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  Protein Modification  DOI: 10.1002/anie.201207089 Protein Organic Chemistry and Applications forLabeling and Engineering in Live-Cell Systems Yousuke Takaoka, Akio Ojida, and Itaru Hamachi*  Angewandte Chemie Keywords: bioconjugation ·bioorthogonal chemistry ·ligand-directed chemistry ·protein engineering ·protein labeling    AngewandteReviews  I. Hamachi et al. 4088  www.angewandte.org   2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim  Angew. Chem. Int. Ed.  2013 ,  52 , 4088–4106  1.  Introduction The study of protein structure and function is not only of significant importance to fundamental scientific research, butalso critical to the development of biomedical and biotech-nological applications, because proteins are implicated in somany biological systems. The modification of proteins withmolecular probes provides a powerful technique for theelucidation of protein functions. [1,2] For fluorescence bio-imaging studies in live cells, for example, the proteins of interest should be labeled with a fluorescent marker, whichallows for the selective real-time detection of their local-ization, trafficking, and activities. Protein modification withsynthetic molecules is also a valuable technique for theengineering of protein functions to create new biocatalystsand bio-analytical tools. In a recent trend in chemical biologyresearch, there has been an aspiration to conduct proteinmodification under more biological (crude) conditions, suchas in live cells or tissues/bodies, rather than in pure test tubes(in vitro). Several issues need to be addressed for theconstruction of homogenously modified proteins with syn-thetic molecules, including 1) protein selectivity, 2) labeling-site selectivity, 3) control of the labeling-site numbers, and4) expansion of the labeled amino acids. Recently developedhigh-precision protein modification methods can be roughlydivided into two categories, with the first involving the use of a genetic modification system exploiting the expandedgenetic code, and the second being based on the labeling of expressed proteins (post-translational protein modification).The first technique was pioneered by Peter G. Schultz, andinvolves the direct introduction of a non-natural amino acidpossessing a fluorophore or other functionalities into a targetprotein scaffold at a desirable position. [3–6] The application of this technique, however, can some-times be frustrating because of thelimited functionality of the probes andthe restricted number of usable celllines, as well as the insufficient yield of the genetically engineered proteins. In contrast, the techniqueof post-translational protein modification is better suited tofully engage the variety of different functionalities present insynthetic molecules. This method of protein modification,which is driven predominantly by chemistry, allows for theflexible and efficient modification of proteins at any appro-priate time and position in the protein sequence. The proteinsthemselves can be modified with a variety of functionalmolecules, including luminescent dyes, spin-active probes,NMR-active probes, photo-responsive (caged) molecules,polymers, provided the issues mentioned above can beovercome. The main focus of the current Review is post-translational protein modification, and a description of recentprogress in this research area toward expanding the utility of these chemical tools for fundamental biological research andapplications has been provided (Scheme 1). T  he modification of proteins with synthetic probes is a powerful means of elucidating and engineering the functions of proteins bothin vitro and in live cells or in vivo. Herein we review recent progress inchemistry-based protein modification methods and their application in protein engineering, with particular emphasis on the following four  strategies: 1) the bioconjugation reactions of amino acids on the surfaces of natural proteins, mainly applied in test-tube settings; 2) thebioorthogonal reactions of proteins with non-natural functional  groups; 3) the coupling of recognition and reactive sites using anenzyme or short peptide tag–probe pair for labeling natural aminoacids; and 4) ligand-directed labeling chemistries for the selectivelabeling of endogenous proteins in living systems. Overall, thesetechniques represent a useful set of tools for application in chemical biology, with the methods 2–4 in particular being applicable to crude(living) habitats. Although still in its infancy, the use of organicchemistry for the manipulation of endogenous proteins, with subse-quent applications in living systems, represents a worthy challenge for many chemists. From the Contents 1.  Introduction  4089  2.  Bioconjugation of Natural  Amino Acids for ProteinModification and Engineering   4089   3.  Specific Protein Labeling inCells and In Vivo  4093  4.  Selective Endogenous ProteinLabeling in Live-Cell Systems  4098   5.  Summary and Outlook   4102 [*] Dr. Y. Takaoka, Prof. Dr. I. HamachiDepartment of Synthetic Chemistry and Biological ChemistryKyoto UniversityKatsura, Nishikyo-Ku, Kyoto 615-8510 (Japan)E-mail: ihamachi@sbchem.kyoto-u.ac.jpProf. Dr. A. OjidaGraduate School of Pharmaceutical Sciences, Kyushu University3-1-1 Maidashi, Higashi-Ku, Fukuoka 812-8582 (Japan)Prof. Dr. I. HamachiJapan Science and Technology Agency (JST), CREST5 Sanbancho, Chiyoda-ku, Tokyo 102-0075 (Japan) Protein Modification  Angewandte Chemie 4089  Angew. Chem. Int. Ed.  2013 ,  52 , 4088–4106  2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim  2.  Bioconjugation of Natural Amino Acids for Protein Modification and Engineering  2.1.  Bioconjugation Reactions of Natural Amino Acids ona Protein Surface 2.1.1.  Classical Bioconjugation Methods for Proteins From the chemical perspective, all proteins can beregarded as folded polymers which have many nucleophilicfunctional groups on their surfaces. Of the various naturallyoccurring nucleophilic amino acid residues present in pro-teins, the thiol group of cysteine is often used for the site-specific modification of proteins because of its highernucleophilicity and relatively low level of occurrence on thesurfaces of many proteins (only 2.3% genome-wide). If a reactive cysteine does not exist on the protein surface thenonecan be introduced by agenetic pointmutation, whichis aneffective technique for site-specific protein modification. Thethiol group of cysteine can readily undergo alkylation byreaction with  a -haloketones or Michael acceptors, such asmaleimide derivatives (Scheme 2a). Although considerablymore prevalent than cysteine, the  e -amino group of lysine isa popular target for protein modification because of theabundance of methods available for the selective modifica-tion of primary amines. [7] Lysine can react with activatedesters, sulfonyl chlorides, isocyanates, and isothiocyanates toafford the corresponding amides, sulfonamides, ureas, andthioureas, respectively (Scheme 2b). It is worthy of note thatthese reagents can also modify the N termini of proteins. 2.1.2.  New Methods for the Modification of Lysine and CysteineResidues Several new methods for the selective modification of amines and thiols have recently been developed and opti-mized. For example, Francis and McFarland reported a lysine-specific reductive alkylation reaction that proceeded by aniridium-catalyzed transfer hydrogenation (Scheme 3a). [8] Incontrast to the classical technique of reductive alkylationusing sodium cyanoborohydride under acidic conditions, theiridium-mediated process provided high yields of the desiredproducts under neutral pH conditions. Franciss group alsoreported a unique biomimetic transamination of the N termi-nus of a protein. This method involved the condensation of the N-terminal amine of the protein with pyridoxal-5-phosphate, followed by hydrolysis to provide the correspond-ing pyruvamide (Scheme 3b). [9] The resulting protein wasthen further modified by the reaction of the newly generatedketone moiety of the pyruvamide with aminooxy reagents.Fukase et al. reported a new lysine-based labeling method forproteins on the basis of a 6 p -aza-electrocyclization (Sche-me 3c). [10] Using this techniques, a short-lived PET (positronemission tomography) probes can be incorporated into thetarget proteins or hormones and utilized for the in vivoimaging of glycans and glycoconjugates because this reactionis extremely rapid (within 30 min under neutral pH condi-tions). [10,11] Davis et al. recently developed a two-step cysteinemodification method (Scheme 3d), with the first step involv-ing the transformation of cysteine to dehydroalanine bytreatment with  O -mesitylenesulfonylhydroxylamine. Thedehydroalanine residues were then used as “reactive handles”for further modifications, such as a Michael addition withthiol reagents [12] or olefin cross-metathesis reactions with allylsulfides catalyzed by the Hoveyda–Grubbs ruthenium cata-lyst. [13] 2.1.3.  Bioconjugation for Tyrosine and Tryptophane In contrast to Cys and Lys, the remaining canonical18 amino acid residues have been only minimally explored astools for selective modification. Novel methods have beendeveloped for the modification of tyrosine and tryptophaneinvolving transition-metal-mediated processes. In their pio-neering work, Kodadek et al. developed a method for theoxidative coupling of the phenolic groups of two Tyr residuesfor cross-linking two proteins using a ruthenium(II) catalystand a co-oxidant. [14,15] Francis and co-workers explored themodification of tyrosine residues through a three-componentMannich reaction with aldehydes and anilines (Sche-me 4a). [16,17] They also succeeded in modifying Tyr residues Dr. Yousuke Takaoka was born in 1982 inShimane, Japan. He received his PhD (withProf. I. Hamachi) from Kyoto University in2010 and carried out post-doctoral research(Prof. K. Hirose) at the University of Tokyo.He joined the faculty of Kyoto University asan assistant professor in 2011. His researchinterests include supramolecular chemistry,protein engineering, neurobiology, and chemical biology.Prof. Akio Ojida was born in 1968 inFukuoka, Japan. He received his PhD (withProf. K. Kanemitsu) from Kyushu Universityin 1995 and carried out post-doctoral research (Prof. M. Shionoya) at the Institute for Molecular Science. He worked for Takeda Chemical Industries (1997–2001)then became an assistant professor atKyushu University. In 2003 he became anassistant professor at Kyoto University, and a lecturer in 2007. In 2010 he was madea full professor as Kyushu University. Hisinterests focus on chemical biology, especiallyon design of small molecular probes for elucidating biological functions.   Prof. Itaru Hamachi was born in 1960 inFukuoka, Japan. He received his PhD (Prof.I. Tabushi) from Kyoto University in 1988.He joined the faculty of Kyushu University asan assistant professor in 1988, and becamean associate professor at Kyushu Universityin 1992. In 2001, he became a full professor at Kyushu University and then moved toKyoto University in 2005. He is now CREST investigator of JSTand a RSC fellow. Hisresearch interests include biomolecular chemistry, nanobiochemistry and engineer-ing, supramolecular chemistry, and chemical biology.    Angewandte Reviews  I. Hamachi et al. 4090  www.angewandte.org   2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim  Angew. Chem. Int. Ed.  2013 ,  52 , 4088–4106  using  p -allyl palladium complexes (Scheme 4b). [18] Franciset al. reported an interesting bioconjugation reaction fortryptophane, which is the rarest amino acid in proteins,involving a rhodium carbenoid that was generated in situfrom rhodium acetate and a diazo compound (Sche-me 4c). [19,20] 2.2.  Protein Engineering Using the Bioconjugation Method  The majority of the methods of bioconjugation for proteinengineering are conducted in test-tube settings, which pro-vides a variety of different opportunities for manipulating,improving, or mimicking the performances of proteins. Themodification of proteins with polyethylene glycol (PEG)groups, otherwise known as PEGylation, is one of the mostrepresentative applications in protein bioconjugation. [21,22] Scheme 1.  Schematic illustration of the chemical reactions used for specific endogenously expressed protein modifications in miscellaneousconditions including various reactive species (such as proteins, sugars, and peptides). Protein Modification  Angewandte Chemie 4091  Angew. Chem. Int. Ed.  2013 ,  52 , 4088–4106  2013 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim  www.angewandte.org

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