Yichao Huang

Irreversible Site-Specific Hydrazinolysis of Proteins by Use of Sortase†

Sortase-mediated hydrazinolysis of proteins with hydrazine or its derivatives was developed for the production of recombinant protein hydrazides. This process provides an alternative approach for protein semisynthesis through the use of recombinant protein hydrazides as thioester surrogates. It also provides an alternative method for C-terminal modification of proteins with functional units as well as for the preparation of C-to-C fusion proteins.

Quasi-Racemic X-ray Structures of K27-Linked Ubiquitin Chains Prepared by Total Chemical Synthesis

Quasi-racemic crystallography has been used to determine the X-ray structures of K27-linked ubiquitin (Ub) chains prepared through total chemical synthesis. Crystal structures of K27-linked di- and tri-ubiquitins reveal that the isopeptide linkages are confined in a unique buried conformation, which provides the molecular basis for the distinctive function of K27 linkage compared to the other seven Ub chains. K27-linked di- and triUb were found to adopt different structural conformations in the crystals, one being symmetric whereas the other triangular. Furthermore, bioactivity experiments showed that the ovarian tumor family de-ubiquitinase 2 significantly favors K27-linked triUb than K27-linked diUb. K27-linked triUb represents the so-far largest chemically synthesized protein (228 amino acids) that has been crystallized to afford a high-resolution X-ray structure.

Ligation of Expressed Protein α-Hydrazides via Genetic Incorporation of an α-Hydroxy Acid

Expressed protein ligation bridges the gap between synthetic peptides and recombinant proteins and thereby significantly increases the size and complexity of chemically synthesized proteins. Although the intein-based expressed protein ligation method has been extensively used in this regard, the development of new expressed protein ligation methods may improve the flexibility and power of protein semisynthesis. In this study a new alternative version of expressed protein ligation is developed by combining the recently developed technologies of hydrazide-based peptide ligation and genetic code expansion. Compared to the previous intein-based expressed protein ligation method, the new method does not require the use of protein splicing technology and generates recombinant protein α-hydrazides as ligation intermediates that are more chemically stable than protein α-thioesters. Furthermore, the use of an evolved mutant pyrrolysyl-tRNA synthetase(PylRS), ACPK-RS, from M. barkeri shows an improved performance for the expression of recombinant protein backbone oxoesters. By using HdeA as a model protein we demonstrate that the hydrazide-based method can be used to synthesize proteins with correctly folded structures and full biological activity. Because the PylRS-tRNACUAPyl system is compatible with both prokaryotic and eukaryotic cells,the strategy presented here may be readily expanded to manipulate proteins produced in mammalian cells. The new hydrazide-based method may also supplement the intein-based expressed protein ligation method by allowing for a more flexible selection of ligation site.

Genetically encoded alkenyl–pyrrolysine analogues for thiol–ene reaction mediated site-specific protein labeling

A series of alkene-bearing pyrrolysine analogues were synthesized and subsequently incorporated into proteins at two sites by a mutant PylRS–tRNA pair with excellent efficiency. This strategy allowed the site-specific labeling of proteins carrying single or double genetically encoded alkene handles via bioorthogonal thiol–ene ligation reactions.

Step-by-Step Strategy from Achiral Precursors to Polyoxometalates-Based Chiral Organic–Inorganic Hybrids

Using two types of triol ligands, several novel asymmetrically triol-functionalized Anderson organic hybrids have been efficiently synthesized in high purity and good yields via a convenient two-step esterification reaction. These organic-inorganic hybrids are chiral and can be spontaneously resolved with suitable solvents. Their molecular and crystal structures have been confirmed by single-crystal X-ray diffraction studies. Stable solid-state chirality of the corresponding enantiopure crystals has also been confirmed definitively by CD spectra. Interestingly, these organic-inorganic hybrids possess a layer-by-layer structure, forming solvent-accessible nanoscale chiral channels via a 1D infinite helical chain substructure. TGA measurements indicated that the species of the central heteroatoms significantly effects the stability of these compounds.

Detection of covert voice-over Internet protocol communications using sliding window-based steganalysis

The authors describe a reliable and accurate steganalysis method for detecting covert voice-over Internet protocol (VoIP) communication channels. The proposed method utilises a unique sliding window mechanism and an improved regular singular (RS) algorithm for VoIP steganalysis, which detects the presence of least significant bit embedded VoIP streams. With this mechanism, the detection window moves forward one packet or several packets each time to screen VoIP streams. The optimum detection threshold for the proposed detection metric is computed by modelling the distributions of the new metric for stego and cover VoIP streams. Experimental analysis reveals that the proposed method improves the detection time significantly, utilising less memory resources for VoIP steganalysis, thereby enabling real-time detection of stego VoIP streams. The proposed method also provides a significant improvement on precision in detecting multiple covert VoIP channels when compared to the conventional RS method.

Synthesis of l- and d-Ubiquitin by One-Pot Ligation and Metal-Free Desulfurization

Native chemical ligation combined with desulfurization has become a powerful strategy for the chemical synthesis of proteins. Here we describe the use of a new thiol additive, methyl thioglycolate, to accomplish one-pot native chemical ligation and metal-free desulfurization for chemical protein synthesis. This one-pot strategy was used to prepare ubiquitin from two or three peptide segments. Circular dichroism spectroscopy and racemic protein X-ray crystallography confirmed the correct folding of ubiquitin. Our results demonstrate that proteins synthesized chemically by streamlined 9-fluorenylmethoxycarbonyl (Fmoc) solid-phase peptide synthesis coupled with a one-pot ligation-desulfurization strategy can supply useful molecules with sufficient purity for crystallographic studies.

A general and highly regioselective synthesis approach to multi-functionalized organoimido derivatives of Polyoxometalates

Organoimidoylization of Polyoxometalates (POMs) can dramatically modify the electronic structures of POMs and gives rise to novel “value-adding” properties of the POMs for promising material applications including photo-electronic transformation and catalysis. To date, the preparation of multi-functionalized organoimido derivatives of POMs is generally conducted under strict condition and is time-consuming with limited yields. Herein, a series of regioselective polyorganoimido derivatives of POMs, ocatant- [Mo6O13(NAr)3(μ2-NAr)3]2− (Ar = phenyl (1), p-methoxyphenyl (2) and p-ethylphenyl (3)), were synthesized with high selectivity and in good yields via a general and highly regioselective synthesis method, called as the one-octant synthesis protocol. The reaction was monitored by ESI-MS and the as-prepared products were studied by ESI-MS, IR, UV-Vis, EA, 1HNMR, single crystal XRD analysis and DFT calculations. The one-octant synthesis protocol here may serve as an idea method to design novel nanoscale POM-based organic-inorganic multi-functional hybrids.

Protein modification: Standing out from the crowd

The discovery of a tetrapeptide containing a reactive cysteine provides a method to site-selectively modify peptides and proteins, even if other cysteine residues are present in the polypeptide chain.

A semisynthetic Atg3 reveals that acetylation promotes Atg3 membrane binding and Atg8 lipidation

Acetylation of Atg3 regulates the lipidation of the protein Atg8 in autophagy. The molecular mechanism behind this important biochemical event remains to be elucidated. We describe the first semi-synthesis of homogeneous K19/K48-diacetylated Atg3 through sequential hydrazide-based native chemical ligation. In vitro reconstitution experiments with the semi-synthetic proteins confirm that Atg3 acetylation can promote the lipidation of Atg8. We find that acetylation of Atg3 enhances its binding to phosphatidylethanolamine-containing liposomes and to endoplasmic reticulum, through which it promotes the lipidation process.