Cell Reports Physical Science,
Год журнала:
2023,
Номер
4(11), С. 101638 - 101638
Опубликована: Окт. 18, 2023
The
post-translational
modifications
of
histone
H4
play
significant
roles
in
regulation
epigenetic
status.
Reconstituted
nucleosomes
that
mimic
the
native
chromatin
are
valuable
for
modification
research,
which
requires
site-specifically
modified
histone.
Sortase-mediated
ligation
(SML)
has
advantages
easy
preparation
substrates
and
robust
kinetics,
recent
nucleosome-based
deacylation
research
H3
H2B
benefitted
from
SML.
Here
we
report
a
novel
semisynthesis
method
via
SML,
allowed
us
to
prepare
containing
multivalently
investigations
deacetylation
by
sirtuins.
In
addition,
identified
subfamily
PWWP
domain-containing
proteins
as
H4K20me3
readers
photo-crosslinking
multifunctional
nucleosome
probes.
studies
revealed
new
mechanisms
might
not
be
available
using
peptides.
As
result,
is
expected
accelerate
our
understanding
modifications,
will
expand
H4K20
methylation.
Journal of the American Chemical Society,
Год журнала:
2022,
Номер
144(29), С. 13118 - 13126
Опубликована: Июль 18, 2022
Asparaginyl
endopeptidases
(AEPs)
have
recently
been
widely
utilized
for
peptide
and
protein
modification.
Labeling
is
however
restricted
to
termini,
severely
limiting
flexibility
scope
in
creating
diverse
conjugates
as
needed
therapeutic
diagnostic
applications.
Here,
we
use
genetic
code
expansion
site-specifically
modify
target
proteins
with
an
isopeptide-linked
glycylglycine
moiety
that
serves
acceptor
nucleophile
AEP-mediated
transpeptidation
various
probes
containing
a
tripeptidic
recognition
motif.
Our
approach
allows
simple
flexible
labeling
of
recombinant
at
any
internal
site
leaves
minimal,
entirely
peptidic
footprint
(NGG)
the
conjugation
product.
We
show
site-specific
biophysical
probes,
including
dual
N-terminus.
Furthermore,
harness
generation
ubiquitin-
ubiquitin-like-modifier
bearing
native
isopeptide
bond
only
one
point
mutation
linker
region.
Nature Chemistry,
Год журнала:
2024,
Номер
16(9), С. 1481 - 1489
Опубликована: Май 24, 2024
Abstract
Transpeptidases
are
powerful
tools
for
protein
engineering
but
largely
restricted
to
acting
at
backbone
termini.
Alternative
enzymatic
approaches
internal
labelling
require
bulky
recognition
motifs
or
non-proteinogenic
reaction
partners,
potentially
restricting
which
proteins
can
be
modified
the
types
of
modification
that
installed.
Here
we
report
a
strategy
lysine
side
chain
ε-amines
by
repurposing
an
engineered
asparaginyl
ligase,
naturally
catalyses
peptide
head-to-tail
cyclization,
versatile
isopeptide
ligations
compatible
with
peptidic
substrates.
We
find
lysines
adjacent
leucine
residue
mimic
conventional
N-terminal
glycine–leucine
substrate.
This
dipeptide
motif
enables
efficient
intra-
intermolecular
ligation
through
chains,
minimally
leaving
asparagine
C-terminally
linked
via
bond.
The
versatility
this
approach
is
demonstrated
chemoenzymatic
synthesis
peptides
non-native
C
terminus-to-side
topology
and
conjugation
chemically
recombinant
proteins.
Journal of the American Chemical Society,
Год журнала:
2025,
Номер
unknown
Опубликована: Янв. 2, 2025
Enzyme-catalyzed
protein
modifications
have
become
invaluable
in
diverse
applications,
outperforming
chemical
methods
terms
of
precision,
conjugation
efficiency,
and
biological
compatibility.
Despite
significant
advances
ligases,
such
as
sortase
A
OaAEP1,
their
use
heterogeneous
environments
remains
constrained
by
limited
target
sequence
specificity.
In
2021,
Lupas'
group
introduced
Connectase,
a
family
repurposed
archaeal
proteases
for
ligations,
but
its
low
processivity
lack
structural
information
impeded
further
engineering
practical
biophysical
applications.
Here,
we
present
the
X-ray
crystallographic
structures
MmConnectase
(Methanococcus
maripaludis,
MmCET)
both
apo
substrate-bound
forms.
Comparative
analysis
with
inactive
paralogue,
MjCET
janaschi),
reveals
basis
MmCET's
high-precision
ligation
activity.
We
propose
to
N-terminal
substrate
recognition
motifs
suppress
reversible
protease
activity,
enabling
ligations
complex
environments,
serum-containing
cell
cultures.
To
demonstrate
enhanced
single-molecule
unfolding
experiments
showed
that
our
optimized
conjunction
OaAEP1(C247A),
can
perform
stepwise
tandem
leading
well-defined
polymer.
RSC Chemical Biology,
Год журнала:
2025,
Номер
unknown
Опубликована: Янв. 1, 2025
NRPS
modules
are
expressed
and
complexed
with
substrate
analogues
separately
then
ligated
to
stall
at
a
specific
catalytic
step,
investigated
using
crystallography.
The
protein
ligase
Connectase
can
be
used
to
fuse
proteins
small
molecules,
solid
carriers,
or
other
proteins.
Compared
ligases,
it
offers
greater
substrate
specificity,
higher
catalytic
efficiency,
and
catalyzes
no
side
reactions.
However,
its
reaction
is
reversible,
resulting
in
only
50%
fusion
product
from
two
equally
abundant
educts.
Here,
we
present
a
simple
method
reliably
obtain
100%
1:1
conjugation
This
efficient
for
protein-protein
protein-peptide
fusions
at
the
N-or
C-termini.
It
enables
generation
of
defined
completely
labeled
antibody
conjugates
with
one
partner
on
each
chain.
requires
short
incubation
times
amounts
enzyme
effective
even
low
concentrations
temperatures.
With
these
characteristics,
presents
valuable
new
tool
bioengineering.
