Methods and strategies of peptide ligation

Tam, James P.; Xu, Jiaxi; Eom, Khee Dong

doi:10.1002/1097-0282(2001)60:3<194::aid-bip10031>3.0.co;2-8

Cited by 192 publications

(152 citation statements)

References 217 publications

(246 reference statements)

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“…4). Because the K9 site of cH3 (13) was effectively blocked by the acetyl group introduced during SPPS, no transfer of [ 3 H]methyls was observed, as was anticipated (Fig. 4B).…”

Section: Folded Synthetic Histones As Substrates For Histone Modificasupporting

confidence: 55%

“…NCL is a methodology by which two unprotected peptides, bearing a reactive group, either a C-terminal thioester or N-terminal cysteine, can be chemoselectively condensed in aqueous solution at neutral pH to form a peptide bond with a Cys residue at the ligation site (13,(22)(23)(24)(25). Because the majority of histone posttranslational modifications are found on residues within the first 30 aa of their N termini, we devised a modular NCL strategy for histone synthesis whereby peptides corresponding to this region were synthesized by SPPS and then ligated to a recombinant protein fragment corresponding to the histone C-terminal globular domain (Fig.…”

Section: Resultsmentioning

confidence: 99%

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Facile synthesis of site-specifically acetylated and methylated histone proteins: Reagents for evaluation of the histone code hypothesis

Bauman

Davis

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

114

109

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The functional capacity of genetically encoded histone proteins can be powerfully expanded by posttranslational modification. A growing body of biochemical and genetic evidence clearly links the unique combinatorial patterning of side chain acetylation, methylation, and phosphorylation mainly within the highly conserved N termini of histones H2A, H2B, H3, and H4 with the regulation of gene expression and chromatin assembly and remodeling, in effect constituting a ''histone code'' for epigenetic signaling. Deconvoluting this code has proved challenging given the inherent posttranslational heterogeneity of histone proteins isolated from biological sources. Here we describe the application of native chemical ligation to the preparation of full-length histone proteins containing site-specific acetylation and methylation modifications. Peptide thioesters corresponding to histone N termini were prepared by solid phase peptide synthesis using an acid labile Boc͞HF assembly strategy, then subsequently ligated to recombinantly produced histone C-terminal globular domains containing an engineered N-terminal cysteine residue. The ligation site is then rendered traceless by hydrogenolytic desulfurization, generating a native histone protein sequence. Synthetic histones generated by this method are fully functional, as evidenced by their self-assembly into a higher order H3͞H4 heterotetramer, their deposition into nucleosomes by human ISWI-containing (Imitation of Switch) factor RSF (Remodeling and Spacing Factor), and by enzymatic modification by human Sirt1 deacetylase and G9a methyltransferase. Site-specifically modified histone proteins generated by this method will prove invaluable as novel reagents for the evaluation of the histone code hypothesis and analysis of epigenetic signaling mechanisms.

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“…4). Because the K9 site of cH3 (13) was effectively blocked by the acetyl group introduced during SPPS, no transfer of [ 3 H]methyls was observed, as was anticipated (Fig. 4B).…”

Section: Folded Synthetic Histones As Substrates For Histone Modificasupporting

confidence: 55%

Section: Resultsmentioning

confidence: 99%

Facile synthesis of site-specifically acetylated and methylated histone proteins: Reagents for evaluation of the histone code hypothesis

Bauman

Davis

et al. 2003

Proc. Natl. Acad. Sci. U.S.A.

114

109

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“…[17] Nevertheless, despite some notable successes, [18][19] such methods have not found widespread use (yet) after the development of the more simple and versatile chemical ligation methods. [1,[20][21][22][23][24] The size of proteins which can be chemically synthesized has been increased Because the mutually reactive groups (or at least one of them) are functionalities not normally found in peptides, the price to be paid for applying such chemoselective ligation is the formation of an unnatural structure at the ligation site. However, in practice, these unnatural structures are often well tolerated within the context of a folded protein and numerous examples of fully active proteins were prepared using these methods.…”

Section: Chemical Protein Synthesismentioning

confidence: 99%

Diels–Alder Ligation of Peptides and Proteins

Araújo

Palomo

Cramer

et al. 2006

Chemistry A European J

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“…Although chemoselective ligation has been widely used for the synthesis of artificial peptides and proteins of linear, cyclic and branched forms, 33,34,43 our results further illustrate its versatility for preparing protein mimetics of quaternarystructured oligomeric proteins such as gp41. The condition for Thz or Trp ligation is extremely mild and is performed in buffered solutions using unprotected peptides and various forms of the functionalized ILs.…”

Section: Chemoselective Ligationmentioning

confidence: 76%

“…Two ligation methods, thiazolidine (Thz) and tryptophan (Trp) ligation, [33][34][35] were used to produce the 3a mimetics (Figure 2). Both methods utilized an aldehyde-functionalized IL and a specific N-terminal (NT) amino acid as nucleophile present in the monomer.…”

Section: Preparation Of Gp41 Quaternary Protein Mimeticsmentioning

confidence: 99%

Quaternary protein mimetics of gp41 elicit neutralizing antibodies against HIV fusion‐active intermediate state

Sadler

Zhang

et al. 2008

Biopolymers

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During viral entry, the fusogenic state of human immunodeficiency virus Type 1 (HIV‐1) envelope protein gp41 is a quaternary structure consisting of three gp41 glycoproteins, each with two conserved helical domains (N‐HR and C‐HR). Thus far, the examination of monomeric gp41 peptides as an immunologically focused approach to vaccine design has not been successful. Here we report an approach using quaternary protein mimetics (called 3α mimetics) that are based on the gp41 N‐HR and C‐HR domains to closely mimic the fusogenic state and overcome the deficiencies of the monomeric peptide approach for synthetic vaccine design. The 3α mimetics are conveniently prepared by chemoselective ligation of unprotected monomeric peptides to an interstrand linker, and display enhanced conformational stability compared to the corresponding monomers. The 3α mimetics with or without a covalently attached T‐helper epitope were immunogenic and elicited antisera that bound both recombinant gp160, which contains gp41, and HIV‐1 virions and immunoprecipitated recombinant gp41. Anti‐3α mimetic antisera neutralized viral infectivity against R5‐ and X4‐tropic strains of HIV‐1 at 31.5°C. The results suggest that a quaternary protein approach to mimic conserved and functional domains of viral envelope proteins is desirable for HIV vaccine development as such antigens are more likely to produce immunologically‐focused and broadly neutralizing antibody responses. © 2008 Wiley Periodicals, Inc. Biopolymers (Pept Sci) 90: 320–329, 2008.This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com

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Methods and strategies of peptide ligation

Cited by 192 publications

References 217 publications

Facile synthesis of site-specifically acetylated and methylated histone proteins: Reagents for evaluation of the histone code hypothesis

Facile synthesis of site-specifically acetylated and methylated histone proteins: Reagents for evaluation of the histone code hypothesis

Diels–Alder Ligation of Peptides and Proteins

Quaternary protein mimetics of gp41 elicit neutralizing antibodies against HIV fusion‐active intermediate state

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