Renliang Yang

Dual native chemical ligation at lysine.

A thiol group introduced on the gamma-carbon of lysine mediates robust native chemical ligation at both the alpha- and epsilon-amines in two consecutive steps. Desulfurization then affords the final product, in which the lysine residue at the ligation site has an isopeptide bond on its side chain. The method is useful for the synthesis of proteins containing special post-translational modifications on lysine.

The effects of histone H4 tail acetylations on cation-induced chromatin folding and self-association

Understanding the molecular mechanisms behind regulation of chromatin folding through covalent modifications of the histone N-terminal tails is hampered by a lack of accessible chromatin containing precisely modified histones. We study the internal folding and intermolecular self-association of a chromatin system consisting of saturated 12-mer nucleosome arrays containing various combinations of completely acetylated lysines at positions 5, 8, 12 and 16 of histone H4, induced by the cations Na+, K+, Mg2+, Ca2+, cobalt-hexammine3+, spermidine3+ and spermine4+. Histones were prepared using a novel semi-synthetic approach with native chemical ligation. Acetylation of H4-K16, but not its glutamine mutation, drastically reduces cation-induced folding of the array. Neither acetylations nor mutations of all the sites K5, K8 and K12 can induce a similar degree of array unfolding. The ubiquitous K+, (as well as Rb+ and Cs+) showed an unfolding effect on unmodified arrays almost similar to that of H4-K16 acetylation. We propose that K+ (and Rb+/Cs+) binding to a site on the H2B histone (R96-L99) disrupts H4K16 ε-amino group binding to this specific site, thereby deranging H4 tail-mediated nucleosome–nucleosome stacking and that a similar mechanism operates in the case of H4-K16 acetylation. Inter-array self-association follows electrostatic behavior and is largely insensitive to the position or nature of the H4 tail charge modification.

Influence of Histone Tails and H4 Tail Acetylations on Nucleosome–Nucleosome Interactions

Nucleosome-nucleosome interaction plays a fundamental role in chromatin folding and self-association. The cation-induced condensation of nucleosome core particles (NCPs) displays properties similar to those of chromatin fibers, with important contributions from the N-terminal histone tails. We study the self-association induced by addition of cations [Mg(2+), Ca(2+), cobalt(III)hexammine(3+), spermidine(3+) and spermine(4)(+)] for NCPs reconstituted with wild-type unmodified histones and with globular tailless histones and for NCPs with the H4 histone tail having lysine (K) acetylations or lysine-to-glutamine mutations at positions K5, K8, K12 and K16. In addition, the histone construct with the single H4K16 acetylation was investigated. Acetylated histones were prepared by a semisynthetic native chemical ligation method. The aggregation behavior of NCPs shows a general cation-dependent behavior similar to that of the self-association of nucleosome arrays. Unlike nucleosome array self-association, NCP aggregation is sensitive to position and nature of the H4 tail modification. The tetra-acetylation in the H4 tail significantly weakens the nucleosome-nucleosome interaction, while the H4 K→Q tetra-mutation displays a more modest effect. The single H4K16 acetylation also weakens the self-association of NCPs, which reflects the specific role of H4K16 in the nucleosome-nucleosome stacking. Tailless NCPs can aggregate in the presence of oligocations, which indicates that attraction also occurs by tail-independent nucleosome-nucleosome stacking and DNA-DNA attraction in the presence of cations. The experimental data were compared with the results of coarse-grained computer modeling for NCP solutions with explicit presence of mobile ions.

Synthesis of 4-mercapto-L-lysine derivatives: potential building blocks for sequential native chemical ligation.

A general and diastereoselective synthesis of (2S, 4S)-4-mercapto-L-lysine derivative was described. The key features of this synthesis include Zn-mediated diastereoselective Reformatsky reaction and selective reduction of methyl ester with sodium borohydride. Introduction of thiol functional group on lysine side chain proved to be appropriate for dual native chemical ligation. This methodology allows to develop various 4-substituted L-lysine derivatives.

N-to-C sequential ligation using peptidyl N,N-bis(2-mercaptoethyl)amide building blocks.

A novel N- to C-terminus sequential chemical ligation approach has been developed for protein synthesis. Key to this strategy is the relative stability of the N,N-bis(2-mercaptoethyl)amide (BMEA) to the conventional conditions of native chemical ligation. We have also found a new thiol additive for the BMEA-mediated ligation reaction. The usefulness of this approach was demonstrated in the syntheses of a medium-sized peptide and ubiquitin.

A Simple Method for Preparing Peptide C-Terminal Thioacids and Their Application in Sequential Chemoenzymatic Ligation

This chemistry requires a special thioester benzhydryl linkerthat is not commercially available, and the use of hydrogenfluorideforfinaldeprotectionandcleavage.Theyieldisusuallynot high because, being a supernucleophile, the thioacidgroup is susceptible to nucleophilic attacks by the cationicelectrophiles that are released during the final cleavage.Herein, we report a simple and efficient method to producepeptideC-terminal thioacids through hydrothiolysisofthioest-ers (Scheme1). More importantly, because a thioacid groupmight not be recognized and hydrolyzed by subtiligase, itwould be orthogonal to the subtiligase-catalyzed ligationchemistry.

Native chemical ubiquitination using a genetically incorporated azidonorleucine

A robust chemical ubiquitination method was developed. The method employed a genetically incorporated azidonorleucine as an orthogonal lysine precursor for the installation of a Gly residue bearing an Nα-auxiliary which mediated the ligation between ubiquitin(1-75)-thioester and the target protein. To demonstrate our methodology, a model protein, K48-linked diubiquitin, was synthesized with an overall yield of 35%.

ISWI Remodelling of Physiological Chromatin Fibres Acetylated at Lysine 16 of Histone H4

ISWI is the catalytic subunit of several ATP-dependent chromatin remodelling factors that catalyse the sliding of nucleosomes along DNA and thereby endow chromatin with structural flexibility. Full activity of ISWI requires residues of a basic patch of amino acids in the N-terminal ‘tail’ of histone H4. Previous studies employing oligopeptides and mononucleosomes suggested that acetylation of the H4 tail at lysine 16 (H4K16) within the basic patch may inhibit the activity of ISWI. On the other hand, the acetylation of H4K16 is known to decompact chromatin fibres. Conceivably, decompaction may enhance the accessibility of nucleosomal DNA and the H4 tail for ISWI interactions. Such an effect can only be evaluated at the level of nucleosome arrays. We probed the influence of H4K16 acetylation on the ATPase and nucleosome sliding activity of Drosophila ISWI in the context of defined, in vitro reconstituted chromatin fibres with physiological nucleosome spacing and linker histone content. Contrary to widespread expectations, the acetylation did not inhibit ISWI activity, but rather stimulated ISWI remodelling under certain conditions. Therefore, the effect of H4K16 acetylation on ISWI remodelling depends on the precise nature of the substrate.

Chemical Methods for Protein Ubiquitination

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.

Regulation of Nucleosome Stacking and Chromatin Compaction by the Histone H4 N-Terminal Tail–H2A Acidic Patch Interaction

Chromatin folding and dynamics are critically dependent on nucleosome-nucleosome interactions with important contributions from internucleosome binding of the histone H4 N-terminal tail K16-R23 domain to the surface of the H2A/H2B dimer. The H4 Lys16 plays a pivotal role in this regard. Using in vitro reconstituted 12-mer nucleosome arrays, we have investigated the mechanism of the H4 N-terminal tail in maintaining nucleosome-nucleosome stacking and mediating intra- and inter-array chromatin compaction, with emphasis on the role of K16 and the positive charge region, R17-R23. Analytical ultracentrifugation sedimentation velocity experiments and precipitation assays were employed to analyze effects on chromatin folding and self-association, respectively. Effects on chromatin folding caused by various mutations and modifications at position K16 in the H4 histone were studied. Additionally, using charge-quenching mutations, we characterized the importance of the interaction of the residues within the H4 positive charge region R17-R23 with the H2A acidic patch of the adjacent nucleosome. Furthermore, crosslinking experiments were conducted to establish the proximity of the basic tail region to the acidic patch. Our data indicate that the positive charge and length of the side chain of H4 K16 are important for its access to the adjacent nucleosome in the process of nucleosome-nucleosome stacking and array folding. The location and orientation of the H4 R17-R23 domain on the H2A/H2B dimer surface of the neighboring nucleosome core particle (NCP) in the compacted chromatin fiber were established. The dominance of electrostatic interactions in maintaining intra-array interaction was demonstrated.