Total Chemical Synthesis of Di‐ubiquitin Chains

Kumar, K. S. Ajish; Spasser, Liat; Erlich, Lesly A.; Bavikar, Sudhir N.; Brik, Ashraf

doi:10.1002/ange.201003763

Cited by 59 publications

(39 citation statements)

References 35 publications

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“…Moreover, this concept has been extended to include sugar-assisted ligation [30,31] for glycopeptide synthesis, and lysine-mediated peptide and protein ubiquitination. [27,32,33] Results and Discussion…”

Section: Introductionmentioning

confidence: 98%

Chemical Synthesis and Expression of the HIV‐1 Rev Protein

et al. 2011

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The HIV‐1 Rev protein is responsible for shuttling partially spliced and unspliced viral mRNA out of the nucleus. This is a crucial step in the HIV‐1 lifecycle, thus making Rev an attractive target for the design of anti‐HIV drugs. Despite its importance, there is a lack of structural, biophysical, and quantitative information about Rev. This is mainly because of its tendency to undergo self‐assembly and aggregation; this makes it very difficult to express and handle. To address this knowledge gap, we have developed two new highly efficient and reproducible methods to prepare Rev in large quantities for biochemical and structural studies: 1) Chemical synthesis by using native chemical ligation coupled with desulfurization. Notably, we have optimized our synthesis to allow for a one‐pot approach for the ligation and desulfurization steps; this reduced the number of purification steps and enabled the obtaining of desired protein in excellent yield. Several challenges emerged during the design of this Rev synthesis, such as racemization, reduced solubility, formylation during thioester synthesis, and the necessity for using orthogonal protection during desulfurization; solutions to these problems were found. 2) A new method for expression and purification by using a vector that contained an HLT tag, followed by purification with a Ni column, a cation exchange column, and gel filtration. Both methods yielded highly pure and folded Rev. The CD spectra of the synthetic and recombinant Rev proteins were identical, and consistent with a predominantly helical structure. These advances should facilitate future studies that aim at a better understanding of the structure and function of the protein.

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Section: Introductionmentioning

confidence: 98%

Chemical Synthesis and Expression of the HIV‐1 Rev Protein

et al. 2011

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“…The use of δ-mercaptolysine developed by Kumar et al enabled us for the first time the facile site-specific conjugation of ubiquitin into the desired lysine residues of α-syn and other proteins. 93,141 N-terminal ubiquitinated α-syn was prepared by the ligation of two fragments under NCL conditions: a recombinantly expressed α-syn fragment comprising residues 19-140 and bearing an N-terminal Cys α-syn(19-140) and a synthetic peptide thioester comprising the N-terminal residues 1-18 and bearing a protected δ-mercaptolysine (Fig. 9b, bottom panel) at the desired lysine residue, α-syn(1-18)-SR. Ubiquitination was then achieved by addition of the T7-Ub-SR to the full-length α-syn(1-140) presenting the free δ-mercaptolysine to generate the first monoubiquitinated α-syn variant at K6, with a native isopeptide bond.…”

Section: Strategies For the Study Of Ptms At The N-terminus Of α-Synmentioning

confidence: 99%

“…This approach could also be extended to study the effect of phosphorylation, ubiquitination, or sumoylation on exon 1 of the Huntingtin protein or cross-talk between these modifications, all of which occur within the first 16 N-terminal residues of exon 1. linkages. 93,141,145 Through protein semisynthesis, segmental isotopic labeling of one monomer in K48-and K33-linked di-ubiquitin chains was demonstrated, allowing the study of monomer-specific structural and conformational features by nuclear magnetic resonance (NMR). 146 Extending these advances to amyloid proteins will permit the investigation of the effect of the ubiquitin chain length and types of interchain linkages on the function, aggregation, and clearance of these proteins.…”

Section: Strategies For the Study Of Ptms At The N-terminus Of α-Synmentioning

confidence: 99%

Chemical Strategies for Controlling Protein Folding and Elucidating the Molecular Mechanisms of Amyloid Formation and Toxicity

Butterfield

Hejjaoui

Fauvet

et al. 2012

Journal of Molecular Biology

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It has been more than a century since the first evidence linking the process of amyloid formation to the pathogenesis of Alzheimer's disease. During the last three decades in particular, increasing evidence from various sources (pathology, genetics, cell culture studies, biochemistry, and biophysics) continues to point to a central role for the pathogenesis of several incurable neurodegenerative and systemic diseases. This is in part driven by our improved understanding of the molecular mechanisms of protein misfolding and aggregation and the structural properties of the different aggregates in the amyloid pathway and the emergence of new tools and experimental approaches that permit better characterization of amyloid formation in vivo. Despite these advances, detailed mechanistic understanding of protein aggregation and amyloid formation in vitro and in vivo presents several challenges that remain to be addressed and several fundamental questions about the molecular and structural determinants of amyloid formation and toxicity and the mechanisms of amyloid-induced toxicity remain unanswered. To address this knowledge gap and technical challenges, there is a critical need for developing novel tools and experimental approaches that will not only permit the detection and monitoring of molecular events that underlie this process but also allow for the manipulation of these events in a spatial and temporal fashion both in and out of the cell. This review is primarily dedicated in highlighting recent results that illustrate how advances in chemistry and chemical biology have been and can be used to address some of the questions and technical challenges mentioned above. We believe that combining recent advances in the development of new fluorescent probes, imaging tools that enabled the visualization and tracking of molecular events with advances in organic synthesis, and *Corresponding author. E-mail address: hilal.lashuel@epfl.ch.† M.H. and B.F. contributed equally to this work. Abbreviations used: Aβ, amyloid-β; IAPP, islet amyloid polypeptide; ThT, thioflavin T; TFA, trifluoroacetic acid; TEM, transmission electron microscopy; DMDA, N,N-dimethylethylenediamine; PEG, polyethylene glycol; CNB, α-carboxyl-2-nitrobenzyl; LMW, low molecular weight; AD, Alzheimer's disease; PICUP, photoinduced protein cross-linking of unmodified proteins; tfmd, trifluoromethyldiazirine; SPPS, solid-phase peptide synthesis; PTM, posttranslational modification; LB, Lewy body; NCL, native chemical ligation; EPL, expressed protein ligation; TEV, tobacco etch virus; α-syn, α-synuclein; PD, Parkinson's disease; GPI, glycosylphosphatidylinositol; DOPC, dioleoyl-sn-glycero-3-phosphocholine; SUV, small unilamellar vesicle; CPP, cell-penetrating peptide. novel approaches for protein synthesis and engineering provide unique opportunities to gain a molecular-level understanding of the process of amyloid formation. We hope that this review will stimulate further research in this area and catalyze increased collaboration at the interface of chemistry...

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“…Notably, our group reported [8] the first total chemical synthesis of all seven variants of isopeptide-bond linked di-Ub (Ub 2 ) chains in milligram quantities, as well as K48-linked tetra-Ub, [9] thus paving the way for various biochemical and structural studies, opening new, previously unavailable opportunities to characterize the properties of these chains.…”

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confidence: 99%

Segmental Isotopic Labeling of Ubiquitin Chains To Unravel Monomer‐Specific Molecular Behavior

et al. 2011

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Nimm zwei: Durch Kombination von chemischer Synthese eines Ubiquitinmonomers mit rekombinanter Expression seines 15N‐Gegenstücks gelang die Markierung von Ubiquitinen in nichtenzymatisch aufgebauten Diubiquitinketten, was monomerspezifische Studien in Lösung ermöglichte. So konnten für Lys33‐verknüpftes Diubiquitin erstmals die Struktur, Dynamik, Ligandenbindung und Wechselwirkungen innerhalb der Kette charakterisiert werden.

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Total Chemical Synthesis of Di‐ubiquitin Chains

Cited by 59 publications

References 35 publications

Chemical Synthesis and Expression of the HIV‐1 Rev Protein

Chemical Synthesis and Expression of the HIV‐1 Rev Protein

Chemical Strategies for Controlling Protein Folding and Elucidating the Molecular Mechanisms of Amyloid Formation and Toxicity

Segmental Isotopic Labeling of Ubiquitin Chains To Unravel Monomer‐Specific Molecular Behavior

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