Flavin-tag:exploiting a flavin transferase for protein labeling and engineering

Site-specific protein labeling plays a core role in investigating protein function at molecular level. A multitude of protein labelling techniques has been developed which are based on chemical and/or enzymatic methods. Enzyme-based labeling of proteins typically relies on a specific peptide sequence (a “tag”) that can be recognized and modified by a specific protein-modifying enzymes. In nature, some bacteria possess a flavin-transferase (called ApbE) which covalently incorporates an FMN cofactor in the so-called FMN-binding motif of some specific redox enzymes and proteins of which the role still has to be established. Based on this flavin transferase and its conserved recognition motif, we developed an enzymatic site-specific protein labelling method: the Flavin-tag method. This thesis involves the establishment, optimization, and application of the Flavin-tag method. We also expressed and characterized a multi-FMN-binding protein from Streptomyces azureus (SaFMN3) that is predicted to carry three covalently attached FMNs. This has unveiled some of the biochemical properties of such an extracellular multi-FMN-binding protein

Broadening the scope of the Flavin-tag method by improving flavin incorporation and incorporating flavin analogs

Methods for facile site‐selective modifications of proteins are in high demand. We have recently shown that a flavin transferase can be used for site‐specific covalent attachment of a chromo‐ and fluorogenic flavin (FMN) to any targeted protein. Although this Flavin‐tag method resulted in efficient labeling of proteins in vitro, labelling in E. coli cells resulted in partial flavin incorporation. It was also restricted in the type of installed label with only one type of flavin, FMN, being incorporated. Here, we report on an extension of the Flavin‐tag method that addresses previous limitations. We demonstrate that co‐expression of FAD synthetase improves the flavin incorporation efficiency, allowing complete flavin‐labeling of a target protein in E. coli cells. Furthermore, we have found that various flavin derivatives and even a nicotinamide can be covalently attached to a target protein, rendering this method even more versatile and valuable

Flavin-Tag:A Facile Method for Site-Specific Labeling of Proteins with a Flavin Fluorophore

Site-specific protein labeling methods are highly valuable tools for research and applications. We present a new protein labeling method that allows covalent attachment of a chromo-and fluorogenic flavin (FMN) to any targeted protein using a short flavinylation peptide-Tag. We show that this peptide can be as short as 7 residues and can be located at the N-Terminus, C-Terminus, or in internal regions of the target protein. Analogous to kinase-catalyzed phosphorylation, the flavin is covalently attached via a stable phosphothreonyl linkage. The site-specific covalent tethering of FMN is accomplished by using a bacterial flavin transferase. The covalent coupling of FMN was shown to work in Escherichia coli and Saccharomyces cerevisiae cells and could be performed in vitro, rendering the "Flavin-Tag"method a powerful tool for the selective decoration of proteins with a biocompatible redox-Active fluorescent chromophore

Characterization of two bacterial multi-flavinylated proteins harboring multiple covalent flavin cofactors

In recent years, studies have shown that a large number of bacteria secrete multi-flavinylated proteins. The exact roles and properties, of these extracellular flavoproteins that contain multiple covalently anchored FMN cofactors, are still largely unknown. Herein, we describe the biochemical and structural characterization of two multi-FMN-containing covalent flavoproteins, SaFMN3 from Streptomyces azureus and CbFMN4 from Clostridiaceae bacterium. Based on their primary structure, these proteins were predicted to contain three and four covalently tethered FMN cofactors, respectively. The genes encoding SaFMN3 and CbFMN4 were heterologously coexpressed with a flavin transferase (ApbE) in Escherichia coli, and could be purified by affinity chromatography in good yields. Both proteins were found to be soluble and to contain covalently bound FMN molecules. The SaFMN3 protein was studied in more detail and found to display a single redox potential (-184 mV) while harboring three covalently attached flavins. This is in line with the high sequence similarity when the domains of each flavoprotein are compared. The fully reduced form of SaFMN3 is able to use dioxygen as electron acceptor. Single domains from both proteins were expressed, purified and crystallized. The crystal structures were elucidated, which confirmed that the flavin cofactor is covalently attached to a threonine. Comparison of both crystal structures revealed a high similarity, even in the flavin binding pocket. Based on the crystal structure, mutants of the SaFMN3-D2 domain were designed to improve its fluorescence quantum yield by changing the microenvironment of the isoalloxazine moiety of the flavin cofactor. Residues that quench the flavin fluorescence were successfully identified. Our study reveals biochemical details of multi-FMN-containing proteins, contributing to a better understanding of their role in bacteria and providing leads to future utilization of these flavoprotein in biotechnology.</p

Substrate binding tunes the reactivity of hispidin 3-hydroxylase, a flavoprotein monooxygenase involved in fungal bioluminescence

Fungal bioluminescence was recently shown to depend on a unique oxygen-dependent system of several enzymes. However, the identities of the enzymes did not reveal the full biochemical details of this process, as the enzymes do not bear resemblance to those of other luminescence systems, and thus the properties of the enzymes involved in this fascinating process are still unknown. Here, we describe the characterization of the penultimate enzyme in the pathway, hispidin 3-hydroxylase, from the luminescent fungus Mycena chlorophos (McH3H), which catalyzes the conversion of hispidin to 3-hydroxyhispidin. 3-Hydroxyhispidin acts as a luciferin substrate in luminescent fungi. McH3H was heterologously expressed in Escherichia coli and purified by affinity chromatography with a yield of 100 mg/liter. McH3H was found to be a single component monomeric NAD(P)H-dependent FAD-containing monooxygenase having a preference for NADPH. Through site-directed mutagenesis, based on a modeled structure, mutant enzymes were created that are more efficient with NADH. Except for identifying the residues that tune cofactor specificity, these engineered variants may also help in developing new hispidin-based bioluminescence applications. We confirmed that addition of hispidin to McH3H led to the formation of 3-hydroxyhispidin as sole aromatic product. Rapid kinetic analysis revealed that reduction of the flavin cofactor by NADPH is boosted by hispidin binding by nearly 100-fold. Similar to other class A flavoprotein hydroxylases, McH3H did not form a stable hydroperoxyflavin intermediate. These data suggest a mechanism by which the hydroxylase is tuned for converting hispidin into the fungal luciferin.</p

Study on evolution law of mechanical properties of coal samples subjected to freezing and freeze-thaw cycles of liquid nitrogen

The permeability of coal reservoir is generally low in China, how to effectively improve the permeability of coal reservoir is a key and difficult point of coalbed methane exploitation, Liquid nitrogen fracturing technology as a kind waterless fracturing technology has received extensive attention in recent years. In order to reveal the influence of liquid nitrogen freezing and freeze-thaw on the mechanical properties of coal, the temperature distribution of coal samples was monitored by infrared thermal imaging technology, and uniaxial compression and acoustic emission tests were performed on the coal samples after the liquid nitrogen freezing and freezethaw, the P-wave velocity, porosity, acoustic emission and energy evolution characteristics of coal samples before and after the freezing and freeze thaw were analyzed. The research result showed that: ①After 360 min freezing and 12 freeze-thaw cycles, the P-wave velocity of coal samples decreased by 58.2% and 64.7%, respectively. The P-wave velocity does not decrease significantly during the initial freezing and freeze-thaw cycle stages, the velocity gradually decreases with the increase of freezing time and freeze-thaw cycles. ②The temperature of the coal sample gradually decreases with increase of freezing times. The surface temperature of the coal sample drops below -60°C after the liquid nitrogen frozen for 180s, the temperature distribution fluctuations at the center of the coal sample occurs due to the different thermal conduction coefficient of the coal particles. ③After liquid nitrogen freezing and freezethaw, the elastic modulus of coal sample decreases exponentially, while the porosity gradually increases. The increase in porosity of the coal sample after liquid nitrogen freeze-thaw is greater than that after liquid nitrogen freezing. ④The acoustic emission activity of coal samples during uniaxial loading is divided into development phase, active phase and severe phase, the maximum acoustic emission ringing count and cumulative acoustic emission ringing count of coal samples increase with the increase of freezing time and freeze-thaw cycles. ⑤Liquid nitrogen freezing and freeze-thaw will weaken the energy storage limit of coal sample, resulting in the reduction of the total energy, elastic energy and dissipated energy at the peak point during the uniaxial loading process

Fixing flavins: hijacking a flavin transferase for equipping flavoproteins with a covalent flavin cofactor

Most flavin-dependent enzymes contain a dissociable flavin cofactor. We present a new approach for installing a covalent bond between a flavin cofactor and its hosting protein. By using a flavin transferase and carving a flavinylation motif in target proteins, we demonstrate that ‘dissociable’ flavoproteins can be turned into covalent flavoproteins. Specifically, three different FMN-containing proteins were engineered to undergo covalent flavinylation: a light-oxygen-voltage (LOV) domain protein, a mini singlet-oxygen-generator (miniSOG), and a nitroreductase (BtNR). Optimizing the flavinylation motif and expression conditions led to covalent flavinylation of all three flavoproteins. The engineered covalent flavoproteins retained function and often exhibited improved performance such as higher thermostability or catalytic performance. Crystal structures of all three covalent flavoproteins confirmed the designed threonyl-phosphate linkage. The targeted flavoproteins differ in fold and function, indicating that this method of introducing a covalent flavin-protein bond is a powerful new method to create flavoproteins which cannot lose their cofactor, boosting their performance