oaaep1-mediated-enzymatic-synthesis-immobilization-polymerized

OaAEP1-Mediated Enzymatic Synthesis and Immobilization of Polymerized Protein for Single-Molecule Force Spectroscopy

Chemical and bio-conjugation techniques have been developed rapidly in recent years and allow the building of protein polymers. However, a controlled ...

Nanjing University

Metalloproteins play indispensable roles in biology owing to the versatile chemical reactivity of metal centres. However, studying their reactivity in many metalloproteins is challenging, as protein three-dimensional structure encloses labile metal centres, thus limiting their access to reactants and impeding direct measurements. Here we demonstrate the use of single-molecule atomic force microscopy to induce partial unfolding to expose metal centres in metalloproteins to aqueous solution, thus allowing for studying their chemical reactivity in aqueous solution for the first time. As a proof-of-principle, we demonstrate two chemical reactions for the FeS4 centre in rubredoxin: electrophilic protonation and nucleophilic ligand substitution. Our results show that protonation and ligand substitution result in mechanical destabilization of the FeS4 centre. Quantum chemical calculations corroborated experimental results and revealed detailed reaction mechanisms. We anticipate that this novel approach will provide insights into chemical reactivity of metal centres in metalloproteins under biologically more relevant conditions. 

Force-induced chemical reactions on the metal centre in a single metalloprotein molecule.

It is a challenge to synthesize materials that possess the properties of biological muscles-strong, elastic and capable of self-healing. Herein we report a network of poly(dimethylsiloxane) polymer chains crosslinked by coordination complexes that combines high stretchability, high dielectric strength, autonomous self-healing and mechanical actuation. The healing process can take place at a temperature as low as -20 °C and is not significantly affected by surface ageing and moisture. The crosslinking complexes used consist of 2,6-pyridinedicarboxamide ligands that coordinate to Fe(III) centres through three different interactions: a strong pyridyl-iron one, and two weaker carboxamido-iron ones through both the nitrogen and oxygen atoms of the carboxamide groups. As a result, the iron-ligand bonds can readily break and re-form while the iron centres still remain attached to the ligands through the stronger interaction with the pyridyl ring, which enables reversible unfolding and refolding of the chains. We hypothesize that this behaviour supports the high stretchability and self-healing capability of the material.

A highly stretchable autonomous self-healing elastomer.

Aim To report our experience regarding management of cholecystoenteric fistula (CEF) and identify the most effective diagnostic methods and surgical treatment. Methods In total, 10,588 patients underwent laparoscopic cholecystectomy for cholecystolithiasis from January 2000 to December 2014 at the Research Institute of General Surgery, Jinling Hospital (Nanjing, China). Twenty-nine patients were diagnosed with CEF preoperatively or intraoperatively. Data were retrospectively collected on demographics, preoperative diagnostics, intraoperative findings, laparoscopic procedures, complications, and follow-up. Results Twenty-nine patients (female/male ratio, 2.2; mean age, 68.7 years) with CEF were evaluated. Twenty-three (79.3%) patients had a cholecystoduodenal fistula (CDF), four (13.8%) had a cholecystocolonic fistula (CCF), one (3.4%) had a cholecystogastric fistula, and one (3.4%) had a CDF combined with a CCF. Only nine (31.0%) patients obtained a preoperative diagnosis. All patients initially underwent laparoscopic treatment, but five (17.2%) underwent conversion to open surgery; three of these five developed postoperative morbidity or mortality, and the other two had an uneventful postoperative course. Among patients managed successfully by laparoscopy, the hospital stay ranged from 3 to 6 days (mean, 4 days). All patients were asymptomatic at a mean follow-up of 13 months (range, 3-21 months). Conclusion Ultrasound and computed tomography can provide valuable diagnostic clues for CEF. Laparoscopic management of CEF in experienced hands is safe, feasible, and associated with rapid postoperative recovery.

Laparoscopic management of cholecystoenteric fistula: A single-center experience.

Metalloproteins are essential in biology. The incorporation of metal ion into metalloproteins significantly expands protein functionality and enhances protein stability. Over the last few years, atomic force microscopy-based single molecule force spectroscopy (SMFS) has evolved into a unique tool allowing for probing metalloproteins and metalligand bonds one molecule/bond at a time. Mechanical strength of a wide variety of metalligand bonds has been measured in metal-ligand complexes as well as in metalloproteins, providing detailed information of their underlying free energy profiles and the influence of the protein environment on the bond strength. SMFS experiments have directly demonstrated the effect of the metal binding on the mechanical stability of proteins. Moreover, SMFS has enabled the direct observation of the unfolding and folding of metalloproteins, revealing detailed mechanistic insight into the unfolding pathways modulated by the metal center.

Single molecule force spectroscopy: a new tool for bioinorganic chemistry.

In this work, a mixing-calcination method was developed to facilely construct MXene/CuO nanocomposite. CuO and MXene were first dispersed in ethanol with sufficient mixing. After solvent evaporation, the dried mixture was calcinated under argon to produce a MXene/CuO nanocomposite. As characterized by X-ray diffraction (XRD), field-emission scanning electron microscopy (FESEM), and X-ray photoelectron spectra (XPS), CuO nanoparticles (60⁻100 nm) were uniformly distributed on the surface and edge of MXene nanosheets. Furthermore, as evaluated by differential scanning calorimetry (DSC) and thermal gravimetric analysis (TGA), the high-temperature decomposition (HTD) temperature decrease of ammonium perchlorate (AP) upon addition of 1 wt% CuO (hybridized with 1 wt% MXene) was comparable with that of 2 wt% CuO alone, suggesting an enhanced catalytic activity of CuO on thermal decomposition of AP upon hybridization with MXene nanosheets. This strategy could be further applied to construct other MXene/transition metal oxide (MXene/TMO) composites with improved performance for various applications.

A Facile Method to Construct MXene/CuO Nanocomposite with Enhanced Catalytic Activity of CuO on Thermal Decomposition of Ammonium Perchlorate.

There is often a trade-off between mechanical properties (modulus and toughness) and dynamic self-healing. Here we report the design and synthesis of a polymer containing thermodynamically stable whilst kinetically labile coordination complex to address this conundrum. The Zn-Hbimcp (Hbimcp = 2,6-bis((imino)methyl)-4-chlorophenol) coordination bond used in this work has a relatively large association constant (2.2 × 10) but also undergoes fast and reversible intra- and inter-molecular ligand exchange processes. The as-prepared Zn(Hbimcp)-PDMS polymer is highly stretchable (up to 2400% strain) with a high toughness of 29.3 MJ m, and can autonomously self-heal at room temperature. Control experiments showed that the optimal combination of its bond strength and bond dynamics is responsible for the material's mechanical toughness and self-healing property. This molecular design concept points out a promising direction for the preparation of self-healing polymers with excellent mechanical properties. We further show this type of polymer can be potentially used as energy absorbing material.

Thermodynamically stable whilst kinetically labile coordination bonds lead to strong and tough self-healing polymers.

Knowledge of the molecular mechanisms of specific bacterial virulence factors can significantly contribute to antibacterial drug discovery.  is a Gram-negative microaerophilic bacterium that infects almost half of the world's population, leading to gastric disorders and even gastric cancer.  expresses a series of virulence factors in the host, among which high-temperature requirement A (HtrA) is a newly identified serine protease secreted by HtrA cleaves the extracellular domain of the epithelial cell surface adhesion protein E-cadherin and disrupts gastric epithelial cell junctions, allowing  to access the intercellular space. Here we report the first crystal structure of HtrA at 3.0 Å resolution. The structure revealed a new type of HtrA protease trimer stabilized by unique N-terminal domain swapping distinct from other known HtrA homologs. We further observed that truncation of the N terminus completely abrogates HtrA trimer formation as well as protease activity. In the presence of unfolded substrate, HtrA assembled into cage-like 12-mers or 24-mers. Combining crystallographic, biochemical, and mutagenic data, we propose a mechanistic model of how HtrA recognizes and cleaves the well-folded E-cadherin substrate. Our study provides a fundamental basis for the development of anti- agents by using a previously uncharacterized HtrA protease as a target.

The unique trimeric assembly of the virulence factor HtrA from  occurs via N-terminal domain swapping.

The recent development of chemical and bio-conjugation techniques allows for the engineering of various protein polymers. However, most of the polymerization process is difficult to control. To meet this challenge, we develop an enzymatic procedure to build polyprotein using the combination of a strict protein ligase OaAEP1 (Oldenlandia affinis asparaginyl endopeptidases 1) and a protease TEV (tobacco etch virus). We firstly demonstrate the use of OaAEP1-alone to build a sequence-uncontrolled ubiquitin polyprotein and covalently immobilize the coupled protein on the surface. Then, we construct a poly-metalloprotein, rubredoxin, from the purified monomer. Lastly, we show the feasibility of synthesizing protein polymers with rationally-controlled sequences by the synergy of the ligase and protease, which are verified by protein unfolding using atomic force microscopy-based single-molecule force spectroscopy (AFM-SMFS). Thus, this study provides a strategy for polyprotein engineering and immobilization.

Enzymatic biosynthesis and immobilization of polyprotein verified at the single-molecule level.

Protein (un)folding is a complex and essential process. With the rapid development of single-molecule techniques, we can detect multiple and transient proteins (un)folding pathways/intermediates. However, the observation of multiple multistep (>2) unfolding scenarios for a single protein domain remains limited. Here, we chose metalloprotein with relatively stable and multiple metal-ligand coordination bonds as a system for such a purpose. Using AFM-based single-molecule force spectroscopy (SMFS), we successfully demonstrated the complex and multistep protein unfolding scenarios of the β-domain of a human protein metallothionein-3 (MT). MT is a protein of ~60 amino acids (aa) in length with 20 cysteines for various metal binding, and the β-domain (βMT) is of ~30 aa with an MS metal cluster. We detected four different types of three-step protein unfolding scenarios from the Cd-βMT, which can be possibly explained by the rupture of Cd-S bonds in the complex CdS metal cluster. In addition, complex unfolding scenarios with four rupture peaks were observed. The Cd-S bonds ruptured in both single bond and multiple bonds modes. Our results provide not only evidence for multistep protein unfolding phenomena but also reveal unique properties of metalloprotein system using single-molecule AFM.

Multistep Protein Unfolding Scenarios from the Rupture of a Complex Metal Cluster CdS.

The iron-amino acid interactions Fe-O(Glu/Asp), Fe-N(His), and Fe-S(Cys) are the three major iron-ligand bonds in proteins. To compare their properties in proteins, we used atomic force microscopy (AFM)-based single-molecule force spectroscopy to investigate a superoxide reductase (Fe(III)-SOR) with all three types of bonds forming an Fe(His)CysGlu center. We first found that Apo-SOR without bound iron showed multiple unfolding pathways only from the β-barrel core. Then, using Holo-SOR with a ferric ion, we found that a single Fe-O(Glu) bond can tightly connect the flexible N-terminal fragment to the β-barrel and stabilize the whole protein, showing a complete protein unfolding scenario, while the single Fe-N(His) bond was weak and unable to provide such a stabilization. Moreover, when multiple Fe-N bonds are present, a similar stabilization effect can be achieved. Our results showed that the iron-ligand bond modulates protein structure and stability in different modes at the single-bond level.

Single-Molecule Force Spectroscopy Reveals that Iron-Ligand Bonds Modulate Proteins in Different Modes.

The highly porous luminescent metal-organic frameworks (MOFs) can act as fluorescent probes for the detection of nitro compounds and can also serve as containers and energy transfer platforms to construct the host-guest systems. Herein, two new three-dimensional MOFs with high porosity were prepared successfully by the electron-rich tetrakis(4-pyridylphenyl)ethylene (tppe) as ligands. Compound  shows the high sensitivity and selectivity toward nitro-antibiotics in an aqueous media, particularly showing the best detection efficiency for furazolidone (FZD) among the reported luminescent sensors. The highly efficient fluorescence quenching toward FZD may be attributed to the electron and energy transfer. Compound  has naphthalene-2,7-dicarboxylic acid (2,7-npd) and tppe as dual linkers, and the energy transfer between 2,7-npd and tppe leads to the emission band in a large scale. It is worth noting that the single-phased white-light materials can be obtained by the in situ encapsulation of different concentration of sulforhodamine 101 (SR101) into compound  matrix.

Tetraphenylethylene-Decorated Metal-Organic Frameworks as Energy-Transfer Platform for the Detection of Nitro-Antibiotics and White-Light Emission.

Iron-sulfur (FeS) clusters are essential metal cofactors involved in a wide variety of biological functions. Their catalytic efficiency, biosynthesis, and regulation depend on FeS stability in aqueous solution. Here, molecular modeling is used to investigate the hydrolysis of an oxidized (ferric) mononuclear FeS cluster by bare dissociation and water substitution mechanisms in neutral and acidic solution. First, approximate electronic structure descriptions of FeS reactions by density functional theory are validated against high-level wave function CCSD(T) calculations. Solvation contributions are included by an all-atom model with hybrid quantum chemical/molecular mechanical (QM/MM) potentials and enhanced sampling molecular dynamics simulations. The free energy profile obtained for FeS cluster hydrolysis indicates that the hybrid functional M06 together with an implicit solvent correction capture the most important aspects of FeS cluster reactivity in aqueous solution. Then, 20 reaction channels leading to two consecutive Fe-S bond ruptures were explored with this calibrated model. For all protonation states, nucleophilic substitution with concerted bond breaking and forming to iron is the preferred mechanism, both kinetic and thermodynamically. In neutral solution, proton transfer from water to the sulfur leaving group is also concerted. Dissociative reactions show higher barriers and will not be relevant for FeS reactivity when exposed to solvent. These hydrolysis mechanisms may help to explain the stability and catalytic mechanisms of FeS clusters of multiple sizes and proteins.

Modeling the Hydrolysis of Iron-Sulfur Clusters.

This study aimed to elucidate the potential role of long non-coding RNA PlncRNA-1 in the septic acute kidney injury (AKI). The expression of PlncRNA-1 in the serum of patients with septic AKI patient was detected. We then established lipopolysaccharide (LPS)-induced septic AKI model in NRK-52E cells to investigate the effects of the overexpression of PlncRNA-1 on cell proliferation, apoptosis, and autophagy. In addition, the regulatory relationship between PlncRNA-1 and B-cell lymphoma 2 (BCL2) was explored to further elucidate the regulatory mechanism of PlncRNA-1 in septic AKI. PlncRNA-1 is downregulated in the serum of patients with septic AKI and in LPS-induced septic AKI cells. The overexpression of PlncRNA-1 considerably increases proliferation and inhibits apoptosis and autophagy of LPS-induced septic AKI cells. In addition, PlncRNA-1 can promote BCL2 expression, and the overexpression of BCL2 enhances proliferation and inhibits apoptosis and autophagy of LPS-induced septic AKI cells. Our findings reveal that the overexpression of PlncRNA-1 may promote cell proliferation and inhibit apoptosis and autophagy in septic AKI by regulating BCL2 expression. PlncRNA-1 may serve as a potential biomarker or target for the diagnosis and treatment of septic AKI.

Article	Year
Force-induced chemical reactions on the metal centre in a single metalloprotein molecule. Nature communications\| PubMed ID: 26108369	2015
A highly stretchable autonomous self-healing elastomer. Nature chemistry\| PubMed ID: 27219708	2016
Laparoscopic management of cholecystoenteric fistula: A single-center experience. The Journal of international medical research\| PubMed ID: 28417651	2017
Single molecule force spectroscopy: a new tool for bioinorganic chemistry. Current opinion in chemical biology\| PubMed ID: 29223008	2018
A Facile Method to Construct MXene/CuO Nanocomposite with Enhanced Catalytic Activity of CuO on Thermal Decomposition of Ammonium Perchlorate. Materials (Basel, Switzerland)\| PubMed ID: 30518073	2018
Thermodynamically stable whilst kinetically labile coordination bonds lead to strong and tough self-healing polymers. Nature communications\| PubMed ID: 30858371	2019
The unique trimeric assembly of the virulence factor HtrA from occurs via N-terminal domain swapping. The Journal of biological chemistry\| PubMed ID: 30936204	2019
Enzymatic biosynthesis and immobilization of polyprotein verified at the single-molecule level. Nature communications\| PubMed ID: 31235796	2019
Multistep Protein Unfolding Scenarios from the Rupture of a Complex Metal Cluster CdS. Scientific reports\| PubMed ID: 31324867	2019
Single-Molecule Force Spectroscopy Reveals that Iron-Ligand Bonds Modulate Proteins in Different Modes. The journal of physical chemistry letters\| PubMed ID: 31433648	2019
Tetraphenylethylene-Decorated Metal-Organic Frameworks as Energy-Transfer Platform for the Detection of Nitro-Antibiotics and White-Light Emission. Inorganic chemistry\| PubMed ID: 31556291	2019
Modeling the Hydrolysis of Iron-Sulfur Clusters. Journal of chemical information and modeling\| PubMed ID: 31790241	2019
Long non-coding RNA PlncRNA-1 regulates cell proliferation, apoptosis, and autophagy in septic acute kidney injury by regulating BCL2. International journal of clinical and experimental pathology\| PubMed ID: 31938114	2018

Peng Zheng

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