Structural Basis for the Site-Specific Incorporation of Lysine Derivatives into Proteins

Posttranslational modifications (PTMs) of proteins determine their structure-function relationships, interaction partners, as well as their fate in the cell and are crucial for many cellular key processes. For instance chromatin structure and hence gene expression is epigenetically regulated by acetylation or methylation of lysine residues in histones, a phenomenon known as the 'histone code'. Recently it was shown that these lysine residues can furthermore be malonylated, succinylated, butyrylated, propionylated and crotonylated, resulting in significant alteration of gene expression patterns. However the functional implications of these PTMs, which only differ marginally in their chemical structure, is not yet understood. Therefore generation of proteins containing these modified amino acids site specifically is an important tool. In the last decade methods for the translational incorporation of non-natural amino acids using orthogonal aminoacyl-tRNA synthetase (aaRS):tRNAaaCUA pairs were developed. A number of studies show that aaRS can be evolved to use non-natural amino acids and expand the genetic code. Nevertheless the wild type pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei readily accepts a number of lysine derivatives as substrates. This enzyme can further be engineered by mutagenesis to utilize a range of non-natural amino acids. Here we present structural data on the wild type enzyme in complex with adenylated epsilon-N-alkynyl-,epsilon-N-butyryl-,epsilon-N-crotonyl- and epsilon-N-propionyl-lysine providing insights into the plasticity of the PylRS active site. This shows that given certain key features in the non-natural amino acid to be incorporated, directed evolution of this enzyme is not necessary for substrate tolerance

Synthesis of epsilon-N-propionyl-, epsilon-N-butyryl-, and epsilon-N-crotonyl-lysine containing histone H3 using the pyrrolysine system

Recently new lysine modifications were detected in histones and other proteins. Using the pyrrolysine amber suppression system we genetically inserted three of the newamino acids epsilon-N-propionyl-, epsilon-N-butyryl-, and epsilon-N-crotonyl-lysine site specifically into histone H3. The lysine at position 9 (H3 K9), which is known to be highly modified in chromatin, was replaced by these unnatural amino acids

Sulfonyl azide-mediated norbornene aziridination for orthogonal peptide and protein labeling

We describe a new bioconjugation reaction based on the aziridination of norbornenes using electron-deficient sulfonyl azides. The reaction enables to attach various useful tags to peptides and proteins under mild conditions

Synthesis of C3-Substituted N1-tert-Butyl 1,2,4-Triazinium Salts via the Liebeskind–Srogl Reaction for Fluorogenic Labeling of Live Cells

We recently described the development and application of new bioorthogonal conjugation, the triazinium ligation. To explore the wider application of this reaction, in this work we introduce a general method for synthesizing C3-substituted triazinium salts based on the Liebeskind–Srogl cross-coupling reaction and catalytic thioether reduction. These methods enabled the synthesis of triazinium derivatives for investigating the effect of different substituents on the ligation kinetics and stability of the compounds under biologically relevant conditions. Finally, we demonstrate that the combination of coumarin fluorophore attached to position C3 with a C5-(4-methoxyphenyl) substituent, yields a fluorogenic triazinium probes suitable for no-wash, live-cell labeling. The developed methodology represents a promising synthetic approach to the late-stage modification of triazinium salts, potentially widening their application in bioorthogonal reactions

Structural basis for the site-specific incorporation of lysine derivatives into proteins.

Posttranslational modifications (PTMs) of proteins determine their structure-function relationships, interaction partners, as well as their fate in the cell and are crucial for many cellular key processes. For instance chromatin structure and hence gene expression is epigenetically regulated by acetylation or methylation of lysine residues in histones, a phenomenon known as the 'histone code'. Recently it was shown that these lysine residues can furthermore be malonylated, succinylated, butyrylated, propionylated and crotonylated, resulting in significant alteration of gene expression patterns. However the functional implications of these PTMs, which only differ marginally in their chemical structure, is not yet understood. Therefore generation of proteins containing these modified amino acids site specifically is an important tool. In the last decade methods for the translational incorporation of non-natural amino acids using orthogonal aminoacyl-tRNA synthetase (aaRS):tRNAaaCUA pairs were developed. A number of studies show that aaRS can be evolved to use non-natural amino acids and expand the genetic code. Nevertheless the wild type pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei readily accepts a number of lysine derivatives as substrates. This enzyme can further be engineered by mutagenesis to utilize a range of non-natural amino acids. Here we present structural data on the wild type enzyme in complex with adenylated ε-N-alkynyl-, ε-N-butyryl-, ε-N-crotonyl- and ε-N-propionyl-lysine providing insights into the plasticity of the PylRS active site. This shows that given certain key features in the non-natural amino acid to be incorporated, directed evolution of this enzyme is not necessary for substrate tolerance

An Optimized Protocol for the Synthesis of Peptides Containing trans-Cyclooctene and Bicyclononyne Dienophiles as Useful Multifunctional Bioorthogonal Probes

Despite the great advances in solid-phase peptide synthesis (SPPS), the incorporation of certain functional groups into peptide sequences is restricted by the compatibility of the building blocks with conditions used during SPPS. In particular, the introduction of highly reactive groups used in modern bioorthogonal reactions into peptides remains elusive. Here, we present an optimized synthetic protocol enabling installation of two strained dienophiles, trans-cyclooctene (TCO) and bicyclononyne (BCN), into different peptide sequences. The two groups enable fast and modular post-synthetic functionalization of peptides, as we demonstrate in preparation of peptide-peptide and peptide-drug conjugates. Due to the excellent biocompatibility, the click-functionalization of the peptides can be performed directly in live cells. We further show that the introduction of both clickable groups into peptides enables construction of smart, multifunctional probes that can streamline complex chemical biology experiments such as visualization and pull-down of metabolically labeled glycoconjugates. The presented strategy will find utility in construction of peptides for diverse applications, where high reactivity, efficiency and biocompatibility of the modification step is critical.Fil: la Venia, Agustina. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Rosario. Instituto de Química Rosario. Universidad Nacional de Rosario. Facultad de Ciencias Bioquímicas y Farmacéuticas. Instituto de Química Rosario; Argentina. Czech Academy of Sciences. Institute of Organic Chemistry and Biochemistry; República ChecaFil: Dzijak, Rastislav. Czech Academy of Sciences. Institute of Organic Chemistry and Biochemistry; República ChecaFil: Rampmaier, Robert. Czech Academy of Sciences. Institute of Organic Chemistry and Biochemistry; República ChecaFil: Vrabel, Milan. Czech Academy of Sciences. Institute of Organic Chemistry and Biochemistry; República Chec

Comparison of the binding pockets of PylRS and LysRS.

<p>Two-dimensional plot <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0096198#pone.0096198-Wallace1" target="_blank">[52]</a> of residues interacting with Lys (A), Pyl (B) and Kalk (C). Van-der-Waals contacts are shown as red half-spheres. Surface representation of the binding pockets of LysRS (D) and PylRS (E) with Lys, Pyl and Kalk, respectively. (F) Superposition of Pyl (grey), Kalk (orange), Kbu (green), Kcr (blue) and Kpr (red). The surface of the binding pocket is shown as mesh, with the charge distribution indicated by coloring (red  =  negative, blue  =  positive). The surrounding residues are drawn as sticks. (PDB codes PylRS: 2Q7H and LysRS: 3A74).</p

Lysine, pyrrolysine and analogs used in this study.

<p>Lysine, pyrrolysine and analogs used in this study.</p