Basic Residues at Position 11 of α-Conotoxin LvIA Influence Subtype Selectivity between α3β2 and α3β4 Nicotinic Receptors via an Electrostatic Mechanism.

Understanding the determinants of α-conotoxin (α-CTX) selectivity for different nicotinic acetylcholine receptor (nAChR) subtypes is a prerequisite for the design of tool compounds to study nAChRs. However, selectivity optimization of these small, disulfide-rich peptides is difficult not only because of an absence of α-CTX/nAChR co-structures but also because it is challenging to predict how a mutation to an α-CTX will alter its potency and selectivity. As a prototypical system to investigate selectivity, we employed the α-CTX LvIA that is 25-fold selective for the α3β2 nAChR over the related α3β4 nAChR subtype, which is a target for nicotine addiction.

Insight on the Order of Regioselective Ultrafast Formation of Disulfide Bonds in (Antimicrobial) Peptides and Miniproteins.

Disulfide-rich peptides and proteins are among the most fascinating bioactive molecules. The difficulties associated with the preparation of these targets have prompted the development of various chemical strategies. Nevertheless, the production of these targets remains very challenging or elusive.

General synthetic strategy for regioselective ultrafast formation of disulfide bonds in peptides and proteins.

Despite six decades of efforts to synthesize peptides and proteins bearing multiple disulfide bonds, this synthetic challenge remains an unsolved problem in most targets (e.g., knotted mini proteins).

Palladium-Mediated Direct Disulfide Bond Formation in Proteins Containing S-Acetamidomethyl-cysteine under Aqueous Conditions.

One of the applied synthetic strategies for correct disulfide bond formation relies on the use of orthogonal Cys protecting groups. This approach requires purification before and after the deprotection steps, which prolongs the entire synthetic process and lowers the yield of the reaction. A major challenge in using this approach is to be able to apply one-pot synthesis under mild conditions and aqueous media.

Palladium prompted on-demand cysteine chemistry for the synthesis of challenging and uniquely modified proteins.

Organic chemistry allows for the modification and chemical preparation of protein analogues for various studies. The thiolate side chain of the Cys residue has been a key functionality in these ventures. In order to generate complex molecular targets, there is a particular need to incorporate orthogonal protecting groups of the thiolated amino acids to control the directionality of synthesis and modification site.

Chemical chromatin ubiquitylation.

Histone modifications dynamically regulate chromatin structure and function, thereby mediating many processes that require access to DNA. Chemical protein synthesis has emerged as a powerful approach for generating homogeneously modified histone analogues in workable amounts for subsequent incorporation into nucleosome arrays for biochemical, functional and structural studies. This short review focuses on the strength of total chemical protein synthesis and semisynthetic approaches to generate ubiquitylated histones in their native or non-native forms and the utility of these analogues to decode the role of ubiquitylation in epigenetics.

Total chemical synthesis of histones and their analogs, assisted by native chemical ligation and palladium complexes.

Chemical synthesis of histones allows precise control of the installation of post-translational modifications via the coupling of derivatized amino acids. Shortcomings of other approaches for obtaining modified histones for epigenetic studies include heterogeneity of the obtained product and difficulties in incorporating multiple modifications on the same histone. In this protocol, unprotected peptide fragments are prepared by Fmoc solid-phase synthesis and coupled in aqueous buffers via native chemical ligation (NCL; in NCL, a peptide bond is formed between a peptide with an N-terminal Cys and another peptide having a C-terminal thioester).

Palladium-Assisted Removal of a Solubilizing Tag from a Cys Side Chain To Facilitate Peptide and Protein Synthesis.

Reversible attachment of solubilizing tags to hydrophobic peptides to facilitate their purification and ligation is an essential yet challenging task in chemical protein synthesis. The efficient palladium-assisted removal of the solubilizing tag linked to the Cys side chain is reported. The strategy was applied for the efficient preparation of histone protein H4 from two fragments via one-pot operation of ligation, removal of the solubilizing tag, and desulfurization.

Molecular determinants of tetramerization in the KcsA cytoplasmic domain.

The cytoplasmic C-terminal domain (CTD) of KcsA, a bacterial homotetrameric potassium channel, is an amphiphilic domain that forms a helical bundle with four-fold symmetry mediated by hydrophobic and electrostatic interactions. Previously we have established that a CTD-derived 34-residue peptide associates into a tetramer in a pH-dependent manner (Kamnesky et al., JMB 2012;418:237-247).

The distal C-terminal region of the KcsA potassium channel is a pH-dependent tetramerization domain.

The intracellular C-terminal domain (CTD) of KcsA, a bacterial homotetrameric potassium channel, is a 40-residue-long segment that natively adopts a helical bundle conformation with 4-fold symmetry. A hallmark of KcsA behavior is pH-induced conformational change, which leads to the opening of the channel at acidic pH. Previous studies have reached conflicting conclusions as to the role of the CTD in this transition.