3-Mercaptopropionic acid-mediated synthesis of peptide and protein thioesters†
†Electronic supplementary information (ESI) available: Experimental procedures for the production of 1, all model peptides and proteins and their reactions with MPA including HPLC, LC-MS and NMR characterisation of selected products. See DOI: 10.1039/b815888f

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Abstract

Peptides and proteins fragment in the presence of 3-mercaptopropionic acid to afford thioesters which can be used in native chemical ligation reactions.

Abstract

Peptides and proteins fragment sequence-specifically in the presence of 3-mercaptopropionic acid to afford thioesters which can be used in native chemical ligation reactions.

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Most cited references 13

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Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation.

Robert K McGinty, Jaehoon Kim, Champak Chatterjee … (2008)

Numerous post-translational modifications of histones have been described in organisms ranging from yeast to humans. Growing evidence for dynamic regulation of these modifications, position- and modification-specific protein interactions, and biochemical crosstalk between modifications has strengthened the 'histone code' hypothesis, in which histone modifications are integral to choreographing the expression of the genome. One such modification, ubiquitylation of histone H2B (uH2B) on lysine 120 (K120) in humans, and lysine 123 in yeast, has been correlated with enhanced methylation of lysine 79 (K79) of histone H3 (refs 5-8), by K79-specific methyltransferase Dot1 (KMT4). However, the specific function of uH2B in this crosstalk pathway is not understood. Here we demonstrate, using chemically ubiquitylated H2B, a direct stimulation of hDot1L-mediated intranucleosomal methylation of H3 K79. Two traceless orthogonal expressed protein ligation (EPL) reactions were used to ubiquitylate H2B site-specifically. This strategy, using a photolytic ligation auxiliary and a desulphurization reaction, should be generally applicable to the chemical ubiquitylation of other proteins. Reconstitution of our uH2B into chemically defined nucleosomes, followed by biochemical analysis, revealed that uH2B directly activates methylation of H3 K79 by hDot1L. This effect is mediated through the catalytic domain of hDot1L, most likely through allosteric mechanisms. Furthermore, asymmetric incorporation of uH2B into dinucleosomes showed that the enhancement of methylation was limited to nucleosomes bearing uH2B. This work demonstrates a direct biochemical crosstalk between two modifications on separate histone proteins within a nucleosome.

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Protein synthesis by native chemical ligation: expanded scope by using straightforward methodology.

Tilman M Hackeng, Matt Griffin, Timothy Dawson (1999)

The total chemical synthesis of proteins has great potential for increasing our understanding of the molecular basis of protein function. The introduction of native chemical ligation techniques to join unprotected peptides next to a cysteine residue has greatly facilitated the synthesis of proteins of moderate size. Here, we describe a straightforward methodology that has enabled us to rapidly analyze the compatibility of the native chemical ligation strategy for X-Cys ligation sites, where X is any of the 20 naturally occurring amino acids. The simplified methodology avoids the necessity of specific amino acid thioester linkers or alkylation of C-terminal thioacid peptides. Experiments using matrix-assisted laser-desorption ionization MS analysis of combinatorial ligations of LYRAX-C-terminal thioester peptides to the peptide CRANK show that all 20 amino acids are suitable for ligation, with Val, Ile, and Pro representing less favorable choices because of slow ligation rates. To illustrate the method's utility, two 124-aa proteins were manually synthesized by using a three-step, four-piece ligation to yield a fully active human secretory phospholipase A(2) and a catalytically inactive analog. The combination of flexibility in design with general access because of simplified methodology broadens the applicability and versatility of chemical protein synthesis.

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Substituents on quinone methides strongly modulate formation and stability of their nucleophilic adducts.

E Rokita, Mauro Freccero, Emily E. Weinert … (2006)

Electronic perturbation of quinone methides (QM) greatly influences their stability and in turn alters the kinetics and product profile of QM reaction with deoxynucleosides. Consistent with the electron-deficient nature of this reactive intermediate, electron-donating substituents are stabilizing and electron-withdrawing substituents are destabilizing. For example, a dC N3-QM adduct is made stable over the course of observation (7 days) by the presence of an electron-withdrawing ester group that inhibits QM regeneration. Conversely, a related adduct with an electron-donating methyl group is very labile and regenerates its QM with a half-life of approximately 5 h. The generality of these effects is demonstrated with a series of alternative quinone methide precursors (QMP) containing a variety of substituents attached at different positions with respect to the exocyclic methylene. The rates of nucleophilic addition to substituted QMs measured by laser flash photolysis similarly span 5 orders of magnitude with electron-rich species reacting most slowly and electron-deficient species reacting most quickly. The reversibility of QM reaction can now be predictably adjusted for any desired application.

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Author and article information

Journal

Journal ID (nlm-ta): Chem Commun (Camb)

Title: Chemical Communications (Cambridge, England)

Publisher: Royal Society of Chemistry

ISSN (Print): 1359-7345

ISSN (Electronic): 1364-548X

Publication date (Print): 28 January 2009

Publication date (Electronic): 5 November 2008

Issue: 4

Pages: 407-409

Affiliations

[a ] Department of Chemistry, University College London, 20 Gordon Street, London, UK WC1H 0AJ. Email: d.macmillan@ 123456ucl.ac.uk ; Tel: 020-7679 4684

Article

Publisher ID: b815888f

DOI: 10.1039/b815888f

PMC ID: 2898641

PubMed ID: 19137167

SO-VID: 0d526848-65e6-4719-a93d-793edfdf5afa

License:

This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License ( http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

History

Date received : 10 September 2008

Date accepted : 10 October 2008

Notes

†Electronic supplementary information (ESI) available: Experimental procedures for the production of 1, all model peptides and proteins and their reactions with MPA including HPLC, LC-MS and NMR characterisation of selected products. See DOI: 10.1039/b815888f

3-Mercaptopropionic acid-mediated synthesis of peptide and protein thioesters†

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Most cited references 13

Chemically ubiquitylated histone H2B stimulates hDot1L-mediated intranucleosomal methylation.

Protein synthesis by native chemical ligation: expanded scope by using straightforward methodology.

Substituents on quinone methides strongly modulate formation and stability of their nucleophilic adducts.

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