56a Sunday, March 3, 2019
simulations. Our results will provide more information for the design of drug
and novel therapeutic methods.
281-Pos
Structural Studies of Membrane Proteins using Pulsed EPR Spectroscopy
Gary A. Lorigan1, Indra D. Sahu1, Daniel L. Drew1, Gunjan Dixit2,
Tanbir Ahammad1.
1
Dept Chem/Biochem, Miami Univ, Oxford, OH, USA, 2Cell Molecular and
Structural Biology, Miami University Ohio, Oxford, OH, USA.
Solid-state NMR and EPR spectroscopy are important biophysical techniques
that are being using to study membrane proteins. CW and pulsed EPR spec-
troscopic techniques coupled with site-directed spin-labeling (SDSL) can
provide important structural information on complicated biological systems
such as membrane proteins. Strategically placed spin-labels alter relaxation
times of NMR active nuclei and yield pertinent structural information. EPR
techniques such as Double Electron-Electron Resonance (DEER) and Elec-
tron Spin Echo Envelope Modulation (ESEEM) are powerful structural
biology tools. The DEER technique can be used to measure distances between
2 spin labels from 20 to 70 Å. However, the application of DEER spectros-
copy to study membrane proteins can be difficult due to short phase memory
times (Tm) and weak DEER modulation in more biologically relevant proteo-
liposomes when compared to water soluble proteins or membrane proteins in
detergent micelles. The combination of these factors often leads to broad dis-
tance distributions, poor signal to noise, and limitations in the determination
of longer distances. The short phase memory times are typically due to un-
even distributions of spin-labeled protein within the lipid bilayer, which cre-
ates local inhomogeneous pockets of high spin concentrations. Approaches to
overcome these limitations and improve the quality of DEER measurements
for membrane proteins will be discussed: lipodisq nanoparticles, bi-
functional spin labels (BSL), and Q-band pulsed EPR spectroscopy. ESEEM
data will be used to probe the secondary structure of membrane proteins.
ESEEM can be thought of as EPR detected NMR. CW-EPR spectra of
spin-labeled membrane proteins will be used to investigate dynamics and
the immersion depth in a lipid bilayer. A variety of different membrane pro-
teins will be probed with these state-of-the art magnetic resonance
techniques.
282-Pos
Structural and Functional Role of the Surface-Exposed Loops of Ail Dur-
ing Complement-Mediated Evasion by Y. pestis
Luz Marina Meneghini, Chandan Singh, Kyungsoo Shin, L. Miya Fujimoto,
Ye Tian, Francesca M. Marassi.
Sanford Burnham Prebys Medical Discovery Inst, La Jolla, CA, USA.
Yersinia pestis, the causative agent of plague, promotes its proliferation in the
host by expressing various bacterial proteins that inhibit the host innate im-
mune system. One of these is attachment invasion locus (Ail), which is an
eight-stranded b-barrel outer membrane protein with four surface exposed
extracellular loops. Ail is essential for Y. pestis pathogenesis and proliferation
as it recruits host serum proteins to inhibit the complement-dependent bacte-
riolysis and inflammatory response, and regulates host cell attachment and
delivery of Yersinia outer proteins to host tissue. Recently, we demonstrated
that Y. pestis can also recruit vitronectin (Vn) from the human serum in Ail-
dependent manner. Vn is a 459-residue multi-domain protein that interacts
with various molecules to promote cell adhesion and migration, and inhibits
formation of the membrane attack complex, indicating that the interaction be-
tween Ail and Vn may be responsible for promoting Y. pestis survival and
pathogenesis. Notably, previous works have indicated that serum proteins
such as Vn are recruited by Ail via its extracellular loops. Thus, we hypoth-
esized that Ail’s extracellular loops may also be responsible for binding to
Vn. To characterize this interaction, amino acids in each extracellular loop
of Ail were substituted for alanine to individually characterize the loops
for Vn binding. The structure and function of each Ail loop mutant was char-
acterized in a native like environment inside membrane (detergent micelle
and nanodiscs) by solution state NMR and ELISA binding assays. These
studies identify specific extracellular loops of Ail that are important for bind-
ing to Vn, which will guide future developments of therapeutics aimed at
combating Y. pestis infections.
283-Pos
Nanodiscs as a Platform for the Study of Human P-Glycoprotein in a
Membrane Environment
Sabrina Lusvarghi, Suresh Ambudkar.
LCB/CCR/NCI/NIH, Bethesda, MD, USA.
Multidrug resistance of cancer cells and pathogens is a significant clinical
problem. A major contributor to drug resistance in cancer cells is overex-
pression of P-glycoprotein (P-gp), a member of the ATP-binding cassette
superfamily. P-gp is an ATP-dependent efflux pump with broad substrate
specificity responsible for effluxing xenobiotic substances such as toxins
or drugs from the cell. Absorption by the body of orally administered drugs
is reduced by the presence of P-gp, which facilitates their elimination from
the body. P-gp consists of two transmembrane domains and two nucleotide-
binding domains. Our laboratory has extensively characterized this protein
biochemically using a variety of systems, including native membranes, pu-
rified in detergent micelles or in proteoliposomes. Here we demonstrate the
advantages of using nanodiscs for studying and characterizing P-gp. Nano-
discs are synthetic membrane model systems that have been widely used
to study membrane proteins. They are structurally similar to high-density
lipoproteins and are composed of a belt or membrane scaffolding protein
derived from apolipoprotein-A1 and phospholipids. Nanodiscs are consid-
ered optimal membrane mimetic systems because size, composition,
and specific functional modifications can be controlled on the nanometer
scale. The incorporation of P-gp into nanodiscs was optimized to achieve
highly homogenous preparations. We demonstrate that the nanodiscs
better resemble the behavior of P-gp in membranes when compared to
protein purified in detergent micelles. Finally, P-gp nanodiscs were used
to study conformational changes associated with the ATP hydrolysis cycle.
The UIC2 antibody, which binds to the extracellular region of
P-gp, was found to dissociate during ATP hydrolysis, indicating that the
extracellular loops change in conformation as the molecule hydrolyzes
ATP on the opposite side of the protein. All in all, we demonstrate that
upon optimization, nanodiscs are a valuable platform for studying P-
glycoprotein.
284-Pos
Structural Study for Membrane Protein using Solution X-Ray Scattering
Contrast Variation
Xiaobing Zuo.
X-ray Science Divison, Argonne Natl Lab, Lemont, IL, USA.
Membrane protein often needs lipid bilayer membrane to stabilize its struc-
ture and to fulfill its functions. However, the presence of lipid bilayer mem-
brane makes the structural study on the protein very difficult. Here
we proposed to use X-ray scattering contrast variation method in which
membrane protein (with lipid membrane) samples will be measured in a se-
ries of solutions with various sucrose concentration. X-ray scattering
intensity measures the scattering length/capability difference between sam-
ple/solute and buffer solution/solvent. Since X-ray scattering lengths of pro-
tein and lipid are different and scattering length of sucrose buffer changes
along with its concentration, the ratio of X-ray scattering contribution
from protein part and lipid part varies along with the sucrose concentration.
In such way, we can separate the scattering contribution of protein part from
that of lipid. We also simultaneously fit the data sets collected from the
sucrose concentration series measurements and obtain the 3-D low re-
solution structure for the protein and the lipid at the same time. Comparing
to neutron scattering contrast variation method, this X-ray contrast method
is less expensive and can provide wider q range due to the availability
of high flux X-ray sources. A few membrane protein examples will be
presented. Figure. SAXS data for membrane protein in various sucrose
solution.
In this presentation, I will also discuss the capabilities of beamline 12-ID-B of
the Advanced Photon Source at Argonne National Laboratory.
Acknowledgement: Use of the Advanced Photon Source, an Office of Science
User Facility operated for the U.S. Department of Energy (DOE) Office of
Science by Argonne National Laboratory, was supportedby the U.S. DOE un-
der Contract No. DE-AC02-06CH11357.
285-Pos
Structure and Mechanisms of an Anion Transporter Family
Robert M. Stroud, Jonathan Leano, Samir Batarni, Robert Edwards.
Dept Biochem/Biophys, Univ Calif San Francisco, San Francisco, CA, USA.
Members of the solute carrier 17 family use divergent mechanisms to
concentrate organic anions. Membrane potential drives uptake of the prin-
cipal excitatory neurotransmitter glutamate into synaptic vesicles, whereas
closely related proteins use electroneutral cotransport to drive efflux from
the lysosome. To identify the common features of ionic coupling by the
SLC17 family, we determined the structure of E. coli D-galactonate/Hþ
symporter DgoT in two states: one open to the cytoplasmic side, and the
other open to the periplasmic side with substrate bound. The structures
identify a proton translocation pathway conserved from bacteria to mam-
mals. Functional analysis suggests that a transition in the role of Hþ
from flux coupling to allostery may underlie the divergence in energy
source.