Bai, Ruiliang

Basser, Peter J.

Elsevier - Publisher Connector 

Multi-Component Water Dynamics and Exchange in Brain Cortical Tissue Probed via In-Vitro D-T2 2D Correlation NMR 

Wednesday, February 11, 2015 615astorage, and release of the neurotransmitter acetylcholine and its postsynaptic
activation of the nicotinic acetylcholine receptor. Many lines of evidence sup-
port the idea that in addition to acetylcholine, additional neurotransmitters and/
or neuromodulators are also released from cholinergic synapses. Utilizing a
single-vesicle imaging approach, we have found that individual synaptic vesi-
cles from the electric ray possess neurotransmitter transporters for glutamate,
ATP, and acetylcholine. In addition to those transporters, cholinergic synaptic
vesicles from the electric ray also possess an orphaned transporter from the
bile acid transport family, SLC10A4. We are now expanding our research to
identify the molecules transported by SLC10A4 using multiple biochemical
approaches and a mouse knockout model.
Magnetic Resonance Spectroscopy, Imaging, and
EPR Spectroscopy
3101-Pos Board B531
Multi-ComponentWater Dynamics and Exchange in Brain Cortical Tissue
Probed via In-Vitro D-T2 2D Correlation NMR
Ruiliang Bai1,2, Peter J. Basser1.
1National Institutes of Health, Bethesda, MD, USA, 2University of Maryland,
College Park, MD, USA.
Water self-diffusion in brain cortical tissue has been shown to have two com-
ponents by diffusion NMR measurement. This phenomenon is tissue
microstructure-related and is essential for understanding the diffusion NMR
signals’ changes associated with some physiological processes, such as acute
stroke, neuronal activation, etc. However, the biophysical mechanisms explain-
ing multi-component diffusion inside cortical tissue and possible exchange
among components are still poorly understood.
Here we propose using a Diffusion (D) – Transverse Relaxation (T2) 2D corre-
lation NMRmethod to characterize the multi-component water dynamics in the
biological tissue. In the first dimension, the diffusion is measured by a spin echo
in a strong static magnetic field gradient, which can efficiently shorten the diffu-
sion time to be less than 1.6ms, making the effect of exchange negligible. In the
second dimension, a CPMG pulse sequence is used to measure the T2 of each
diffusion component. Here, exchange will affect the NMR signal evolution dur-
ing this long T2 encoding period. In this 2DD-T2 NMRmethod, each dynamics-
different water component can be characterized by its specific (D, T2) combina-
tion and the exchange process among different components can also be quanti-
fied simultaneously. Another unique aspect of this work is that our NMR data is
obtained using a single-sided NMR system with permanent magnet.
Furthermore, temperature-dependent D-T2 experiments were performed on
cortical tissue from fixed rat brain. The self-diffusion constant of both the
fast and slow diffusion components and the exchange between them are slowed
as the temperature decreases, whereas the slow diffusion component shows
higher translational activation energy. More interesting and surprising, the rela-
tive fraction of each component is also observed to have a strong dependence
on the temperature, which indicates this bi-component diffusion is diffusion-
distance dependent.
3102-Pos Board B532
Probing the Secondary Structure of Membrane Proteins with the Pulsed
EPR Technique: Electron Spin Ehco Envelope Modulation (ESEEM)
Lishan Liu, Gary Lorigan.
Miami University, Oxford, OH, USA.
Despite the importance and large number of membrane proteins, relatively
limited structural information is known about them. New biophysical tech-
niques are needed to probe their structural properties. Their hydrophobic nature
and low expression yields cause difficulties for traditional structural techniques
such as x-ray crystallography and solution NMR. ESEEM spectroscopy indi-
rectly observes NMR transitions through an electron spin coupled to a nearby
NMR active nucleus. ESEEM can detect weak dipolar interactions between a
NMR active nucleus and a spin label out to a distance of approximately 8 A˚.
The modulation depth for weakly coupled nuclei is scaled by 1/r6. A novel
approach is being developed to probe the secondary structure of membrane pro-
teins and peptides qualitatively utilizing the three-pulse Electron Spin Echo
Envelope Modulation (ESEEM) pulse sequence. In order to demonstrate the
practicality of this biophysical technique, the M2delta subunit of AChR (a-he-
lical) and KIGAKI(b-sheet) peptides were incorporated into phospholipids bi-
celle to probe their secondary structure with ESSEM spectroscopy. Utilizing
site-directed spin-labeling (SDSL) coupled with deuterated amino acid labeling
of the peptides, the corresponding ESEEM spectra reveal characteristic patterns
for a-helix and b-sheet structures. This ESSEM secondary structural approach
can be used with different deuterated amino acids and provide pertinent qual-
itative structural information on membrane proteins in a short period of time
(10 minutes) with small amounts of sample (30 mg).3103-Pos Board B533
Spin-Labeled Uni-Lamellar Vesicles as an Oxygen Sensitive Analyte for
Measurement of Cellular Respiration using Rat Dopaminergic Neuronal
Cells
Laxman Mainali, Jason W. Sidabras, Theodore Camenisch,
Jeannette Vasquez-Vivar, James Hyde, Witold K. Subczynski.
Biophysics, Medical College of Wisconsin, Milwaukee, WI, USA.
Small uni-lamellar vesicles were prepared from 1-palmitoyl-2-oleoylphospha-
tidylcholine (POPC) with 1% spin label of 1-palmitoyl-2-(16-doxylstearoyl)
phosphatidylcholine (16-PC). POPC membranes are in the fluid phase in the
physiological temperature range, which ensures high solubility of oxygen in
the membrane center where the 16-PC nitroxide moiety is located. The nitro-
xide moiety is isolated from cellular reductants and paramagnetic ions that
might interfere with spin-label oximetry measurements. This analyte is stable
in cell suspensions and can be used in studies as long as 400 min. The saturation
recovery EPR approach, possibly for the first time, was used to study cellular
respiration of rat dopaminergic neuronal cells. It is widely accepted that this
approach is the most direct way to carry out oximetric studies. The preliminary
experiments were carried out at X-band for closed chamber geometry. Oximet-
ric measurements were performed for cell concentrations from 750 to 5000
cells in one mL of culture media. Results confirmed that the oxygen consump-
tion rate was linearly proportional to the number of cells. Furthermore at the
lower cell concentration, we found a breaking point in the rate which is not
due to diffusion limitation but is due to the change in cell metabolism because
there is not enough oxygen, consistent with Michaelis-Menten kinetics. In the
developed analyte, the same hydrocarbon environment, which dissolves oxy-
gen very well, always surrounds nitroxide moieties of spin-label molecules.
Therefore, the partial pressure of oxygen influence the EPR spectrum of
spin-labels in the analyte. Such microscopic analytes are readily and uniformly
distributed within the sample, thus giving a rapid response to changes in oxygen
partial pressure. The improved oximetry methodology developed here presents
a high impact opportunity for non-invasive assessment of disease mechanisms
and response to therapeutic intervention.
3104-Pos Board B534
Probing the Protein-Protein Interactions between KCNQ1 and KCNE1
using Electron Paramagnetic Resonance (EPR) Spectroscopy
Andrew F. Craig, Indra D. Sahu, Rongfy Zhang, Megan M. Dunagan,
Kunkun Wang, Robert M. McCarrick, Gary A. Lorigan.
Chemistry, Miami University, Oxford, OH, USA.
KCNQ1 is a membrane-bound protein composed of 676 amino acids with a mo-
lecular weight of 75 kD and contains 6 transmembrane helices. KCNQ1 is a
voltage-gated potassium channel primarily involved in the transport of potas-
sium in the human heart. The activity of KCNQ1 is modulated by the integral
membrane protein KNCE1 (also known as MinK). This potassium modulation
is critical for repolarization in the heart, specifically in the left ventricle. Muta-
tions in the KCNQ1 and KCNE1 genes have been linked to genetic disorders
such as long QT syndrome, sudden infant death syndrome (SIDS), as well as
deafness. However, most current studies between KNCQ1 and KCNE1 have
been from an electrophysiological or functional standpoint, which leaves struc-
tural and dynamic information on the proteins unknown. EPR spectroscopy is a
powerful structural biology tool that can be used to probe both protein struc-
tures as well as protein-protein interactions between KCNQ1 and KCNE1.
Currently, we have overexpressed full length KCNE1 and the C-terminal
domain of KCNQ1. In this study, we used both continuous-wave (CW) EPR
and the pulsed EPR technique of double electron-electron resonance (DEER)
to probe the structural and dynamic parameters of the interaction of KCNQ1
and KCNE1.
3105-Pos Board B535
Characterization of a Bifunctional Spin Label for the Structure and
Dynamics of a Membrane Protein using CW-EPR Spectroscopy
Lauren M. Bottorf, Indra D. Sahu, Lishan Liu, Gary A. Lorigan.
Chemistry and Biochemistry, Miami University, Oxford, OH, USA.
Electron Paramagnetic Resonance (EPR) spectroscopy is a powerful struc-
tural biology tool when combined with site-directed spin labeling (SDSL)
to study the structure and dynamics of peptides and proteins. The most widely
used spin label for SDSL is methanthiosulfonate (MTSL), however the flex-
ibility of this spin label introduces greater uncertainties in the EPR measure-
ments obtained for determining structure, side chain dynamics, and backbone
motion of membrane protein systems. Most recently, a new bifunctional spin
label (BSL) 3,4-Bis-(methanethiosulfonylmethyl)-2,2,5,5-tetramethyl-2,5-di-
hydro-1H-pyrrol-1-yloxy, has been introduced to overcome some of the lim-
itations associated with the MTSL spin label and has been invaluable in
determining protein dynamics and inter-residue distances due to its restricted
internal motion and fewer size restrictions. While BSL has been successful in


file:///data/remote/core/dit/data/elsevier/pdf/f77/aHR0cDovL2FwaS5lbHNldmllci5jb20vY29udGVudC9hcnRpY2xlL3BpaS9zMDAwNjM0OTUxNDA0NTU4NQ==.pdf

Multi-Component Water Dynamics and Exchange in Brain Cortical Tissue Probed via In-Vitro D-T2 2D Correlation NMR

Abstract

Similar works

Full text

Available Versions

Elsevier - Publisher Connector

Elsevier - Publisher Connector