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.

Photosensitizers, Zn protoporphyrin IX and Zn chlorin e6, are completely inserted into each heme pocket of a hexameric apohemoprotein. The fluorescence quenching efficiencies upon addition of methyl viologen are 2.3 and 2.6 fold-higher than those of the partially photosensitizer-inserted proteins, respectively, indicating that the energy migration occurs within the proteins.

Malignant tumor is the most common cause of death, and great efforts have been made to develop new ways for the cancer treatments and the rapid diagnoses. The cancer treatments including surgery, chemotherapy, radiotherapy, immunotherapy, photodynamic therapy (PDT), etc., are in widespread clinical use. Since these treatments are often accompanied with serious side effects, improvements are important to diminish the undesirable side effects. We have focused on the improvement of photodynamic therapy (PDT). In the PDT procedure, the photosensitizer is administrated to patients by intravenous injection, tumor is irradiated with light at the highest uptake of the photosensitizer by tumor, and singlet oxygen produced by the excited photosensitizer results in tumor apoptosis [1][2][3][4]. Therefore, PDT is promising as the less toxic cancer treatment than chemotherapy if the selective uptake of the photosensitizer by tumor is achieved. ABSTRACT: The glucopyranoside-conjugated porphyrins, H 2 TPP{p-O-(CH 2 ) 2 -O-OAcGlc} (1), [InTPP{p-O-(CH 2 ) 2 -O-OAcGlc}]NO 3 (2), H 2 TPP{p-O-(CH 2 ) 2 -O-Glc} (3), [InTPP{p-O-(CH 2 ) 2 -O-Glc}]-NO 3 (4) and ZnTPP{p-O-(CH 2 ) 2 -O-OAcGlc} (5) were synthesized, and characterized by 1 H NMR, 13 C NMR, ESI-MS, UV-vis spectroscopies and elemental analyses. In the 1 H NMR spectrum of 2, two sets of signals were observed for H-atoms of the phenyl group of porphyrin, indicating that 2 has the axial chirality due to a NO 3 ion coordinating to the indium atom. Abilities of the singlet oxygen production of these porphyrins, investigated by using 1,3-diphenylisobenzofuran (DPBF) as a quencher, were higher than those of the free-based and zinc porphyrins, reflecting the heavy atom effect. The photodynamic properties of these porphyrin derivatives were investigated against COLO 679. All of the glucopyranosideconjugated porphyrins exhibited the high photocytotoxicity compared with Laserphyrin ® . Above all, 4 exhibited the highest photocytotoxicity, coinciding with the high abilities of this complex for the singlet oxygen production and the cell permeability.

Protein assemblies are being investigated as a new-class of biomaterials. A supramolecular assembly of a mutant hexameric tyrosine coordinated hemoprotein (HTHP) modified with a pyrene derivative is described. Cysteine was first introduced as a site-specific reaction point at position V44 which is located at the bottom surface of the cylindrical structure of HTHP. [Formula: see text]-(1-pyrenyl)maleimide was then reacted with the mutant. The modification was confirmed by MALDI-TOF mass spectrometry and UV-vis absorption spectroscopy, indicating that approximately 90% cysteine residues are attached via the pyrene derivative. Size exclusion chromatography (SEC) measurements for pyrene-attached HTHP include a single peak which elutes earlier than the unmodified HTHP. Further investigation by SEC and dynamic light scattering (DLS) measurements indicate the desired size corresponding to the dimer of the hemoprotein hexamers. The multivalent effect of pyrene–pyrene interactions including hydrophobic and [Formula: see text]–[Formula: see text] stacking interactions appears to be responsible for including formation of the stable dimer of the hexamers. Interestingly, the assembly dissociates to the hexamer by removal of heme. In the case of the apo-form of pyrene-attached HTHP, the pyrene moiety appears to be incorporated into the heme pocket because the modification point is located at the adjacent residue of the Tyr45 coordinating to heme in the holo-form of HTHP. Subsequent addition of heme into the apo-form of pyrene-attached HTHP regenerates the dimer of the hexamers. The present study demonstrates a unique heme-dependent system in which HTHP is assembled to form a dimer of hexamers in the presence of heme and disassembled by removal of heme.

Hexameric hemoprotein (HTHP) is employed as a scaffold protein for the supramolecular assembly and activation of the apoptotic signalling enzyme caspase‐9, using short DNA elements as modular recruitment domains. Caspase‐9 assembly and activation on the HTHP platform due to enhanced proximity is followed by combinatorial inhibition at high scaffold concentrations. The DNA recruitment domains allow for reversible switching of the caspase‐9 assembly and activity state using short modulatory DNA strands. Tuning of the recruitment domain affinity allows for generating kinetically trapped active enzyme complexes, as well as for dynamic repositioning of caspases over scaffold populations and inhibition using monovalent sink platforms. The conceptual combination of a highly structured multivalent protein platform with modular DNA recruitment domains provides emergent biomimicry properties with advanced levels of control over protein assembly.

To convert an originally tyrosine-coordinated heme to histidine-coordinated heme in hexameric tyrosine-coordinated hemoprotein, HTHP, Tyr45, a residue coordinating to the heme cofactor, and Arg25 located in the distal site are replaced with Phe45 and His25, respectively in each of the subunits of the protein. The obtained HTHP mutant (HTHP[Formula: see text] was characterized by SDS-PAGE, ESI-TOF MS, dynamic light scattering measurements and size exclusion chromatography. These analyses indicate that HTHP[Formula: see text] maintains its stable hexameric structure with the altered ligation of each of the heme cofactors. Comparison of UV-vis absorption spectra of the ferric-, ferrous-, CO- and CN-forms of HTHP[Formula: see text] with those of several well-known His-ligated hemoproteins indicates that heme is coordinated by the His25 residue. The reaction of HTHP[Formula: see text] with cumene hydroperoxide produces both cumyl alcohol and acetophenone in a 2.3:1 ratio, indicating that heterolytic O–O bond cleavage dominantly occurs to form the two-electron oxidized species known as compound I. Peroxidase activity of HTHP[Formula: see text] is found to follow Michaelis–Menten kinetics. The [Formula: see text] values of HTHP[Formula: see text] for H[Formula: see text]O[Formula: see text]-dependent oxidation of ABTS and guaiacol are 10- and 100-fold higher, respectively, than those of wild type HTHP (HTHP[Formula: see text]. The [Formula: see text]/[Formula: see text] values of HTHP[Formula: see text] for both substrates are increased 30-fold relative to that of HTHP[Formula: see text]. Moreover, HTHP[Formula: see text] is capable of promoting catalytic sulfoxidation of thioanisole with H[Formula: see text]O[Formula: see text] with a turnover number ca. 2-fold higher than that of HTHP[Formula: see text]. The present findings demonstrate that proximal His ligation to the heme is significantly effective to increase the peroxidase activity in the HTHP matrix.

An assembly of multiple photosensitizers is demonstrated by development of a hexameric hemoprotein (HTHP) scaffold as a light harvesting model to replicate the successive energy transfer occuring within photosensitizer assemblies of natural systems. In our model, six zinc protoporphyrin IX (ZnPP) molecules are arrayed at the heme binding site of HTHP by supramolecular interactions and five fluorescein (Flu) molecules and one Texas Red (Tex) molecule as donor and acceptor photosensitizers, respectively, are attached to the HTHP protein surface with covalent linkages. The flow of excited energy from photoexcited Flu to Tex occurs via two pathways: direct energy transfer from Flu to Tex (path 1) and energy transfer via ZnPP (path 2). Steady state and time-resolved fluorescence measurements reveal that the energy transfer ratio of these pathways (path 1 : path 2) is 39 : 61. These findings indicate that the excited energy originating at five Flu and six ZnPP molecules is collected at one Tex molecule as a funnel-like bottom for light harvesting. The present system using the hexameric hemoprotein scaffold is a promising candidate for construction of an artificial light harvesting system having multiple photosensitizers to promote efficient use of solar energy.

Many metalloproteins can undergo 3D domain swapping. This future article summarizes in vitro and in vivo formation of supramolecular metalloproteins through 3D domain swapping.