16 research outputs found
Self-assembly and characterization of small and monodisperse dye nanospheres in a protein cage
Phthalocyanines (Pc) are dyes in widespread use in materials science and nanotechnology, with numerous applications in medicine, photonics, electronics and energy conversion. With the aim to construct biohybrid materials, we here prepared and analyzed the structure of two Pc- loaded virus- like particles (VLP) with diameters of 20 and 28 nm (i.e., T = 1 and T= 3 icosahedral symmetries, respectively). Our cryoelectron microscopy (cryo- EM) studies show an unprecedented, very high level of Pc molecule organization within both VLP. We found thaT = 10 nm diameter nanospheres form inside the T = 1 VLP by self- assembly of supramolecular Pc stacks. Monodisperse, self- assembled organic dye nanospheres were not previously known, and are a consequence of capsid- imposed symmetry and size constraints. The Pc cargo also produces major changes in the protein cage structure and in the mechanical properties of the VLP. Pc- loaded VLP are potential photosensitizer/ carrier systems in photodynamic therapy (PDT), for which their mechanical behaviour must be characterized. Many optoelectronic applications of Pc dyes, on the other hand, are dependent on dye organization at the nanoscale level. Our multidisciplinary study thus opens the way towards nanomedical and nanotechnological uses of these functional molecules
Assembling Enzymatic Cascade Pathways inside Virus-Based Nanocages Using Dual-Tasking Nucleic Acid Tags
Self-Sorting of Foreign Proteins in a Bacterial Nanocompartment
Nature uses bottom-up
approaches for the controlled assembly of
highly ordered hierarchical structures with defined functionality,
such as organelles, molecular motors, and transmembrane pumps. The
field of bionanotechnology draws inspiration from nature by utilizing
biomolecular building blocks such as DNA, proteins, and lipids, for
the (self-) assembly of new structures for applications in biomedicine,
optics, or electronics. Among the toolbox of available building blocks,
proteins that form cage-like structures, such as viruses and virus-like
particles, have been of particular interest since they form highly
symmetrical assemblies and can be readily modified genetically or
chemically both on the outer or inner surface. Bacterial encapsulins
are a class of <i>nonviral</i> protein cages that self-assemble <i>in vivo</i> into stable icosahedral structures. Using teal fluorescent
proteins (TFP) engineered with a specific native C-terminal docking
sequence, we report the molecular self-sorting and selective packaging
of TFP cargo into bacterial encapsulins during <i>in vivo</i> assembly. Using native mass spectrometry techniques, we show that
loading of either monomeric or dimeric TFP cargo occurs with unprecedented
high fidelity and exceptional loading accuracy. Such self-assembling
systems equipped with self-sorting capabilities would open up exciting
opportunities in nanotechnology, for example, as artificial (molecular
storage or detoxification) organelles or as artificial cell factories
for <i>in situ</i> biocatalysis
Photoprogramming Allostery in Human Serum Albumin
Developing
strategies to interfere with allosteric interactions
in proteins not only promises to deepen our understanding of vital
cellular processes but also allows their regulation using external
triggers. Light is particularly attractive as a trigger being spatiotemporally
selective and compatible with the physiological environment. Here,
we engineered a hybrid protein in which irradiation with light opens
a new allosteric communication route that is not inherent to the natural
system. We select human serum albumin, a promiscuous protein responsible
for transporting a variety of ligands in plasma, and show that by
covalently incorporating a synthetic photoswitch to subdomain IA we
achieve optical control of the ligand binding in subdomain IB. Molecular
dynamics simulations confirm the allosteric nature of the interactions
between IA and IB in the engineered protein. Specifically, upon illumination,
photoconversion of the switch is found to correlate with a less-coordinated
motion of the two subdomains and an increased flexibility of the binding
pocket in subdomain IB, whose fluctuations are cooperatively enhanced
by the presence of ligands, ultimately facilitating their release.
Our combined experimental and computational work demonstrates how
harnessing artificial molecular switches enables photoprogramming
the allosteric regulation of binding activities in such a prominent
protein
Assembling Enzymatic Cascade Pathways inside Virus-Based Nanocages Using Dual-Tasking Nucleic Acid Tags
The
packaging of proteins into discrete compartments is an essential
feature for cellular efficiency. Inspired by Nature, we harness virus-like
assemblies as artificial nanocompartments for enzyme-catalyzed cascade
reactions. Using the negative charges of nucleic acid tags, we develop
a versatile strategy to promote an efficient noncovalent co-encapsulation
of enzymes within a single protein cage of cowpea chlorotic mottle
virus (CCMV) at neutral pH. The encapsulation results in stable 21–22
nm sized CCMV-like particles, which is characteristic of an icosahedral <i>T</i> = 1 symmetry. Cryo-EM reconstruction was used to demonstrate
the structure of <i>T</i> = 1 assemblies templated by biological
soft materials as well as the extra-swelling capacity of these <i>T</i> = 1 capsids. Furthermore, the specific sequence of the
DNA tag is capable of operating as a secondary biocatalyst as well
as bridging two enzymes for co-encapsulation in a single capsid while
maintaining their enzymatic activity. Using CCMV-like particles to
mimic nanocompartments can provide valuable insight on the role of
biological compartments in enhancing metabolic efficiency
Structural Characterization of Native and Modified Encapsulins as Nanoplatforms for in Vitro Catalysis and Cellular Uptake
Recent years have witnessed the emergence of bacterial semiorganelle encapsulins as promising platforms for bio-nanotechnology. To advance the development of encapsulins as nanoplatforms, a functional and structural basis of these assemblies is required. Encapsulin from Brevibacterium linens is known to be a protein-based vessel for an enzyme cargo in its cavity, which could be replaced with a foreign cargo, resulting in a modified encapsulin. Here, we characterize the native structure of B. linens encapsulins with both native and foreign cargo using cryo-electron microscopy (cryo-EM). Furthermore, by harnessing the confined enzyme (i.e., a peroxidase), we demonstrate the functionality of the encapsulin for an in vitro surface-immobilized catalysis in a cascade pathway with an additional enzyme, glucose oxidase. We also demonstrate the in vivo functionality of the encapsulin for cellular uptake using mammalian macrophages. Unraveling both the structure and functionality of the encapsulins allows transforming biological nanocompartments into functional systems
Structural Characterization of Native and Modified Encapsulins as Nanoplatforms for in Vitro Catalysis and Cellular Uptake
Predicting the Loading of Virus-Like Particles with Fluorescent Proteins
The virus-like particle (VLP) of the Cowpea Chlorotic Mottle Virus (CCMV) has often been used to encapsulate foreign cargo. Here we show two different rational design approaches, covalent and noncovalent, for loading teal fluorescent proteins (TFP) into the VLP. The covalent loading approach allows us to gain control over capsid loading on a molecular level. The achieved loading control is used to accurately predict the loading of cargo into CCMV VLP. The effects of molecular confinement were compared for the differently loaded VLPs created with the covalent method. We see that the loading of more than 10 fluorescent proteins in the 18 nm internal cavity of the CCMV capsid gives rise to a maximum efficiency of homo-FRET between the loaded proteins, as measured by fluorescence anisotropy. This shows that already at low levels of VLP loading molecular crowding starts to play a role