ, Prescher JA, Bertozzi CR, Tirrell DA: Discovery of aminoacyl-tRNA synthetase activity through cell-surface display of noncanonical amino acidsProceedings of the National Academy of Sciences 2006, 103: 10180-10185.

Dieterich DC, Link AJ, Graumann J, Tirrell DA, Schuman EM: Selective identification of newly synthesized proteins in mammalian cells using bioorthogonal non-canonical amino acid tagging (BONCAT)Proceedings of the National Academy of Sciences 2006, 103: 9482-9487.

Link AJ, Tirrell DA: Reassignment of Sense Codons In vivoMethods 2005, 36: 291-298.

Link AJ, Vink MKS, Tirrell DA: Presentation and Detection of Azide Functionality in 
Bacterial Cell Surface ProteinsJournal of the American Chemical Society 2004, 126: 10598-10602.

Link AJ, Mock ML, Tirrell DA: Non-canonical Amino Acids in Protein EngineeringCurrent Opinion in Biotechnology 2003, 14: 603-609.

Link AJ, Tirrell DA: Cell Surface Labeling of Escherichia coli via Copper(I)- Catalyzed [3+2] CycloadditionJournal of the American Chemical Society 2003, 125: 11164-11165.

Bradley AZ, Link AJ, Biswas K, Kahne D, Schwartz J, Jones M, Zhu ZD, Platz MS: Hydrogen Abstraction on Photolysis of a NaphthocarboraneTetrahedron Letters 2000, 41: 8691-8694.