8 research outputs found
A Fluorogenic TMP-Tag for High Signal-to-Background Intracellular Live Cell Imaging
Developed to complement the use of
fluorescent proteins in live
cell imaging, chemical tags enjoy the benefit of modular incorporation
of organic fluorophores, opening the possibility of high photon output
and special photophysical properties. However, the theoretical challenge
in using chemical tags as opposed to fluorescent proteins for high-resolution
imaging is background noise from unbound and/or nonspecifically bound
ligand-fluorophore. We envisioned we could overcome this limit by
engineering fluorogenic trimethoprim-based chemical tags (TMP-tags)
in which the fluorophore is quenched until binding with <i>E.
coli</i> dihydrofolate reductase (eDHFR)-tagged protein displaces
the quencher. Thus, we began by building a nonfluorogenic, covalent
TMP-tag based on a proximity-induced reaction known to achieve rapid
and specific labeling both <i>in vitro</i> and inside of
living cells. Here we take the final step and render the covalent
TMP-tag fluorogenic. In brief, we designed a trimeric TMP–fluorophore–quencher
molecule (TMP-Q-Atto520) with the quencher attached to a leaving group
that, upon TMP binding to eDHFR, would be cleaved by a cysteine residue
(Cys) installed just outside the binding pocket of eDHFR. We present
the <i>in vitro</i> experiments showing that the eDHFR:L28C
nucleophile cleaves the TMP-Q-Atto520 rapidly and efficiently, resulting
in covalent labeling and remarkable fluorescence enhancement. Most
significantly, while only our initial design, TMP-Q-Atto520 achieved
the demanding goal of not only labeling highly abundant, localized
intracellular proteins but also less abundant, more dynamic cytoplasmic
proteins. These results suggest that the fluorogenic TMP-tag can significantly
impact high-resolution live cell imaging and further establish the
potential of proximity-induced reactivity and organic chemistry more
broadly as part of the growing toolbox for synthetic biology and cell
engineering
d‑Amino Acid-Mediated Translation Arrest Is Modulated by the Identity of the Incoming Aminoacyl-tRNA
A complete understanding
of the determinants that restrict d-amino acid incorporation
by the ribosome, which is of interest
to both basic biologists and the protein engineering community, remains
elusive. Previously, we demonstrated that d-amino acids are
successfully incorporated into the C-terminus of the nascent polypeptide
chain. Ribosomes carrying the resulting peptidyl-d-aminoacyl-tRNA
(peptidyl-d-aa-tRNA) donor substrate, however, partition
into subpopulations that either undergo translation arrest through
inactivation of the ribosomal peptidyl-transferase center (PTC) or
remain translationally competent. The proportion of each subpopulation
is determined by the identity of the d-amino acid side chain.
Here, we demonstrate that the identity of the aminoacyl-tRNA (aa-tRNA)
acceptor substrate that is delivered to ribosomes carrying a peptidyl-d-aa-tRNA donor further modulates this partitioning. Our discovery
demonstrates that it is the pairing of the peptidyl-d-aa-tRNA
donor and the aa-tRNA acceptor that determines the activity of the
PTC. Moreover, we provide evidence that both the amino acid and tRNA
components of the aa-tRNA acceptor contribute synergistically
to the extent of arrest. The results of this work deepen our understanding
of the mechanism of d-amino acid-mediated translation arrest
and how cells avoid this precarious obstacle, reveal similarities
to other translation arrest mechanisms involving the PTC, and provide
a new route for improving the yields of engineered proteins containing d-amino acids
Fluorescence Polarization Assay for Small Molecule Screening of FK506 Biosynthesized in 96-Well Microtiter Plates
The fluorescence
polarization (FP) assay has been widely used to
study enzyme kinetics, antibody–antigen interactions, and other
biological interactions. We propose that the FP assay can be adapted
as a high-throughput and potentially widely applicable screen for
small molecules. This is useful in metabolic engineering, which is
a promising approach to synthesizing compounds of pharmaceutical,
agricultural, and industrial importance using bioengineered strains.
There, the development of high-yield strains is often a costly and
time-consuming process. This problem can be addressed by generating
and testing large mutant strain libraries. However, a current key
bottleneck is the lack of high-throughput screens to detect the small
molecule products. The FP assay is quantitative, sensitive, fast,
and cheap. As a proof of principle, we established the FP assay to
screen for FK506 (tacrolimus) produced by <i>Streptomyces tsukubaensis</i>, which was cultivated in 96-well plates. An ultraviolet mutagenized
library of 160 colonies was screened to identify strains showing higher
FK506 productivities. The FP assay has the potential to be generalized
to detect a wide range of other small molecules
A Heritable Recombination System for Synthetic Darwinian Evolution in Yeast
Genetic recombination is central to the generation of
molecular
diversity and enhancement of evolutionary fitness in living systems.
Methods such as DNA shuffling that recapitulate this diversity mechanism <i>in vitro</i> are powerful tools for engineering biomolecules
with useful new functions by directed evolution. Synthetic biology
now brings demand for analogous technologies that enable the controlled
recombination of beneficial mutations in living cells. Thus, here
we create a Heritable Recombination system centered around a library
cassette plasmid that enables inducible mutagenesis <i>via</i> homologous recombination and subsequent combination of beneficial
mutations through sexual reproduction in <i>Saccharomyces cerevisiae</i>. Using repair of nonsense codons in auxotrophic markers as a model,
Heritable Recombination was optimized to give mutagenesis efficiencies
of up to 6% and to allow successive repair of different markers through
two cycles of sexual reproduction and recombination. Finally, Heritable
Recombination was employed to change the substrate specificity of
a biosynthetic enzyme, with beneficial mutations in three different
active site loops crossed over three continuous rounds of mutation
and selection to cover a total sequence diversity of 10<sup>13</sup>. Heritable Recombination, while at an early stage of development,
breaks the transformation barrier to library size and can be immediately
applied to combinatorial crossing of beneficial mutations for cell
engineering, adding important features to the growing arsenal of next
generation molecular biology tools for synthetic biology
Second-Generation Covalent TMP-Tag for Live Cell Imaging
Chemical tags are now viable alternatives to fluorescent
proteins
for labeling proteins in living cells with organic fluorophores that
have improved brightness and other specialized properties. Recently,
we successfully rendered our TMP-tag covalent with a proximity-induced
reaction between the protein tag and the ligand-fluorophore label.
This initial design, however, suffered from slow <i>in vitro</i> labeling kinetics and limited live cell protein labeling. Thus,
here we report a second-generation covalent TMP-tag that has a fast
labeling half-life and can readily label a variety of intracellular
proteins in living cells. Specifically, we designed an acrylamide-trimethoprim-fluorophore
(A-TMP-fluorophore v2.0) electrophile with an optimized linker for
fast reaction with a cysteine (Cys) nucleophile engineered just outside
the TMP-binding pocket of Escherichia coli dihydrofolate reductase (eDHFR) and developed an efficient chemical
synthesis for routine production of a variety of A-TMP-probe v2.0
labels. We then screened a panel of eDHFR:Cys variants and identified
eDHFR:L28C as having an 8-min half-life for reaction with A-TMP-biotin
v2.0 <i>in vitro</i>. Finally, we demonstrated live cell
imaging of various cellular protein targets with A-TMP-fluorescein,
A-TMP-Dapoxyl, and A-TMP-Atto655. With its robustness, this second-generation
covalent TMP-tag adds to the limited number of chemical tags that
can be used to covalently label intracellular proteins efficiently
in living cells. Moreover, the success of this second-generation design
further validates proximity-induced reactivity and organic chemistry
as tools not only for chemical tag engineering but also more broadly
for synthetic biology
Cooperative Vinculin Binding to Talin Mapped by Time-Resolved Super Resolution Microscopy
The dimeric focal adhesion protein
talin contains up to 22 cryptic
vinculin binding sites that are exposed by unfolding. Using a novel
method to monitor the in situ dynamics of the talin dimer stretch,
we find that in contrast to several prevalent talin dimer models the
integrin-binding talin N-termini are separated by 162 ± 44 nm
on average whereas as expected the C-terminal dimerization domains
colocalize and are mobile. Using vinculin tagged by DHFR-TMP Atto655
label, we found that optimal vinculin and vinculin head binding occurred
when talin was stretched to 180 nm, while the controls did not bind
to talin. Surprisingly, multiple vinculins bound within a single second
in narrowly localized regions of the talin rod during stretching.
We suggest that talin stretches as an antiparallel dimer and that
activates vinculin binding in a cooperative manner, consistent with
the stabilization of folded talin by other binding proteins
Conditional Glycosylation in Eukaryotic Cells Using a Biocompatible Chemical Inducer of Dimerization
Conditional Glycosylation in Eukaryotic Cells Using a Biocompatible Chemical Inducer of Dimerizatio
Identification of PDE6D as a Molecular Target of Anecortave Acetate <i>via</i> a Methotrexate-Anchored Yeast Three-Hybrid Screen
Glaucoma and age-related macular degeneration are ocular
diseases
targeted clinically by anecortave acetate (AA). AA and its deacetylated
metabolite, anecortave desacetate (AdesA), are intraocular pressure
(IOP)-lowering and angiostatic cortisenes devoid of glucocorticoid
activity but with an unknown mechanism of action. We used a methotrexate-anchored
yeast three-hybrid (Y3H) technology to search for binding targets
for AA in human trabecular meshwork (TM) cells, the target cell type
that controls IOP, a major risk factor in glaucoma. Y3H hits were
filtered by competitive Y3H screens and coimmunoprecipitation experiments
and verified by surface plasmon resonance analysis to yield a single
target, phosphodiesterase 6-delta (PDE6D). PDE6D is a prenyl-binding
protein with additional function outside the PDE6 phototransduction
system. Overexpression of PDE6D in mouse eyes caused elevated IOP,
and this elevation was reversed by topical ocular application of either
AA or AdesA. The identification of PDE6D as the molecular binding
partner of AA provides insight into the role of this drug candidate
in treating glaucoma