2 research outputs found
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
Computational Design of Catalytic Dyads and Oxyanion Holes for Ester Hydrolysis
Nucleophilic catalysis is a general strategy for accelerating
ester
and amide hydrolysis. In natural active sites, nucleophilic elements
such as catalytic dyads and triads are usually paired with oxyanion
holes for substrate activation, but it is difficult to parse out the
independent contributions of these elements or to understand how they
emerged in the course of evolution. Here we explore the minimal requirements
for esterase activity by computationally designing artificial catalysts
using catalytic dyads and oxyanion holes. We found much higher success
rates using designed oxyanion holes formed by backbone NH groups rather
than by side chains or bridging water molecules and obtained four
active designs in different scaffolds by combining this motif with
a Cys-His dyad. Following active site optimization, the most active
of the variants exhibited a catalytic efficiency (<i>k</i><sub>cat</sub>/<i>K</i><sub>M</sub>) of 400 M<sup>–1</sup> s<sup>–1</sup> for the cleavage of a <i>p</i>-nitrophenyl
ester. Kinetic experiments indicate that the active site cysteines
are rapidly acylated as programmed by design, but the subsequent slow
hydrolysis of the acyl-enzyme intermediate limits overall catalytic
efficiency. Moreover, the Cys-His dyads are not properly formed in
crystal structures of the designed enzymes. These results highlight
the challenges that computational design must overcome to achieve
high levels of activity