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.
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.
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.
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