15 research outputs found
1-O-Sulfatobastadins-1 and -2 from Ianthella basta (Pallas).Antagonists of the yR1-FKBP12 Ca2+ Channel
Two new sulfate monoesters of hemibastadins-1 and -2 were isolated from the marine sponge Ianthella basta (Pallas) from Guam. A third new compound was tentatively assigned the structure 34-O-sulfatobastadin-9. The 1-O-sulfatohemibastadins-1 and –2 were antagonists of the RyR1-FKBP12 Ca2+ channel under conditions where the known compound bastadin-5 exhibits potent agonism (EC50 2μM)
Quantifying Hormone Disruptors with an Engineered Bacterial Biosensor
Endocrine disrupting
compounds are found in increasing amounts
in our environment, originating from pesticides, plasticizers, and
pharmaceuticals, among other sources. Although the full impact of
these compounds is still under study, they have already been implicated
in diseases such as obesity, diabetes, and cancer. The list of chemicals
that disrupt normal hormone function is growing at an alarming rate,
making it crucially important to find sources of contamination and
identify new compounds that display this ability. However, there is
currently no broad-spectrum, rapid test for these compounds, as they
are difficult to monitor because of their high potency and chemical
dissimilarity. To address this, we have developed a new detection
strategy for endocrine disrupting compounds that is both fast and
portable, and it requires no specialized skills to perform. This system
is based on a native estrogen receptor construct expressed on the
surface of <i>Escherichia coli</i>, which enables both the
detection of many detrimental compounds and signal amplification from
impedance measurements due to the binding of bacteria to a modified
electrode. With this approach, sub-ppb levels of estradiol and ppm
levels of bisphenol A are detected in complex solutions. Rather than
responding to individual components, this system reports the total
estrogenic activity of a sample using the most relevant biological
receptor. As an applied example, estrogenic chemicals released from
a plastic baby bottle following microwave heating were detectable
with this technique. This approach should be broadly applicable to
the detection of chemically diverse classes of compounds that bind
to a single receptor
Genetically encoded sensors of protein hydrodynamics and molecular proximity
The specialized light organ of the ponyfish supports the growth of the bioluminescent symbiont Photobacterium leiognathi. The bioluminescence of P. leiognathi is generated within a heteromeric protein complex composed of the bacterial luciferase and a 20-kDa lumazine binding protein (LUMP), which serves as a Förster resonance energy transfer (FRET) acceptor protein, emitting a cyan-colored fluorescence with an unusually long excited state lifetime of 13.6 ns. The long fluorescence lifetime and small mass of LUMP are exploited for the design of highly optimized encoded sensors for quantitative fluorescence anisotropy (FA) measurements of protein hydrodynamics. In particular, large differences in the FA values of the free and target-bound states of LUMP fusions appended with capture sequences of up to 20 kDa are used in quantitative FA imaging and analysis of target proteins. For example, a fusion protein composed of LUMP and a 5-kDa G protein binding domain is used as an FA sensor to quantify the binding of the GTP-bound cell division control protein 42 homolog (Cdc42) (21 kDa) in solution and within Escherichia coli. Additionally, the long fluorescence lifetime and the surface-bound fluorescent cofactor 6,7-dimethyl-8- (1′-dimethyl-ribityl) lumazine in LUMP are utilized in the design of highly optimized FRET probes that use Venus as an acceptor probe. The efficiency of FRET in a zero-length LUMP-Venus fusion is 62% compared to ∼31% in a related CFP-Venus fusion. The improved FRET efficiency obtained by using LUMP as a donor probe is used in the design of a FRET-optimized genetically encoded LUMP-Venus substrate for thrombin
Mechanism of Lithium Diisopropylamide-Mediated Ortholithiation of 1,4-Bis(trifluoromethyl)benzene under Nonequilibrium Conditions: Condition-Dependent Rate Limitation and Lithium Chloride-Catalyzed Inhibition
Lithiation
of 1,4-bisÂ(trifluoromethyl)Âbenzene with lithium diisopropylamide
in tetrahydrofuran at −78 °C occurs under conditions at
which the rates of aggregate exchanges are comparable to the rates
of metalation. Under such nonequilibrium conditions, a substantial
number of barriers compete to be rate limiting, making the reaction
sensitive to trace impurities (LiCl), reactant concentrations, and
isotopic substitution. Rate studies using the perdeuterated arene
reveal odd effects of LiCl, including catalyzed rate acceleration
at lower temperature and catalyzed rate inhibition at higher temperatures.
The catalytic effects are accompanied by corresponding changes in
the rate law. A kinetic model is presented that captures the critical
features of the LiCl catalysis, focusing on the influence of LiCl-catalyzed
re-aggregation of the fleeting monomer that can reside above, at,
or below the equilibrium population without catalyst
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Structural and Biochemical Studies of Actin in Complex with Synthetic Macrolide Tail Analogues
The actin filament-binding and filament-severing activities of the aplyronine, kabiramide, and reidispongiolide families of marine macrolides are located within the hydrophobic tail region of the molecule. Two synthetic tail analogues of aplyronine C (SF-01 and GC-04) are shown to bind to G-actin with dissociation constants of (285±33) and (132±13) nM, respectively. The crystal structures of actin complexes with GC-04, SF-01, and kabiramide C reveal a conserved mode of tail binding within the cleft that forms between subdomains (SD) 1 and 3. Our studies support the view that filament severing is brought about by specific binding of the tail region to the SD1/SD3 cleft on the upper protomer, which displaces loop-D from the lower protomer on the same half-filament. With previous studies showing that the GC-04 analogue can sever actin filaments, it is argued that the shorter complex lifetime of tail analogues with F-actin would make them more effective at severing filaments compared with plasma gelsolin. Structure-based analyses are used to suggest more reactive or targetable forms of GC-04 and SF-01, which may serve to boost the capacity of the serum actin scavenging system, to generate antibody conjugates against tumor cell antigens, and to decrease sputum viscosity in children with cystic fibrosis
Lithium Diisopropylamide-Mediated Lithiation of 1,4-Difluorobenzene under Nonequilibrium Conditions: Role of Monomer‑, Dimer‑, and Tetramer-Based Intermediates and Lessons about Rate Limitation
Lithiation
of 1,4-difluorobenzene with lithium diisopropylamide
(LDA) in THF at −78 °C joins the ranks of a growing number
of metalations that occur under conditions in which the rates of aggregate
exchanges are comparable to the rates of metalation. As such, a substantial
number of barriers vie for rate limitation. Rate studies reveal that
rate-limiting steps and even the choice of reaction coordinate depend
on subtle variations in concentration. Deuteration shifts the rate-limiting
step and markedly alters the concentration dependencies and overall
rate law. This narrative is less about ortholithiation per se and
more about rate limitation and the dynamics of LDA aggregate exchange
Lithium Diisopropylamide-Mediated Ortholithiation of 2‑Fluoropyridines: Rates, Mechanisms, and the Role of Autocatalysis
Lithium diisopropylamide (LDA)-mediated
ortholithiations of 2-fluoropyridine
and 2,6-difluoropyridine in tetrahydrofuran at −78 °C
were studied using a combination of IR and NMR spectroscopic and computational
methods. Rate studies show that a substrate-assisted deaggregation
of LDA dimer occurs parallel to an unprecedented tetramer-based pathway.
Standard and competitive isotope effects confirm post-rate-limiting
proton transfer. Autocatalysis stems from ArLi-catalyzed deaggregation
of LDA proceeding via 2:2 LDA–ArLi mixed tetramers. A hypersensitivity
of the ortholithiation rates to traces of LiCl derives from LiCl-catalyzed
LDA dimer–monomer exchange and a subsequent monomer-based ortholithiation.
Fleeting 2:2 LDA–LiCl mixed tetramers are suggested to be key
intermediates. The mechanisms of both the uncatalyzed and catalyzed
deaggregations are discussed. A general mechanistic paradigm is delineated
to explain a number of seemingly disparate LDA-mediated reactions,
all of which occur in tetrahydrofuran at −78 °C