BIOPHEST
Organizing Committee: Logan Ahlstrom, Brian Anderson, Michael Brown, Martha Cassetti, Udeep
Chawla, Koen Visscher
Time
Event
8:00 AM-8:55 AM
Breakfast (Room 103a, BIO5)
9:00 AM-10:45 AM
Morning session #1 (Chair: Logan Ahlstrom)
Trivikram Molugu (UA) “In vitro evolved non-aggregating and thermostable
lipase.”
Allan Friesen (ASU) “Energetics of biological electron transfer in cytochrome
B562.”
Elana Stennett (ASU) “Photobleaching and blinking of tetramethylrhodamine on
DNA induced by paramagnetic cations.”
Brian Anderson (UA) “Single molecule force spectroscopy of arrhythmogenic
cardiomyopathy-linked titin mutation implicates reduced Ig domain stability.”
Ashini Bolia (ASU) “BP-Dock: a flexible docking scheme for exploring proteinligand interactions.”
Melanie Dannemeyer (ASU) “Combined atomic force and confocal fluorescence
microscopy for elasticity measurements on living cells.”
Daniel Martin (ASU) “Protein dynamics, electron transfer, and solvation theory.”
10:45 AM-11:00AM
11:00 AM- 1:00PM
Coffee Break
Morning session #2 (Chair: Brian Anderson)
K.J. Mallikarjunaiah (UA) "Membrane structure deformation and its area
compressibility modulus: 2H NMR and SAXS."
Angelo Di Bernardo (ASU) “Electronic and vibrational properties of magnetic
core-shell nanoparticles.”
Nicholas Laude (UA) “Hybrid microfluidics and conducting polymer electrodes
for quantitative analysis of biological small molecules.”
Anindya Roy (ASU) “De novo design and synthesis of artificial metal binding
peptide motifs: towards a functional mimic of [Fe-Fe] hydrogenases.”
Jack Rory Staunton (ASU) “Measuring the elasticity of cells embedded in 3D
matrices.”
Chad Park (UA) “Activated oligomerization of SgrAl.”
Stephanie Cope (ASU) “Conformation and dynamics of monomeric IAPP:
implications for amyloid aggregation.”
Dmitry Matyushov (ASU) “Dia-electric proteins in solution.”
1:00 PM-2:00PM
Lunch Break
2
2:00 PM-4:00 PM
Afternoon session #1 (Chair: Jack Rory Staunton)
Ming Zhao (UA) “Fourier multi-color lifetime excitation-emission matrix imaging.”
Manas Chakraborty (ASU) “Investigating the assembly pathway of ADP-bound
RuBisCO activase by Fluorescence Fluctuation Spectroscopy.”
Eric Monroe (UA) “Developing an electrochemical oxidation method for probing
protein structure via hydroxyl radical footprinting mass spectrometry.”
Agnieszka Kuriata (ASU) “Self-assembly of rubisco activase.”
Xu Xiaolin (UA) “Solid-state deuterium NMR spectroscopy reveals structural
changes of mixed-lipid bilayers under osmotic pressure.”
James Zook (ASU) "High resolution NMR reveals secondary structure and
folding of amino acid transporter from the outer chloroplast membrane."
Jennifer Binder (ASU) “Monitoring the dimerization of GpA and ME1 using
FRET.”
Christopher Atcherley (UA) “Delayed timing voltammetry for measuring
adsorption kinetics.”
4:00 PM-4:15 PM
4:15 PM-6:15 PM
Coffee Break
Afternoon session #2 (Chair: Stephanie Cope)
Fernanda Camacho-Alanis (ASU) “Combining focused ion beam milling and
optical lithography to fabricate microfluidic devices for DNA and protein
dielectrophoresis.”
Sara Sizemore (ASU) “Transient tertiary contact formation in the CGRP
neuropeptide revealed by nanosecond laser spectroscopy.”
Suchithranga Perera (UA) “Steric and electronic factors govern torsional energy
landscape of retinal in rhodopsin activation.”
Bryant Doss (ASU) “Combined AFM nanoindentation and finite element analysis
on soft heterogeneous materials.”
Mowei Zhou (UA) “Probing protein quaternary structures by surface collision,
ion mobility, and mass spectrometry.”
Suratna Hazra (ASU) “Kinetics study of rubisco activase.”
Asuka Nakano (ASU) “Streaming dielectrophoresis of proteins in a microfluidic
platform.”
Logan Ahlstrom (UA) “Adding dynamical insight to Cro Dimer X-Ray structures
by solution and crystal molecular dynamics simulation.”
6:15 PM-6:30 PM
Closing Remarks
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ABSTRACT LIST
9:00AM
Trivikram Molugu
[email protected]
"In vitro evolved non-aggregating and thermostable lipase." Trivikram R. Molugu, Kamal
MZ, Ahmad S, Vijayalakshmi A, Deshmukh MV, Sankaranarayanan R, and Rao NM
Chemistry and Biochemistry, University of Arizona
We have created a robust variant of a lipase from Bacillus subtilis named "6B" using
multiple rounds of in vitro evolution. The melting temperature and optimum activity
temperature of 6B is 78 °C and 65 °C, respectively, which is ~22 °C and 30 °C higher than
that of wild-type lipase. Most significantly, 6B does not aggregate upon heating. Physical
basis of remarkable thermostability and non-aggregating behavior of 6B was explored using
X-ray crystallography, NMR and differential scanning calorimetry. Our structural
investigations highlight the importance of tightening of mobile regions of the molecule such
as loops and helix termini to attain higher thermostability. Accordingly, NMR studies
suggest a very rigid structure of 6B lipase. Our study suggest that better anchoring of the
loops with the rest of the protein molecule through mutations particularly on the sites that
perturb/disturb the exposed hydrophobic patches can simultaneously increase protein
stability and aggregation resistance [JMB(2011)28,p726].
9:15AM Allan Friesen
[email protected]
"Energetics of biological electron transfer in cytochrome B562." Allan Friesen and Dmitry
Matyushov
Chemistry and Biochemistry, Arizona State University
We present molecular dynamics simulations of the redox protein cytochrome B562 in
water. Several recent studies have indicated that non-Gaussian fluctuations of the electric
potential at the redox site lead to lower free energy barriers for biological electron
transfer. In this cytochrome, the heme sits in a pocket near the surface of the protein, ligated
by a methionine sulfur and a histidine nitrogen. We show that the electrostatic fluctuations at
the redox site are sensitive to changes in the rigidity of the coordination around the heme
iron, indicating a possible tuning mechanism for energetics of biological electron transfer.
9:30AM Elana Stennett
[email protected]
"Photobleaching and blinking of tetramethylrhodamine on DNA induced by paramagnetic
cations." Elana Stennett and Marcia Levitus
Chemistry and Biochemistry, Arizona State University
Work on a manganese-dependent protein exhibited fluorescence fluctuations of
tetramethylrhodamine (TAMRA) in the presence of manganese but not magnesium. This
project seeks to understand the origin of the fluctuations and why they occur in the presence
of certain cations. Fluorescence correlation spectroscopy was used to investigate the
photophysical behavior of an internally labeled TAMRA-DNA. In the presence of
manganese, a sub-millisecond fluctuation is present in the correlation decay of TAMRADNA that is not present in the presence of magnesium. Flash photolysis experiments showed
the fluorescence fluctuationsis from the formation of a triplet. The enhanced triplet formation
in manganese is likely because of the paramagnetic cation causing enhanced spin-orbit
coupling interactions, as well as the close proximity of the dye to the cation due to the
presence of DNA. This work has important implications for single molecule measurements
where photophysical behavior can complicate data analysis.
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9:45AM Brian Anderson
[email protected]
"Single molecule force spectroscopy of arrhythmogenic cardiomyopathy-linked titin
mutation implicates reduced Ig domain stability." Brian Anderson and Henk Granzier
Physics, University of Arizona
Titin plays crucial roles in sarcomere organization and cardiac elasticity by acting as an
intrasarcomeric molecular spring. A mutation in the 10th immunoglobulin(Ig)-like domain in
titin’s spring region is associated with arrhythmogenic cardiomyopathy (AC), a disease
characterized by ventricular arrhythmias leading to cardiac arrest and sudden death. Titin is
the first sarcomeric protein linked to AC. To characterize the disease mechanism we have
used atomic force microscopy to directly measure the effects that the disease-linked point
mutation (T16I) has on the mechanical and kinetic stability of Ig10 at the single molecule
level. The mutation decreases the force needed to unfold Ig10 and increases its rate of
unfolding fourfold. We also found that T16I Ig10 is more prone to degradation, presumably
due to compromised local protein structure. Overall, the disease-linked mutation weakens the
structural integrity of titin’s Ig10 domain and suggests an Ig domain instability based genesis
of AC development.
10:00AM Ashini Bolia
[email protected]
"BP-Dock: a flexible docking scheme for exploring protein-ligand interactions." Ashini Bolia
and Banu Ozkan
Chemistry and Biochemistry, Arizona State University
Protein functionality is frequently associated with the interaction between proteins and small
molecule ligands or peptides. Current molecular docking approaches serve as an important
tool in modeling such protein-ligand interactions but are inadequate and unable to
incorporate protein binding site flexibility due to the large conformational space that needs
sampling. Our flexible docking approach BP-Dock (Backbone Perturbation-Dock) can
integrate both backbone and side chain conformational changes in protein while simulating
such protein-ligand interactions unlike conventional docking approaches that rely only on
ligand flexibility. BP-Dock is based on a coarse grained approach called Perturbation
Response Scanning (PRS) that couples elastic network model with linear response theory to
predict conformational changes upon binding. Currently, we are modeling cyanovirindimannose interactions and reengineering CV-N mutants to optimize the oligo-mannoside
binding site to further improve its affinity.
10:15AM Melanie Dannemeyer
[email protected]
“Combined atomic force and confocal fluorescence microscopy for elasticity measurements
on living cells.” Melanie Dannemeyer and Robert Ros
Physics, Arizona State University
The combination of atomic force microscopy (AFM) and fluorescence microscopy technique
has a wide range of applications in biophysics and nanosciences. In this talk, I will introduce
a setup combining AFM with a single molecule sensitive confocal laser scanning microscope
(CLSM). Alignment of the AFM tip with the confocal volume, as well as the synchronization
of the two instruments, allows simultaneous and correlated acquisition of AFM and
fluorescence data. Besides standard AFM and CLSM imaging, the setup allows us to
combine imaging with nanoindentation experiments as it is of great interest for the
determination of the cellular structure and mechanics. As an example, I will show elasticity
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measurements on non-tumorigenic and metastatic breast cancer cells, as well as the influence
of microtubule stains on these mechanical properties.
10:30AM Daniel Martin
[email protected]
"Dissipative electro-elastic network model (DENM) of protein electrostatics.” Daniel Martin
and Dmitry Matyushov
Physics, Arizona State University
We have developed a dissipative electro-elastic network model to describe the dynamics and
statistics of electrostatic fluctuations at active sites of proteins. The model combines the
harmonic network of residue beads with overdamped dynamics of the normal modes of the
network characterized by two friction coefficients. The electrostatic component is introduced
to the model through atomic charges of the protein force field. The overall effect of the
electrostatic fluctuations of the network is recorded through the frequency-dependent
response functions of the electrostatic potential and electric field at the protein active
site. The model is tested against loss spectra of residue displacements and the electrostatic
potential and electric field at the heme’s iron from all-atom molecular dynamics simulations
of three hydrated globular proteins.
11:00AM K.J. Mallikarjunaiah
[email protected]
"Membrane structure deformation and its area compressibility modulus: 2H NMR and
SAXS." K.J. Mallikarjunaiah and Michael F. Brown
Chemistry and Biochemistry, University of Arizona
Cellular functions relay on molecular structure and dynamics of biomembranes, and
intermembrane forces play a significant role in biological processes. The sensitivity of lipid
bilayer structure to osmotic pressure [1] and temperature enable us to understand the
influences of non-specific lipid-protein interactions on functions of cellular membranes.
NMR lipid order parameters are very sensitive to changes in cross-sectional area per
molecule. Measurements of membrane structural parameters such as bilayer thickness and
area per lipid employ a mean-torque analysis of deuterium NMR order parameters. The
elastic area compressibility modulus of bilayers is determined (KA = 142 ± 30 mJm-2) for
DMPC by employing pressure techniques in combination with NMR and vapor pressure
osmometry methods. The present NMR study demonstrates the ability to distinguish different
regimes of intermembrane forces. It suggests that the undulations dominate at intermediate
intermembrane distances where protrusions dominate at short distances. [1] K.J.
Mallikarjunaiah et al. (2011) BJ 100, 98--107.
11:15AM Angelo Di Bernardo
[email protected]
"Electronic and vibrational properties of magnetic core-shell nanoparticles.” Angelo Di
Bernardo and Vladimiro Mujica
Chemistry and Biochemistry, Arizona State University
Magnetic nanoparticle hyperthermia (MNH) is a promising cancer therapy, wherein magnetic
nanoparticles (MNPs) are used to destroy tumor cells by heating induction in the presence of
external magnetic fields. The issue at hand is to develop more efficient MNPs through a
thorough understanding of the factors responsible for magnetic losses. In our study we have
focused on core/shell Fe@TiO2 MNPs which combine the magnetic nature of the core with
the high biocompatibility, fluorescent properties and photocatalytic activity of the shell. After
determining the optimized geometry of these nanocomposites with spin unrestricted TD-
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DFT, their Raman activity has also been evaluated to identify the most relevant modes for
energy transfer occurring in MNH. Modeling the hysteresis cycle of these MNPs as a
transition from a low-spin to a high-spin state, a new strategy for the determination of their
magnetic susceptibility using TD-DFT has been proposed.
11:30AM Nicholas Laude
[email protected]
"Hybrid microfluidics and conducting polymer electrodes for quantitative analysis of
biological small molecules." Nicholas Laude and Michael Heien
Chemistry and Biochemistry, University of Arizona
Sensitive and robust detection schemes for capillary electrophoresis separations of attomole
quantities of biological materials are needed. Hybrid microfluidics and conducting polymer
electrodes allow for a high coloumetric efficiency electrochemical detection platform which
can be readily coupled to capillary electrophoresis and flow injection systems. These
systems are cheap to manufacture, biocompatible, and are sensitive enough to be used with
existing separation-based assays or in order to improve efficiency in detection. Exploration
of new assay formats including on-line derivatization for electrochemical detection following
separation are also made possible through these hybrid systems. Continued development of
this technology will lead to improvements in analytical study of amino acid and monoamine
neurotransmitters at the single cell and tissue level.
11:45AM Anindya Roy
[email protected]
"De novo design and synthesis of artificial metal binding peptide motifs: towards a
functional mimic of [Fe-Fe] hydrogenases." Anindya Roy and Giovanna Ghirlanda
Chemistry and Biochemistry, Arizona State University
In search for clean, dependable and sufficient amount of alternative non polluting, carbon
neutral fuel, hydrogen is arguably one of the best choices. In nature, hydrogenases that
catalyze the reversible conversion of hydrogen to protons are metalloenzymes often with two
distinct subunits for functioning. One carries a bimetallic center and other one contains an
accessory [4Fe-4S] center. We are utilizing designed peptides to develop artificial model
system capable of performing the same reversible proton reduction. For the [Fe-S]
component, we have chosen DSD, a de novo designed domain swapped bis-[4Fe-4S] cluster
binding dimeric protein as our preliminary scaffold to formulate a multi-cofactor electron
transfer chain. We are also developing peptide framework to house a di-iron organometallic
complex modeled after hydrogenase catalytic site. Integration of the two components in a
complex peptide based scaffold will bring together a biological cofactor for effective electron
transfer and an effective proton reduction catalyst, respectively.
12:00PM Jack Rory Staunton
[email protected]
"Measuring the elasticity of cells embedded in 3D matrices." Jack Rory Staunton and Robert
Ros
Physics, Arizona State University
While breast cancer tumors are around fourfold stiffer than normal tissue, the elasticity of
cancerous breast cells has previously been shown to be about twice that of normal
counterparts. This tissue stiffening and concurrent cell softening demonstrates the importance
of the interplay between cells and their microenvironment. Unfortunately, most cell biology
and biophysics research studies use cells on 2D substrates like polystyrene, even though cell
motility, adhesion and morphology are all fundamentally different in 3D microenvironments,
7
which more closely mimic physiological conditions. Our objective is to measure the
elasticity of cells embedded in 3D matrices using a combination of AFM indentation,
confocal fluorescence microscopy, and finite element model simulations. We will discuss
some experimental constraints and sample preparation methods and present preliminary
results on metastatic breast cancer cells embedded in 3D collagen I hydrogels.
12:15PM Chad Park
[email protected]
"Activated oligomerization of SgrAI." Chad Park
Chemistry and Biochemistry, University of Arizona
SgrAI, a sequence specific endonuclease, has been discovered to function using a unique
molecular pathway involving activation by oligomerization. The dimeric form of SgrAI
binds and cleaves its primary site sequence very slowly, however under activating conditions
SgrAI will oligomerize and exhibit a 200 fold increase in DNA cleavage activity as well as
expanded sequence specificity. The oligomers of SgrAI have been characterized by
analytical ultracentrifugation and electron microscopy, and exhibit a braided rod like
structure heterogeneous in length. The unusual biochemical properties of SgrAI may have
been selected to provide protection from invading phage DNA in an organism with a
relatively large bacterial genome.
12:30PM Stephanie Cope
[email protected]
"Conformation and dynamics of monomeric IAPP: implications for amyloid aggregation."
Stephanie Cope and Sara Vaiana
Physics, Arizona State University
Islet amyloid polypeptide (IAPP) is an intrinsically disordered protein with hormone
properties related to glucose uptake and regulation. It is implicated in the pathogenesis of
diabetes type II, because of its deposition in the form of insoluble amyloid fibers within the
beta cells of the pancreas, where insulin is produced. The ability of the protein to aggregate
into amyloid fibers or into other oligomeric states depends on the conformational and
dynamical properties of individual peptides, as well as on their ability to form favorable
inter-protein interactions. Using a combination of techniques able to probe structural and
dynamical properties of IDPs at the intra- and inter-protein level (including nanosecond
laser-pump spectroscopy) we compare an aggregating, a non-aggregating and an inhibitor
sequence variant of hIAPP. Our results suggest a dual role of chain rigidity on IAPP
aggregation.
12:45PM Dmitry Matyushov
[email protected]
"Dia-electric proteins in solution." Dmitry Matyushov
Physics, Arizona State University
We show, by analyzing dielectric absorption data, that proteins can display dia-electric
response in solution, i.e. repel from a strong electric field (J. Chem. Phys. 136, 085102
(2012)).
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2:00PM
Ming Zhao
[email protected]
"Fourier multi-color lifetime excitation-emission matrix imaging." Ming Zhao and Leilei
Peng
Optical Sciences, University of Arizona
We report a Fourier transform lifetime spectroscopy method that simultaneously measures
fluorescence lifetime and intensity on multiple excitation and emission channels in 45.5
microseconds. We combined the lifetime spectroscopy method with confocal microscopy and
scanning–laser optical tomography to perform lifetime excitation-emission matrix (LEEM)
3D imaging in cells and small organisms such as zebrafish. Multi-channel LEEM images
enables quantitative analysis of multicolor Förster resonance energy transfer, which opens
the door to in vivo study of complex protein-to-protein interactions and multi-modal
physiological sensing from the cellular level to the whole organism level.
2:15PM
Manas Chakraborty
[email protected]
"Investigating the assembly pathway of ADP-bound RuBisCO activase by Fluorescence
Fluctuation Spectroscopy." Manas Chakraborty and Marcia Levitus
Chemistry and Biochemistry, Arizona State University
Ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) is an enzyme that catalyzes
the carboxylation of the substrate Ribulose-1,5-bisphosphate . The notorious inefficiency of
RuBisCO to catalyze carboxylation is due to inhibition by various metabolites. RuBisCO
activase, an ancillary enzyme is needed to foster the activity of RuBisCO. Activase has been
recognized as a member of the AAA+ family of the ATPases. It facilitates the removal of
firmly bound sugar phosphates thereby restoring RuBisCO activity. The stoichiometry and
oligomerization kinetics of fluorescently tagged RuBisCO activase was investigated in a
wide range of concentrations using Fluorescence Correlation Spectroscopy (FCS) in
conjunction with Photon counting Histogram (PCH) analysis. Experiments revealed that
Activase exists as a monomer at sub-micro molar concentrations, and assembles into
oligomers (possible hexamers) at higher concentrations. The analysis of the concentrationdependent diffusion coefficient revealed that the binding between the subunits occurs in steps
involving intermediates.
2:30PM
Eric Monroe
[email protected]
“Developing an electrochemical oxidation method for probing protein structure via hydroxyl
radical footprinting mass spectrometry." Eric Monroe and Michael Heien
Chemistry and Biochemistry, University of Arizona
The structural dynamics of a protein plays an integral role in its biological function and
interactions. One method to examine protein structure utilizes mass spectrometry to study
proteins that have been covalently labeled by hydroxyl radicals produced by the photolysis of
hydrogen peroxide or the radiolysis of water by high-power synchrotron radiation, both
rather costly endeavors. Here we present a facile, inexpensive methodology for the
production of hydroxyl radicals through the electrochemical oxidation of hydrogen peroxide.
Radicals are produced at a glassy-carbon electrode and react with amino acid side chains of
the protein of interest. Initial experiments were performed with model proteins in bulk
solution to optimize the oxidation and analysis strategies. The method is currently being
incorporated into microfluidic devices to improve the production and temporal control of
hydroxyl radicals for these footprinting experiments.
9
2:45PM
Agnieszka Kuriata
[email protected]
"Self-assembly of rubisco activase." Agnieszka Kuriata and Rebekka Wachter
Chemistry and Biochemistry, Arizona State University
Rubisco is an abundant enzyme that catalyzes the carboxylation of the substrate ribulose-1,5bisphosphate by atmospheric CO2. The carbon fixation is inhibited by several sugar
phosphates or substituted with a side reaction- photorespiration. The Rubisco activase (Rca)
reactivates dead-end Rubisco complexes by facilitating the release of tight-binding inhibitors
from the active sites. To better understand the subunit stoichiometry of Rca assemblies, we
are using Fluorescence Correlation Spectroscopy (FCS) and Photon Counting Histogram
(PCH) analysis to develop a thermodynamic self-association model. For these studies,
recombinantly produced Cotton short-form activase is C-terminally labeled with an ALEXA
fluorophore. The PCH results indicate that activase exists as a monomer at nanomolar
concentrations, and the FCS results provide evidence that activase self-associates to
hexameric and higher order assembly states in the low micromolar range. The concentrationdependence of the apparent diffusion coefficient supports a step-wise assembly model with
multiple intermediate states.
3:00PM
Xu Xiaolin
[email protected]
"Solid-state deuterium NMR spectroscopy reveals structural changes of mixed-lipid bilayers
under osmotic pressure." Xiaolin Xu, Muwei Zheng, K. J. Mallikarjunaiah and Michael F.
Brown
Physics, University of Arizona
Cellular membrane properties are sensitive to pressure, temperature, and dehydration as well
as lipid composition [1]. Solid-state 2H NMR can provide detailed information regarding
how structure and associated dynamics of lipid bilayers are affected by external factors.
Utilizing 2H NMR, we have studied mixed-lipid membrane bilayers under osmotic pressure
to understand lipid-protein interactions, as well as to determine viscoelastic properties and
their dependence upon membrane composition. Interpreting 2H NMR spectra with the
mean-torque-model [2], we find that addition of detergents leads to swelling of the lipids,
which is counteracted by applying osmotic pressure. By contrast, the stiffening action of
cholesterol reduces swelling of multilamellar dispersions, and is reinforced by osmotic
pressure. We conclude that membrane deformation involving lipid-protein interactions can
play key roles in biological functions of pressure-sensitive proteins and channels. [1] K.J.
Mallikarjunaiah et al. (2011) BJ 100, 98-107. [2] H.I. Petrache et al. (2000) BJ 79, 31723192.
3:15PM
James Zook
[email protected]
"High resolution NMR reveals secondary structure and folding of amino acid transporter
from the outer chloroplast membrane." James Zook and Petra Fromme
Chemistry and Biochemistry, Arizona State University
Membrane proteins are a growing interest in structural biology. Over 50% of drug targets
are membrane proteins making them medicinally important. They are also major players in
photosynthesis and thus are vital in alternative energy research. However, membrane protein
structures are underrepresented in the protein database (1%) considering that 20%-30% of
the genome encodes for membrane proteins. There are few membrane protein structures
because crystallization for x-ray diffraction studies is difficult. Nuclear Magnetic Resonance
(NMR) spectroscopy provides a crystal-free method for obtaining high resolution data. This
10
study presents the results of NMR spectroscopy of the integral membrane protein: Outer
Envelope Protein, 16kDa (OEP16). Several multiple dimension NMR experiments were
used to achieve >99% of backbone residue assignments. Chemical shifts provided secondary
structure and estimated order parameters (S2) via TALOS+. Relaxation data provided
insight into the intramolecular motion. Titration experiments provided insight into the
structure and function of OEP16.
3:30PM
Jennifer Binder
[email protected]
"Monitoring the dimerization of GpA and ME1 using FRET." Jennifer Binder and Marcia
Levitus
Chemistry and Biochemistry, Arizona State University
The association of transmembrane helical proteins to homodimers is driven by a handful of
sequence motifs, the most common of which is GXXXG. We sought to understand whether
heterodimers could be created via additional interactions that we introduced between the
peptides. We designed a buried salt bridge in the transmembrane domain of a well-known
dimeric membrane protein, GpA, as well as a structurally related protein, ME1. We mutated
Thr 87, which is not part of the dimerization interface, to diaminopropionic acid (Dap) on
one of the helices and to aspartic acid on the other. Dap and aspartic acid interact
electrostatically only in a narrow pH window. We characterized the pH-dependent
association of the peptides when incorporated into micelles using fluorescence resonance
energy transfer (FRET). This allowed us to establish the pH profile for heterodimer
formation and to measure the strength of the interaction.
3:45PM
Christopher Atcherley
[email protected]
"Delayed timing voltammetry for measuring adsorption kinetics." Christopher Atcherley and
Michael Heien
Chemistry and Biochemistry, University of Arizona
The equilibrium constant for the adsorption of dopamine and serotonin was determined using
delayed-timing voltammetry at cylindrical carbon-fiber microelectrodes. In this work, a
single electrode and relay were used to switch between two waveforms at a single electrode
at discrete time points. A triangle waveform (400V/s -0.4V-1.3V) was applied to the
electrode when the relay was closed; when the switch was open, a constant -0.4V signal was
applied to the electrode. The current for the oxidation of dopamine and serotonin was
integrated and it was found that there was an increase in the measured concentration when
compared to the application of the continuous waveform. Additionally, finite-difference
simulations were performed using Comsol Multiphysics 4.0 to model the surface
accumulation, and the charge of the adsorbed dopamine that would be measured given the
flow rate, concentration, and equilibrium constant.
4:15PM
Fernanda Camacho-Alanis
[email protected]
"Combining focused ion beam milling and optical lithography to fabricate microfluidic
devices for DNA and protein dielectrophoresis." Fernanda Camacho-Alanis, Lin Gan, and
Alexandra Ros
Chemistry and Biochemistry, Arizona State University
Dielectrophoresis (DEP) is a technique relying on electric field gradients to sort
biomolecules. One of the challenges is to enhance the DEP force without increasing the
applied voltages to avoid Joule heating effects. Our approach is to modify the insulating post
11
geometry inside the channel using focused ion beam milling (FIBM). The experiments were
performed by recording the movements of particles with fluorescence microscopy. We
demonstrate that adding rectangular nano-posts with FIBM in combination with triangular
micro-posts using optical lithography, λ-DNA DEP trapping is obtained. In the case of
proteins (bovine serum albumin), although DEP trapping is not observed, however,
streaming DEP leads to a high concentration of proteins. Numerical simulations using a
convection-diffusion model taking into account diffusion, electrokinesis and
dielectrophoresis agree well with the experimental results. This study indicates that the novel
fabrication process has the potential to improve applications for dielectrophoric separation,
concentration and fractionation of biomolecules.
4:30PM
Sara Sizemore
[email protected]
"Transient tertiary contact formation in the CGRP neuropeptide revealed by nanosecond
laser spectroscopy." Sara Sizemore and Sara Vaiana
Physics, Arizona State University
Calcitonin gene-related peptide (CGRP) is an intrinsically disordered, 37 residue
neuropeptide that acts as a potent vasodilator, which is of considerable interest in migrane
research. It is a member of the calcitonin peptide (Ct) family, together with amylin,
calcitonin and adrenomedullin. These are genetically and structurally related intrinsically
disordered hormone peptides that are able to bind to each other’s receptors, though with
varying degrees of affinity. They contain highly conserved sequence elements that have been
shown to affect the secondary structural preferences of these peptides. The effect of such
conserved elements on tertiary structure has not been experimentally explored. We use a
nanosecond-resolved spectroscopic technique based on tryptophan triplet quenching by
cystine to detect tertiary contact formation in CGRP under varying solvent and temperature
conditions. We compare our results to those found for other members of Ct peptide family
which differ in mean hydrophobicity and net charge per residue.
4:45PM
Suchithranga Perera
[email protected]
"Steric and electronic factors govern torsional energy landscape of retinal in rhodopsin
activation." Suchithranga Perera, Blake Mertz, Michael Lu, Andrey Struts, Scott E. Feller,
and Michael F. Brown
Chemistry and Biochemistry, University of Arizona
Molecular dynamics (MD) simulations of the rhodopsin are of great interest in membrane
biophysics. Methyl group dynamics of retinal within the binding pocket of rhodopsin play a
crucial role in activation. Solid-state 2H-NMR studies have shown three distinct rotational
energy barriers for the C5-, C9- and C13- methyl groups as opposed to the standard value (12
kJ/mol) currently used in the CHARMM retinal force-field. This study presents quantum
mechanical calculations (MP2/6-31G**) of torsional energy surfaces for the above three
methyl groups for retinal model compounds. The calculations yielded three unique rotational
energy barriers for C5-, C9-, and C13- methyl rotations consistent with the 2H-NMR
inversion recovery experiments. This work illustrates steric and electronic influences on
retinal methyl dynamics, which implies the need for improved dihedral force-fields for
rhodopsin MD simulations. [1] B. Mertz et al. (2011) Biophys. J. 101, L17-L19. [2] A.V.
Struts et al. (2011) Nat. Struct. Mol. Biol. 18, 392-394.
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5:00PM
Bryant Doss
[email protected]
"Combined AFM nanoindentation and finite element analysis on soft heterogeneous
materials.” Bryant Doss and Robert Ros
Physics, Arizona State University
The raw data analysis of AFM nanoindentation experiments on cells typically rely on heavy
assumptions which may both invalidate the quantitative data and neglect features of the
sample which are heterogeneous in nature. In particular, it is of great interest to extract
accurate elastic moduli for systems of cells embedded in a thin collagen matrix or subcellular
regions in cells. To overcome this, we employ finite element analysis (FEA) to numerically
simulate force-indentation curves to match the experimental data. These simulations are
capable of imposing proper geometric constraints and use data from confocal laser scanning
microscopy to reconstruct the 3D model of the sample. I will show the combined AFM/FEA
approach on model systems of glass beads embedded in thin polyacrylamide gels using large
radius silicon tips.
5:15PM
Mowei Zhou
[email protected]
“Probing protein quaternary structures by surface collision, ion mobility, and mass
spectrometry.” Mowei Zhou, Shai Dagan, and Vicki Wysocki
Chemistry and Biochemistry, University of Arizona
Subunit organization of protein complexes can be studied with tandem mass spectrometry by
disruption of the quaternary structure in a controlled manner. The major challenge is to
overcome the undesired unfolding of protein subunits in the commonly used gas collision
activation which usually results in the loss of information for the native conformation. We
present here the advantage of using surface collision to dissociate protein complexes into
substructures which are informative to their native conformations. With collisional cross
sections of protein ions after activation measured from ion mobility experiments, C-reactive
protein pentamer is shown to undergo significant structural rearrangement and unfolding
upon gas collisions but dissociated rapidly into compact subunits without remarkable
structural change by surface collision. In another experiment, surface collision of serum
amyloid P decamers revealed difference in subunit packing between precursor ions with
different conformations separated by ion mobility.
5:30PM Suratna Hazra
[email protected]
"Kinetics study of rubisco activase." Suratna Hazra and Rebekka Wachter
Chemistry and Biochemistry, Arizona State University
Rubisco activase (RCA) is a chemo-mechanical motor protein that activates Rubisco by
catalyzing the removal of inhibitory sugar phosphates. The mechanism and ATP requiremnt
of Rubisco activation is still not known. The kinetic study of ATP hydrolysis, ADP inhibition
and the study of cooperativity are very important in elucidating the mechanism of Rubisco
activation. The ATPase activity can be studied either by monitoring ADP production or Pi
production. To properly calculate the kinetic parameters, a continuous assay is essential. To
understand the effect of the stromal ATP/ADP ratio on RCA turnover and inhibition,
measurement of inorganic phosphate is necessary. In the presence of Pi, the substrate 2amino-6-mercapto-7-methyl purine riboside is converted enzymatically to 2-amino6mercapto-7-methyl purine by purnine nucleoside phosphorylase (PNP)ref. This assay has
been standardized for RCA and compared with the continuous ADP measurement assay. The
preliminary value of kcat is 22.08±1.01min-1 and Km 0.189±0.03mM for tobacco RCA.
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5:45PM Asuka Nakano
[email protected]
"Streaming dielectrophoresis of proteins in a microfluidic platform .” Asuka Nakano and
Alexandra Ros
Chemistry and Biochemistry, Arizona State University
Dielectrophresis (DEP) has been employed as a novel selectivity technique to manipulate
biomolecules. Our work demonstrates the detailed study of various factors influencing the
DEP response of proteins employing molecules. Utilizing the insulator-based DEP (iDEP) in
a tailored elastomer-based microfluidic platform, we investigated DEP streaming
concentration of immunoglubulin G (IgG) in various buffer conditions differing in pH and
conductivity. Streaming DEP was only observed at pH 6.5~8, and the maximum DEP
streaming was found at pH 8 at lowest employed conductivity of 0.01 S/m. This
experimentally obtained concentration profiles were in excellent qualitative accordance with
numerical simulations performed with the assumption of monomeric IgG species.
Furthermore, we observe micelle induced negative iDEP streaming for proteins exhibiting a
micelle former, which is also observed with excellent qualitative agreement in numerical
simulations. Our study provides valuable information to improve a novel protein iDEP
device for separation, pre-concentration and fractionation.
6:00PM Logan Ahlstrom
[email protected]
"Adding dynamical insight to Cro Dimer X-Ray structures by solution and crystal molecular
dynamics simulation." Logan Ahlstrom and Osamu Miyashita
Chemistry and Biochemistry, University of Arizona
X-ray crystallography is the most robust method for solving protein structure. However,
packing forces in the crystal lattice select just a snapshot of the conformational ensemble,
whereas in solution proteins are flexible and can adopt different conformations. Solution and
crystal molecular dynamics (MD) simulation offer an approach by which to add dynamical
insight to protein X-ray images. Here we examine the variation in global conformation
observed in several solved crystal structures of the λ Cro dimer. Network analysis and a free
energy surface constructed from Replica Exchange MD show some reported structures
correspond to stable states in solution. Yet other conformations, while accessible, lie higher
in free energy, indicating the effect of crystal packing. The Cro dimer NMR models disagree
with MD and X-ray. Subsequent crystal MD estimated the strength of packing interfaces in
the lattice, showing the influence of crystal form and mutation in stabilizing different dimer
conformations.
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Notes
We gratefully thank BIO5 and the Department of Chemistry
and Biochemistry at the University of Arizona for support of
Biophest 2012.
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