Associated Faculty, Chemical and Biological Engineering

Contact

ajlink@princeton.edu
609-258-7191
Hoyt Chemical Laboratory, 207
Link Lab

Research Area

Biochemistry, Biophysics & Structural Biology

Research Focus

Protein engineering and chemical biology



Research
Selected Publications
Nature has evolved and designed proteins to perform an exquisite array of tasks, but in the pursuit of biotechnological interests, these proteins must often be improved, altered, or even completely redesigned. In the post-genomic era, protein sequence information is abundant and readily available, and structural biology efforts are rapidly increasing the amount of protein structure information. However, the level of intricacy and complexity of most proteins is still such that rational design efforts are often unsuccessful in imparting an improved or new function to a protein. Fortunately, protein engineers can utilize an experimental algorithm that mimics Darwinian evolution to introduce new functions into proteins. In this algorithm, termed directed evolution, thousands or even millions of protein variants are generated by the introduction of mutations to the gene encoding the protein of interest. The library of protein variants is then screened to identify those members of the population with the highest levels of function or activity: a molecular survival of the fittest. One of the major focuses of the Link group is to apply directed evolution to medically relevant proteins.

Engineering high-affinity inhibitors of anti-apoptotic proteins

The molecular cause of several cancers is an imbalance between pro-apoptotic and anti-apoptotic proteins. It is postulated that the excess anti-apoptotic protein sequesters all of the pro-apoptotic protein thus preventing execution of apoptosis (programmed cell death). One potential treatment for cancers of this type is a high-affinity competitive inhibitor to the anti-apoptotic protein. We are pursuing several different natural proteins as scaffolds for such an inhibitor as well as completely de novo library designs.

Evolving higher efficacy antimicrobial peptides

Some classes of antimicrobial peptides are used by microbes as a defense mechanism against other species. These peptides represent an avenue of treatment for multidrug resistant (MDR) bacterial infections that has not been thoroughly explored yet. We will utilize naturally occurring antimicrobial peptides and apply directed evolution in order to engineer molecules with higher efficacies and broader spectra of activity. Synthetic biology principles are also being investigated in order to generate novel methods of delivering antimicrobial peptides to infection sites.

Biotechnological uses of E. coli: understanding the cellular response

Protein engineers often use host organisms such as E. coli simply as factories with little regard for the physiological state of the cell. We plan to use transcriptional reporters along with genomic and proteomic approaches (including BONCAT) to catalog the response of the cell to biotechnological uses of E. coli such as heterologous protein expression and unnatural amino acid incorporation. The knowledge and insights gained from these large-scale studies will subsequently be used to inform strain engineering experiments to develop host strains of E. coli that are more useful in biotech contexts.
Analyzing differential proteomes with BONCAT (Bio-Orthogonal Non-Canonical Amino Acid Tagging)




Elashal HE, Koos JD, Cheung-Lee WLing, Choi B, Cao L, Richardson MA, et al. Biosynthesis and characterization of fuscimiditide, an aspartimidylated graspetide. Nat Chem. 2022 ;. PubMed
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Wan X, A Link J, Brynildsen MP. Translational Fusion to Hmp Improves Heterologous Protein Expression. Microorganisms. 2022 ;10(2). PubMed
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Schr?der HV, Stadlmeier M, Wühr M, A Link J. The Shuttling Cascade in Lasso Peptide Benenodin-1 is Controlled by Non-Covalent Interactions. Chemistry. 2022 ;28(5):e202103615. PubMed
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Do T, A Link J. Protein Engineering in Ribosomally Synthesized and Post-translationally Modified Peptides (RiPPs). Biochemistry. 2022 ;. PubMed
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Do T, Thokkadam A, Leach R, A Link J. Phenotype-Guided Comparative Genomics Identifies the Complete Transport Pathway of the Antimicrobial Lasso Peptide Ubonodin in . ACS Chem Biol. 2022 ;17(8):2332-2343. PubMed
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Cao L, Do T, A Link J. Mechanisms of action of ribosomally synthesized and posttranslationally modified peptides (RiPPs). J Ind Microbiol Biotechnol. 2021 ;48(3-4). PubMed
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Schr?der HV, Zhang Y, A Link J. Dynamic covalent self-assembly of mechanically interlocked molecules solely made from peptides. Nat Chem. 2021 ;13(9):850-857. PubMed
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Cao L, Beiser M, Koos JD, Orlova M, Elashal HE, Schr?der HV, et al.. Cellulonodin-2 and Lihuanodin: Lasso Peptides with an Aspartimide Post-Translational Modification. J Am Chem Soc. 2021 ;143(30):11690-11702. PubMed
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Nerger BA, Jaslove JM, Elashal HE, Mao S, Ko?mrlj A, A Link J, et al.. Local accumulation of extracellular matrix regulates global morphogenetic patterning in the developing mammary gland. Curr Biol. 2021 ;31(9):1903-1917.e6. PubMed
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Montalbán-López M, Scott TA, Ramesh S, Rahman IR, van Heel AJ, Viel JH, et al.. New developments in RiPP discovery, enzymology and engineering. Nat Prod Rep. 2021 ;38(1):130-239. PubMed
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Baltz RH, Kao K, A Link J, Marsili E, Reguera G, Shao Z, et al. Introduction to Special Issue on?"Frontiers in Industrial Microbiology and Biotechnology 2020". J Ind Microbiol Biotechnol. 2020 ;47(9-10):621-622. PubMed
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Cheung-Lee WLing, Parry ME, Zong C, Cartagena AJaramillo, Darst SA, Connell ND, et al. Discovery of Ubonodin, an Antimicrobial Lasso Peptide Active against Members of the Burkholderia cepacia Complex. Chembiochem. 2020 ;21(9):1335-1340. PubMed
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Chou WKang, Vaikunthan M, Schr?der HV, A Link J, Kim H, Brynildsen MP. Synergy Screening Identifies a Compound That Selectively Enhances the Antibacterial Activity of Nitric Oxide. Front Bioeng Biotechnol. 2020 ;8:1001. PubMed
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Cheung-Lee WLing, A Link J. Genome mining for lasso peptides: past, present, and future. J Ind Microbiol Biotechnol. 2019 ;46(9-10):1371-1379. PubMed
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Cheung-Lee WLing, Cao L, A Link J. Pandonodin: A Proteobacterial Lasso Peptide with an Exceptionally Long C-Terminal Tail. ACS Chem Biol. 2019 ;14(12):2783-2792. PubMed
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Cheung-Lee WLing, Parry ME, Cartagena AJaramillo, Darst SA, A Link J. Discovery and structure of the antimicrobial lasso peptide citrocin. J Biol Chem. 2019 ;294(17):6822-6830. PubMed
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Braffman NR, Piscotta FJ, Hauver J, Campbell EA, A Link J, Darst SA. Structural mechanism of transcription inhibition by lasso peptides microcin J25 and capistruin. Proc Natl Acad Sci U S A. 2019 ;116(4):1273-1278. PubMed
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Piscotta FJ, Jeffrey PD, A Link J. ParST is a widespread toxin-antitoxin module that targets nucleotide metabolism. Proc Natl Acad Sci U S A. 2019 ;116(3):826-834. PubMed
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Zong C, Cheung-Lee WLing, Elashal HE, Raj M, A Link J. Albusnodin: an acetylated lasso peptide from Streptomyces albus. Chem Commun (Camb). 2018 ;54(11):1339-1342. PubMed
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Elashal HE, Cohen RD, Elashal HE, Zong C, A Link J, Raj M. Cyclic and Lasso Peptides: Sequence Determination, Topology Analysis, and Rotaxane Formation. Angew Chem Int Ed Engl. 2018 ;57(21):6150-6154. PubMed
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Zong C, Wu MJ, Qin JZ, A Link J. Lasso Peptide Benenodin-1 Is a Thermally Actuated [1]Rotaxane Switch. J Am Chem Soc. 2017 ;139(30):10403-10409. PubMed
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Cheung WLing, Chen MY, Maksimov MO, A Link J. Lasso Peptide Biosynthetic Protein LarB1 Binds Both Leader and Core Peptide Regions of the Precursor Protein LarA. ACS Cent Sci. 2016 ;2(10):702-709. PubMed
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Arnaudo AM, A Link J, Garcia BA. Bioorthogonal Chemistry for the Isolation and Study of Newly Synthesized Histones and Their Modifications. ACS Chem Biol. 2016 ;11(3):782-91. PubMed
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Zong C, Maksimov MO, A Link J. Construction of Lasso Peptide Fusion Proteins. ACS Chem Biol. 2016 ;11(1):61-8. PubMed
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Chekan JR, Koos JD, Zong C, Maksimov MO, A Link J, Nair SK. Structure of the Lasso Peptide Isopeptidase Identifies a Topology for Processing Threaded Substrates. J Am Chem Soc. 2016 ;138(50):16452-16458. PubMed
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Allen CD, Chen MY, Trick AY, Le DThanh, Ferguson AL, A Link J. Thermal Unthreading of the Lasso Peptides Astexin-2 and Astexin-3. ACS Chem Biol. 2016 ;11(11):3043-3051. PubMed
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Allen CD, A Link J. Self-Assembly of Catenanes from Lasso Peptides. J Am Chem Soc. 2016 ;138(43):14214-14217. PubMed
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Futran AS, Kyin S, Shvartsman SY, A Link J. Mapping the binding interface of ERK and transcriptional repressor Capicua using photocrosslinking. Proc Natl Acad Sci U S A. 2015 ;112(28):8590-5. PubMed
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Maksimov MO, Koos JD, Zong C, Lisko B, A Link J. Elucidating the Specificity Determinants of the AtxE2 Lasso Peptide Isopeptidase. J Biol Chem. 2015 ;290(52):30806-12. PubMed
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A Link J. Biosynthesis: Leading the way to RiPPs. Nat Chem Biol. 2015 ;11(8):551-2. PubMed
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