Synthesis of K48-linked diubiquitin using dual native chemical ligation at lysine

Yang, Renliang; Pasunooti, Kalyan Kumar; Li, Fupeng; Liu, Xuewei; Liu, Chuan‐Fa

doi:10.1039/c0cc01382j

Cited by 79 publications

(50 citation statements)

References 19 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: 99%

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: 99%

Chemical Synthesis and Expression of the HIV‐1 Rev Protein

et al. 2011

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“…Our group [17,18] and Brik's group [19][20][21] have independently reported the γ-and δ-thiolysinemediated ubiquitination methods, respectively. In these two approaches, sitespecific ubiquitination was achieved through chemical ligation involving fulllength ubiquitin(1-76) thioester and the ε-amine of a lysine residue in the peptide Scheme 2 General scheme for the synthesis of K120 ubiquitinated human H2B through N-α-auxiliary-mediated ubiquitination Chemical Methods for Protein Ubiquitinationor protein substrate.…”

Section: Thiolated-lysine-mediated Ubiquitinationmentioning

confidence: 99%

Chemical Methods for Protein Ubiquitination

Yang

Liu

2014

Protein Ligation and Total Synthesis I

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In eukaryotic cells, many proteins undergo extensive post-translational modifications (PTMs) such as methylation, acetylation, phosphorylation, glycosylation, and ubiquitination. Among these, ubiquitination is a particularly interesting PTM from both structural and functional viewpoints. In ubiquitination, the C-terminal carboxyl group of the small ubiquitin protein is attached to the ε-amine of a lysine residue of a substrate protein through an isopeptide bond. Ubiquitination has been shown to be involved in the regulation of many cellular processes including protein degradation and gene expression. And dysfunction of these processes is implicated in many human diseases. Despite many years of intensive research, a large number of protein ubquitination events remain poorly characterized. The challenge lies with the tremendous difficulties in isolating homogeneously modified proteins from biological samples for structural and functional studies. Enzymatic ubiquitination in vitro often has limited practical value due to the large number of substrate-specific E3 ligases and the difficulties in identifying or isolating these enzymes. Chemical approaches to the preparation of ubiquitinated proteins provide a powerful solution, and the development of such approaches has been the subject of intense research by many research laboratories. This review summarizes the methodological developments of protein chemical ubiquitination in recent years.

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“…Toward this goal, we and others have recently developed various chemical methods for making Ub-Ub conjugates [10-14] (reviewed in [15] ). Some of the methods use total chemical synthesis combined with native/isopeptide chemical ligation to generate dimers [10] and longer chains of Ub.…”

mentioning

confidence: 99%

“…Some of the methods use total chemical synthesis combined with native/isopeptide chemical ligation to generate dimers [10] and longer chains of Ub. [16] Although a remarkable achievement, the need to use total chemical synthesis has limited their utility in a biochemical laboratory setting.…”

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confidence: 99%

Nonenzymatic Rubylation and Ubiquitination of Proteins for Structural and Functional Studies

2014

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Uncovering the mechanisms that allow conjugates of ubiquitin (Ub) and/or Ub‐like (UBL) proteins such as Rub1 to serve as distinct molecular signals requires the ability to make them with native connectivity and defined length and linkage composition. A novel, effective, and affordable strategy for controlled chemical assembly of fully natural UBL–Ub, Ub–UBL, and UBL–UBL conjugates from recombinant monomers is presented. Rubylation of Ub and Rub1 and ubiquitination of Rub1 was achieved without E2/E3 enzymes. New residue‐specific information was obtained on the interdomain contacts in naturally‐occurring K48‐linked Rub1–Ub and Ub–Rub1, and K29‐linked Rub1–Ub heterodimers, and their recognition by a K48‐linkage‐specific Ub receptor. The disassembly of these heterodimers by major deubiquitinating enzymes was examined and it was discovered that some deubiquitinases also possess derubylase activity. This unexpected result suggests possible crosstalk between Ub and Rub1/Nedd8 signaling pathways.

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Synthesis of K48-linked diubiquitin using dual native chemical ligation at lysine

Abstract: The dual native chemical ligation at lysine strategy was revised by replacing the acid-labile Cbz protecting group with photolabile NVOC at the 4-mercaptolysine side chain. The optimized strategy was subsequently applied to the synthesis of K48-linked diubiquitin.

Cited by 79 publications

References 19 publications

Chemical Synthesis and Expression of the HIV‐1 Rev Protein

Chemical Synthesis and Expression of the HIV‐1 Rev Protein

Chemical Methods for Protein Ubiquitination

Nonenzymatic Rubylation and Ubiquitination of Proteins for Structural and Functional Studies

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