21 research outputs found
Self-Assembly of Proteinaceous Multishell Structures Mediated by a Supercharged Protein
Engineered variants
of the capsid-forming enzyme lumazine synthase
can exploit electrostatic interactions to encapsulate complementarily
charged guest macromolecules. Here we investigate the effect of ionic
strength and cargo molecules on assembly of AaLS-13, a negatively
supercharged lumazine synthase protein cage, and we show that multishell
structures are produced upon mixing the capsid core with free capsomers
and a positively supercharged variant of the green fluorescent protein
GFPÂ(+36). The assembly process is mediated by favorable electrostatic
interactions between the negatively charged capsid shells/capsomers
and GFPÂ(+36) molecules, and it is therefore strongly dependent on
ionic strength. The mechanism of formation of these assemblages is
likely similar to the assembly of multishell structures of some virus-like
particles, where outer shells organize as nonicosahedral structures
with larger radii of curvature than the templating inner shell. In
contrast to the viral multishell structures, the positively charged
mediator was found to be essential for the assembly of multilayered
structures of different shapes and sizes constituted of AaLS-13 capsomers.
This mediator-bridging approach may be widely applicable to create
protein-based hierarchical nanostructures for various nanotechnology
applications such as drug delivery and bioimaging
Diffusion-Limited Cargo Loading of an Engineered Protein Container
The
engineered bacterial nanocompartment AaLS-13 is a promising
artificial encapsulation system that exploits electrostatic interactions
for cargo loading. In order to study its ability to take up and retain
guests, a pair of fluorescent proteins was developed which allows
spectroscopic determination of the extent of encapsulation by Förster
resonance energy transfer (FRET). The encapsulation process is generally
complete within a second, suggesting low energetic barriers for proteins
to cross the capsid shell. Formation of intermediate aggregates upon
mixing host and guest in vitro complicates capsid loading at low ionic
strength, but can be sidestepped by increasing salt concentrations
or diluting the components. Encapsulation of guests is completely
reversible, and the position of the equilibrium is easily tuned by
varying the ionic strength. These results, which challenge the notion
that AaLS-13 is a continuous rigid shell, provide valuable information
about cargo loading that will guide ongoing efforts to engineer functional
host–guest complexes. Moreover, it should be possible to adapt
the protein FRET pair described in this report to characterize functional
capsid–cargo complexes generated by other encapsulation systems
Diversification of Protein Cage Structure Using Circularly Permuted Subunits
Self-assembling protein cages are
useful as nanoscale molecular
containers for diverse applications in biotechnology and medicine.
To expand the utility of such systems, there is considerable interest
in customizing the structures of natural cage-forming proteins and
designing new ones. Here we report that a circularly permuted variant
of lumazine synthase, a cage-forming enzyme from <i>Aquifex aeolicus</i> (AaLS) affords versatile building blocks for the construction of
nanocompartments that can be easily produced, tailored, and diversified.
The topologically altered protein, cpAaLS, self-assembles into spherical
and tubular cage structures with morphologies that can be controlled
by the length of the linker connecting the native termini. Moreover,
cpAaLS proteins integrate into wild-type and other engineered AaLS
assemblies by coproduction in <i>Escherichia coli</i> to
form patchwork cages. This coassembly strategy enables encapsulation
of guest proteins in the lumen, modification of the exterior through
genetic fusion, and tuning of the size and electrostatics of the compartments.
This addition to the family of AaLS cages broadens the scope of this
system for further applications and highlights the utility of circular
permutation as a potentially general strategy for tailoring the properties
of cage-forming proteins
Enantiocomplementary Synthesis of γ‑Nitroketones Using Designed and Evolved Carboligases
Artificial
enzymes created by computational design and directed
evolution are versatile biocatalysts whose promiscuous activities
represent potentially attractive starting points for divergent evolution
in the laboratory. The artificial aldolase RA95.5-8, for example,
exploits amine catalysis to promote mechanistically diverse carboligations.
Here we report that RA95.5-8 variants catalyze the asymmetric synthesis
of γ-nitroketones via two alternative enantiocomplementary Michael-type
reactions: enamine-mediated addition of acetone to nitrostyrenes,
and nitroalkane addition to conjugated ketones activated as iminium
ions. In addition, a cascade of three aldolase-catalyzed reactions
enables one-pot assembly of γ-nitroketones from three simpler
building blocks. Together, our results highlight the chemical versatility
of artificial aldolases for the practical synthesis of important chiral
synthons
Efficient in Vitro Encapsulation of Protein Cargo by an Engineered Protein Container
An engineered variant of lumazine synthase, a nonviral
capsid protein
with a negatively charged luminal surface, is shown to encapsulate
up to 100 positively supercharged green fluorescent protein (GFP)
molecules in vitro. Packaging can be achieved starting either from
intact, empty capsids or from capsid fragments by incubation with
cargo in aqueous buffer. The yield of encapsulated GFP correlates
directly with the host/guest mixing ratio, providing excellent control
over packing density. Facile in vitro loading highlights the unusual
structural dynamics of this novel nanocontainer and should facilitate
diverse biotechnological and materials science applications
Substrate Sorting by a Supercharged Nanoreactor
Compartmentalization of proteases
enables spatially and temporally
controlled protein degradation in cells. Here we show that an engineered
lumazine synthase protein cage, which possesses a negatively supercharged
lumen, can exploit electrostatic effects to sort substrates for an
encapsulated protease. This proteasome-like nanoreactor preferentially
cleaves positively charged polypeptides over both anionic and zwitterionic
substrates, inverting the inherent substrate specificity of the guest
enzyme approximately 480 fold. Our results suggest that supercharged
nanochambers could provide a simple and potentially general means
of conferring substrate specificity to diverse encapsulated catalysts
Fast Knoevenagel Condensations Catalyzed by an Artificial Schiff-Base-Forming Enzyme
The
simple catalytic motifs utilized by enzymes created by computational
design and directed evolution constitute a potentially valuable source
of chemical promiscuity. Here we show that the artificial retro-aldolase
RA95.5-8 is able to use a reactive lysine in a hydrophobic pocket
to accelerate promiscuous Knoevenagel condensations of electron-rich
aldehydes and activated methylene donors. Optimization of this activity
by directed evolution afforded an efficient enzyme variant with a
catalytic proficiency of 5 × 10<sup>11</sup> M<sup>–1</sup> and a >10<sup>8</sup>-fold catalytic advantage over simple primary
and secondary amines. Divergent evolution of de novo enzymes in this
way could be a promising strategy for creating tailored biocatalysts
for many synthetically useful reactions
Rational Engineering of a Designed Protein Cage for siRNA Delivery
Oligonucleotide
therapeutics have transformative potential in modern
medicine but are poor drug candidates in themselves unless fitted
with compensatory carrier systems. We describe a simple approach to
transform a designed porous protein cage into a nucleic acid delivery
vehicle. By introducing arginine mutations to the lumenal surface,
a positively supercharged capsule is created, which can encapsidate
oligonucleotides in vitro with high binding affinity. We demonstrate
that the siRNA-loaded cage is taken up by mammalian cells and releases
its cargo to induce RNAi and knockdown gene expression. These general
concepts could also be applied to alternative scaffold designs, expediting
the development of artificial protein cages toward delivery applications
Harnessing Protein Symmetry for Enzyme Design
Cyclic protein oligomers are common in Nature. Here we
show that
the central pore of the pentameric ring-forming protein lumazine synthase
from <i>Saccharomyces cerevisiae</i> (ScLS) can be rationally
engineered to catalyze a retro-aldol reaction. The <i>C</i><sub>5</sub>-symmetry of the complex was exploited to equip the protein
tunnel with a ring of five closely spaced lysines adjacent to an apolar
site for substrate binding. The resulting system utilizes amine catalysis
to promote the cleavage of (±)-methodol to 6-methoxy-2-naphthaldehyde
and acetone with a >10<sup>3</sup>-fold rate acceleration. The
ease
of organizing convergent functional groups within a protein pore may
make the tunnels of many symmetric ring-shaped proteins useful starting
points for creating designer enzymes
Modular Protein Cages for Size-Selective RNA Packaging in Vivo
Protein cages have recently emerged
as an important platform for
nanotechnology development. Of the naturally existing protein cages,
viruses are among the most efficient nanomachines, overcoming various
barriers to achieve component replication and efficient self-assembly
in complex biological milieu. We have designed an artificial system
that can carry out the most basic steps of viral particle assembly <i>in vivo</i>. Our strategy is based on patchwork capsids formed
from <i>Aquifex aeolicus</i> lumazine synthase and a circularly
permuted variant with appended cationic peptides. These two-component
protein containers self-assemble <i>in vivo</i>, capturing
endogenous RNA molecules in a size-selective manner. By varying the
number and design of the RNA-binding peptides displayed on the lumenal
surface, the length of guest RNA can be further controlled. Using
a fluorescent aptamer, we also show that short-lived RNA species are
captured by the protein cage. This platform has potential as a model
system for investigating virus assembly, as well as developing RNA
regulation or sampling tools to augment biotechnology