Lysine acetylation and its regulatory enzymes are known to have pivotal roles in mammalian cellular physiology. However, the extent and function of this modification in prokaryotic cells remain largely unexplored, thereby presenting a hurdle to further functional study of this modification in prokaryotic systems. Here we report the first global screening of lysine acetylation, identifying 138 modification sites in 91 proteins from Escherichia coli. None of the proteins has been previously associated with this modification. Among the identified proteins are transcriptional regulators, as well as others with diverse functions. Interestingly, more than 70% of the acetylated proteins are metabolic enzymes and translation regulators, suggesting an intimate link of this modification to energy metabolism. The new dataset suggests that lysine acetylation could be abundant in prokaryotic cells. In addition, these results also imply that functions of lysine acetylation beyond regulation of gene expression are evolutionarily conserved from bacteria to mammals. Furthermore, we demonstrate that bacterial lysine acetylation is regulated in response to stress stimuli.

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 ⁺ , Mg ²⁺ , Ca ²⁺ , cobalt-hexammine ³⁺ , spermidine ³⁺ and spermine ⁴⁺ . 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.

We describe an approach to the synthesis of peptides from segments bearing no protecting groups through an orthogonal coupling method to capture the acyl segment as a thioester that then undergoes an intramolecular acyl transfer to the amine component with formation of a peptide bond. Two orthogonal coupling methods to give the covalent ester intermediate were achieved by either a thiol-thioester exchange mediated by a trialkylphosphine and an alkylthiol or a thioesterification by C alpha-thiocarboxylic acid reacting with a beta-bromo amino acid. With this approach, unprotected segments ranging from 4 to 37 residues were coupled to aqueous solution to give free peptides up to 54 residues long with high efficiency.

We describe the concept and the verification of a chemical ligation approach to the synthesis of proteins using peptide segments with no protecting groups and no activation of the C-terminal alpha-carboxyl group. This approach consists of three steps: (i) aldehyde introduction, in which a masked glycolaldehyde ester is linked to the carboxyl terminus of an unprotected peptide by reverse proteolysis; (ii) ring formation, in which the unmasked aldehyde reacts with the N-terminal alpha-amino group of the second unprotected peptide containing either a cysteine or a threonine residue to form a thiazolidine or oxazolidine ring at an acidic pH; and (iii) rearrangement in which O-acyl ester linkage is transferred to N-acyl amide linkage to form a peptide bond with a pseudoproline structure at higher pH. The feasibility of this scheme was verified by a model study on small compounds and its potential was demonstrated by the synthesis of a 50-residue epidermal growth factor-like peptide containing a preformed disulfide bond.