2 research outputs found
Fast, Ratiometric FRET from Quantum Dot Conjugated Stabilized Single Chain Variable Fragments for Quantitative Botulinum Neurotoxin Sensing
Botulinum neurotoxin (BoNT) presents
a significant hazard under numerous realistic scenarios. The standard
detection scheme for this fast-acting toxin is a lab-based mouse lethality
assay that is sensitive and specific, but slow (ā¼2 days) and
requires expert administration. As such, numerous efforts have aimed
to decrease analysis time and reduce complexity. Here, we describe
a sensitive ratiometric fluorescence resonance energy transfer scheme
that utilizes highly photostable semiconductor quantum dot (QD) energy
donors and chromophore conjugation to compact, single chain variable
antibody fragments (scFvs)
to yield a fast, fieldable sensor for BoNT with a 20ā40 pM
detection limit, toxin quantification, adjustable dynamic range, sensitivity
in the presence of interferents, and sensing times as fast as 5 min.
Through a combination of mutations, we achieve stabilized scFv denaturation
temperatures of more than 60 Ā°C, which bolsters fieldability.
We also describe adaptation of the assay into a microarray format
that offers persistent monitoring, reuse, and multiplexing
<i>Bacillus anthracis</i> Inosine 5ā²-Monophosphate Dehydrogenase in Action: The First Bacterial Series of Structures of Phosphate Ionā, Substrateā, and Product-Bound Complexes
Inosine 5ā²-monophosphate dehydrogenase (IMPDH)
catalyzes
the first unique step of the GMP branch of the purine nucleotide biosynthetic
pathway. This enzyme is found in organisms of all three kingdoms.
IMPDH inhibitors have broad clinical applications in cancer treatment,
as antiviral drugs and as immunosuppressants, and have also displayed
antibiotic activity. We have determined three crystal structures of <i>Bacillus anthracis</i> IMPDH, in a phosphate ion-bound (termed
āapoā) form and in complex with its substrate, inosine
5ā²-monophosphate (IMP), and product, xanthosine 5ā²-monophosphate
(XMP). This is the first example of a bacterial IMPDH in more than
one state from the same organism. Furthermore, for the first time
for a prokaryotic enzyme, the entire active site flap, containing
the conserved Arg-Tyr dyad, is clearly visible in the structure of
the apoenzyme. Kinetic parameters for the enzymatic reaction were
also determined, and the inhibitory effect of XMP and mycophenolic
acid (MPA) has been studied. In addition, the inhibitory potential
of two known <i>Cryptosporidium parvum</i> IMPDH inhibitors
was examined for the <i>B. anthracis</i> enzyme and compared
with those of three bacterial IMPDHs from <i>Campylobacter jejuni</i>, <i>Clostridium perfringens</i>, and <i>Vibrio cholerae</i>. The structures contribute to the characterization of the active
site and design of inhibitors that specifically target <i>B.
anthracis</i> and other microbial IMPDH enzymes