3 research outputs found
Construction of a Rhythm Transfer System That Mimics the Cellular Clock
Creation of an artificial oscillating gene expression
system is
one of the most challenging issues in synthetic biology. Here, we
constructed a simple system to manipulate gene expression patterns
to be circadian, reflecting the intrinsic cellular clock, by fusing
a core clock protein, BMAL1 or CLOCK, with a zinc finger-type DNA
binding domain. Circadian rhythmic gene expression was induced only
when the target gene contained zinc finger-binding sequences. To our
knowledge, this simple approach is the first to manipulate gene expression
patterns into circadian rhythms and would be applicable to various
endogenous genes
Temperature-Resistant Bicelles for Structural Studies by Solid-State NMR Spectroscopy
Three-dimensional structure determination
of membrane proteins
is important to fully understand their biological functions. However,
obtaining a high-resolution structure has been a major challenge mainly
due to the difficulties in retaining the native folding and function
of membrane proteins outside of the cellular membrane environment.
These challenges are acute if the protein contains a large soluble
domain, as it needs bulk water unlike the transmembrane domains of
an integral membrane protein. For structural studies on such proteins
either by nuclear magnetic resonance (NMR) spectroscopy or X-ray crystallography,
bicelles have been demonstrated to be superior to conventional micelles,
yet their temperature restrictions attributed to their thermal instabilities
are a major disadvantage. Here, we report an approach to overcome
this drawback through searching for an optimum combination of bicellar
compositions. We demonstrate that bicelles composed of 1,2-didecanoyl-<i>sn</i>-glycero-3-phosphocholine (DDPC) and 1,2-diheptanoyl-<i>sn</i>-glycero-3-phosphocholin (DHepPC), without utilizing additional
stabilizing chemicals, are quite stable and are resistant to temperature
variations. These <i>temperature-resistant bicelles</i> have
a robust bicellar phase and magnetic alignment over a broad range
of temperatures, between −15 and 80 °C, retain the native
structure of a membrane protein, and increase the sensitivity of solid-state
NMR experiments performed at low temperatures. Advantages of two-dimensional
separated-local field (SLF) solid-state NMR experiments at a low temperature
are demonstrated on magnetically aligned bicelles containing an electron
carrier membrane protein, cytochrome <i>b</i><sub>5</sub>. Morphological information on different DDPC-based bicellar compositions,
varying <i>q</i> ratio/size, and hydration levels obtained
from <sup>31</sup>P NMR experiments in this study is also beneficial
for a variety of biophysical and spectroscopic techniques, including
solution NMR and magic-angle-spinning (MAS) NMR for a wide range of
temperatures
Rapid <sup>13</sup>C Hyperpolarization of the TCA Cycle Intermediate α‑Ketoglutarate via SABRE-SHEATH
α-Ketoglutarate is a key biomolecule involved in
a number
of metabolic pathwaysmost notably the TCA cycle. Abnormal
α-ketoglutarate metabolism has also been linked with cancer.
Here, isotopic labeling was employed to synthesize [1-13C,5-12C,D4]α-ketoglutarate with the future
goal of utilizing its [1-13C]-hyperpolarized state for
real-time metabolic imaging of α-ketoglutarate analytes and
its downstream metabolites in vivo. The signal amplification
by reversible exchange in shield enables alignment transfer to heteronuclei
(SABRE-SHEATH) hyperpolarization technique was used to create 9.7%
[1-13C] polarization in 1 minute in this isotopologue.
The efficient 13C hyperpolarization, which utilizes parahydrogen
as the source of nuclear spin order, is also supported by favorable
relaxation dynamics at 0.4 μT field (the optimal polarization
transfer field): the exponential 13C polarization buildup
constant Tb is 11.0 ± 0.4 s whereas
the 13C polarization decay constant T1 is 18.5 ± 0.7 s. An even higher 13C polarization
value of 17.3% was achieved using natural-abundance α-ketoglutarate
disodium salt, with overall similar relaxation dynamics at 0.4 μT
field, indicating that substrate deuteration leads only to a slight
increase (∼1.2-fold) in the relaxation rates for 13C nuclei separated by three chemical bonds. Instead, the gain in
polarization (natural abundance versus [1-13C]-labeled)
is rationalized through the smaller heat capacity of the “spin
bath” comprising available 13C spins that must be
hyperpolarized by the same number of parahydrogen present in each
sample, in line with previous 15N SABRE-SHEATH studies.
Remarkably, the C-2 carbon was not hyperpolarized in both α-ketoglutarate
isotopologues studied; this observation is in sharp contrast with
previously reported SABRE-SHEATH pyruvate studies, indicating that
the catalyst-binding dynamics of C-2 in α-ketoglutarate differ
from that in pyruvate. We also demonstrate that 13C spectroscopic
characterization of α-ketoglutarate and pyruvate analytes can
be performed at natural 13C abundance with an estimated
detection limit of 80 micromolar concentration × *%P13C. All in all, the fundamental studies reported here
enable a wide range of research communities with a new hyperpolarized
contrast agent potentially useful for metabolic imaging of brain function,
cancer, and other metabolically challenging diseases