Ligation of Soluble but Unreactive Peptide Segments in the Chemical Synthesis of Haemophilus Influenzae DNA Ligase

Abstract

During the total chemicals ynthesis of the watersoluble globular Haemophilus Influenzae DNAl igase (Hin-Lig), we observed the surprising phenomenon of as oluble peptide segment that failed to undergo native chemical ligation. Based on dynamic light scattering and transmission electron microscopye xperiments,w ed etermined that the peptide formed soluble colloidal particles in ah omogeneous solution containing 6 m guanidine hydrochloride.Conventional peptide performance-improving strategies,s uch as installation of at erminal/side-chain Arg tag or O-acyl isopeptide,f ailed to enable the reaction, presumably because of their inability to disrupt the formation of soluble colloidal particles.H owever, ar emovable backbone modification strategy recently developed for the synthesis of membrane proteins did disrupt the formation of the colloids,a nd the desired ligation of this soluble but unreactive system was eventually accomplished. This work demonstrates that an appropriate solution dispersion state,i na ddition to good peptide solubility,i sap rerequisite for successful peptide ligation.

Use of a Removable Backbone Modification Strategy to Prevent Aspartimide Formation in the Synthesis of Asp Lactam Cyclic Peptides†

“…Recently, we developed a method to disrupt the formation of secondary structure by nascent peptides during SPPS and protein ligation by protecting selected amide nitrogen atoms with the RBM building block 2‐hydroxy‐4‐methoxy‐5‐nitrobenzaldehyde. [ 24‐31 ] This strategy is applicable to any secondary amide nitrogen regardless of its steric encumbrance because the building block is attached by reductive amination when the amide nitrogen is still a free amine. The amino acid that is next in the peptide sequence is attached to the phenolic group of the building block, whereupon the protected peptide is obtained by para ‐nitrophenol induced intramolecular O‐to‐N acyl transfer that is a highly efficient process applicable to bulky amino acids.…”

Enabling chemical protein (semi)synthesis via reducible solubilizing tags (RSTs)

“…The resulting residue was refolded by dissolving in 6 M guanidine, followed by 10 times dilution in the refolding buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl). Aer purication by using the size exclusion column and then the streptavidin resin, HMGB1 (21) was successfully obtained in a 22.7% yield by directly treating the resin with 20 mM TCEP in the refolding buffer for 1 h at room temperature. The nal product protein HMGB1 was detected by LC-MS (Fig.…”

“…One of the oen-met problems is the poor solubility of the peptide segments for ligation. [19][20][21] Generally, the ligation can proceed smoothly when the peptides are well dissolved in the ligation solution. However, some peptides with hydrophobic residues or a tendency for selfassembly are very difficult for reversed phase HPLC purication.…”

Advances on structural mechanisms of ubiquitinated nucleosome complexes

“…换成一种非天然氨基酸(δ-巯基赖氨酸), 这种非天然 氨基酸可以以较高的选择性便捷地与泛素分子形成肽 键 [50] . 组蛋白片段之间交联后最后通过一个酰肼中间 体得到完整组蛋白 [43] , 在形成完整组蛋白骨架后进行…”