DNA-Mediated Protein Shuttling between Coacervate-Based Artificial Cells.

The regulation of protein uptake and secretion is crucial for (inter)cellular signaling. Mimicking these molecular events is essential when engineering synthetic cellular systems. A first step towards achieving this goal is obtaining control over the uptake and release of proteins from synthetic cells in response to an external trigger. Herein, we have developed an artificial cell that sequesters and releases proteinaceous cargo upon addition of a coded chemical signal: single-stranded DNA oligos (ssDNA) were employed to independently control the localization of a set of three different ssDNA-modified proteins. The molecular coded signal allows for multiple iterations of triggered uptake and release, regulation of the amount and rate of protein release and the sequential release of the three different proteins. This signaling concept was furthermore used to directionally transfer a protein between two artificial cell populations, providing novel directions for engineering lifelike communication pathways inside higher order (proto)cellular structures.

A Supramolecular Assembly of Hemoproteins Formed in a Star-Shaped Structure via Heme-Heme Pocket Interactions.

Proteins have been used as building blocks to provide various supramolecular structures in efforts to develop nano-biomaterials possessing broad biological functionalities. A series of unique structures have been obtained from the engineering of hemoproteins which contain the iron porphyrin known as heme, as a prosthetic group. This work in developing assembling systems is extended using cytochrome b562, a small electron transfer hemoprotein engineered to include an externally-attached heme moiety. The engineered units, which form a one-dimensional assembly via interprotein heme-heme pocket interactions, are conjugated to an apo-form of hexameric tyrosine-coordinated hemoprotein (apoHTHP) to provide a branching unit promoting the assembly of a star-shaped structure. The incorporation of the heme moiety attached to the protein surface of cytochrome b562 into apoHTHP can be accelerated by elevating the reaction temperature to generate a new assembly. The formation of a new larger assembly structure was confirmed by size exclusion chromatography. The ratio of the heme-containing units in the assemblies was analyzed by UV-Vis spectroscopy and the population of protein units estimated from SDS PAGE suggests the presence of plausible star-shaped structures, which are supported by hydrodynamic diameter data obtained by dynamic light scattering.

Thermoresponsive Micellar Assembly Constructed from a Hexameric Hemoprotein Modified with Poly(N-isopropylacrylamide) toward an Artificial Light-Harvesting System.

Artificial protein assemblies inspired by nature have significant potential in development of emergent functional materials. In order to construct an artificial protein assembly, we employed a mutant of a thermostable hemoprotein, hexameric tyrosine-coordinated heme protein (HTHP), as a building block. The HTHP mutant which has cysteine residues introduced on the bottom surface of its columnar structure was reacted with maleimide-tethering thermoresponsive poly(N-isopropylacrylamide), PNIPAAm, to generate the protein assembly upon heating. The site-specific modification of the cysteine residues with PNIPAAm on the protein surface was confirmed by SDS-PAGE and analytical size exclusion chromatography (SEC). The PNIPAAm-modified HTHP (PNIPAAm-HTHP) is found to provide a 43 nm spherical structure at 60 °C, and the structural changes observed between the assembled and the disassembled forms were duplicated at least five times. High-speed atomic force microscopic measurements of the micellar assembly supported by cross-linkage with glutaraldehyde indicate that the protein matrices are located on the surface of the sphere and cover the inner PNIPAAm core. Furthermore, substitution of heme with a photosensitizer, Zn protoporphyrin IX (ZnPP), in the micellar assembly provides an artificial light-harvesting system. Photochemical measurements of the ZnPP-substituted micellar assembly demonstrate that energy migration among the arrayed ZnPP molecules occurs within the range of several tens of picoseconds. Our present work represents the first example of an artificial light-harvesting system based on an assembled hemoprotein oligomer structure to replicate natural light-harvesting systems.

Cr(VI) bioremediation by active algal-bacterial aerobic granular sludge: Importance of microbial viability, contribution of microalgae and fractionation of loaded Cr.

In this study, chromium (Cr) was used as an example of the most toxic heavy metals that threaten human health, and Cr(VI) bioremediation was implemented by using a new type of aerobic granular sludge (AGS), i.e., algal-bacterial AGS. Results showed that the total Cr removal efficiency by active algal-bacterial AGS was 85.1 ± 0.6% after 6 h biosorption at pH 6 and room temperature, which could be further improved to 93.8 ± 0.4% with external electron donor (glucose) supply. However, inactivation dramatically decreased the total Cr removal efficiency to 29.6 ± 3.5%, and no effect was noticed when external electron donor was provided. With an antibiotic (levofloxacin) or metabolic inhibitor (NaN3) addition, the total Cr removal efficiency of bacterial AGS was inhibited by 16.0% or 10.1%, but this efficiency was maintained in the case of algal-bacterial AGS. Analysis of extracellular polymeric substances (EPS) composition revealed that under Cr(VI) exposure, more loosely bound EPS were secreted by algal-bacterial AGS, favoring Cr(VI) reduction. Results from chemical fractionation indicated that 90.5 ± 4.2% of the loaded Cr on algal-bacterial AGS was in an immobile form, reflecting the low environmental risk of Cr-loaded algal-bacterial AGS after biosorption of hazardous heavy metals from wastewater.

Insight into Cr(VI) biosorption onto algal-bacterial granular sludge: Cr(VI) bioreduction and its intracellular accumulation in addition to the effects of environmental factors.

Hexavalent chromium (Cr(VI)) is one of the typical heavy metals that pose a great threat to the environment. As a novel biotechnology, algal-bacterial aerobic granular sludge (AGS) possesses the merits of both bacterial AGS and algae. This study firstly evaluated Cr(VI) removal via biosorption by algal-bacterial AGS under different operation conditions and then some environmental factors. Results show that the highest Cr(VI) reduction (99.3%) and total Cr removal (89.1%) were achieved within 6 h at pH 2 and 6, respectively. The coexisting oxyanions exhibited slight effects, while both tested natural organic matters (humic acid and tannic acid) and carbon sources promoted Cr(VI) reduction at some appropriate concentrations. The coexistence of metal cations favored Cr(VI) reduction, achieving the highest enhancement of 8.1% by Cu2+ at 5 mg/L, while the total Cr removal was suppressed to some extent. Salinity > 5 g/L severely inhibited both Cr(VI) reduction and total Cr removal. Moreover, the loaded Cr in algal-bacterial AGS was found to be almost in the form of Cr(III), with 66.8% being contributed by intracellular accumulation. This work suggests that Cr(VI) reduction and intracellular accumulation are the main mechanisms involved in Cr(IV) biosorption onto algal-bacterial AGS.