Using Solid State NMR to Understand the Molecular Structure
of Designer Self-Assembling Proteins MAX8 and RADA16
Dr. Anant K. Paravastu
Ashley Cormier
& Biomedical Engineering
Graduate Researcher
FSU-FAMU College of Engineering
FSU-FAMU College of Engineering
Tallahassee, FL 32303
Tallahassee, FL 32303
Introduction
Proteins are a necessary part of everyday life. Nerves, tissues, and
bones all contain proteins we need to survive. Our bodies also make
different proteins that help our cells grow, fight infection, and repair
injuries. The scientists we worked with this summer were interested
in studying designer proteins that
were synthetically produced by man.
Though these proteins are not as
complex as ones that occur naturally,
they are important because they
can be used for repair and
regeneration of cells and nerves.
Our immune systems make complicated T-cell proteins to fight diseases.
Purpose
Our summer mission was to analyze data from NMR spectrometry to
better understand the molecular structures of the designer proteins
MAX8 and RADA16. This would let scientists use computer programs
to create 3-D models of the proteins. Even though scientists are
able to synthesize and experiment with these proteins, they are still
not completely certain about all of their structural characteristics.
Once they understand more about MAX8 and RADA16, they
hypothesize that they will be able to use these proteins to deliver
medicine. These proteins are particularly interesting because they
can self-assemble and then reassemble in new environments. Real
world application of these proteins would allow someone to receive an
injection of MAX8 or RADA16 to assist in nerve regeneration.
Protein Synthesis
Before we could use NMR spectrometry to analyze data about the
proteins, we worked with Dr. Paravastu’s graduate students to
synthesize MAX8 and RADA16. In this process scientists can make
designer proteins by using specific amounts of amino acids. The
amino acids have to follow a particular
sequence for each desired protein. Our
bodies do this naturally through coding
that we are born with in our DNA.
When we assisted in making
RADA16,
first we weighed specific
amounts of
each amino acid (arginine,
Kathy alanine,
weighs the and
delicate powder
aspartic acid). They
form of arginine while Serena keeps
were
placed
in
order on the peptide
a careful eye on the digital reading.
synthesizer, which
injected necessary
amounts solvents that
helped of each
amino acids form the
protein. This
process takes about an
hour per amino
acid, which means a gram
of RADA16
takes about 16 hours to
make. After
this, the protein must be
cleaved and
purified.
The
end
result
is
about .25g
RADA16 is made with 16 amino acids
of RADA16
$1,000.00.
that follow
the RADAwhich
pattern;is worth
Coil?
Megan Crombie
Riversink Elementary School
Crawfordville, FL 32327
The brown portion of the graph shows the
absorption rates of each amino acid during
the decoupling process; scientists infer there
may be a coil in RADA16’s structure.
Chemical
Conclusion and Future Research
Nuclear Magnetic Resonance (NMR)
Next the protein samples are lowered into
the middle of the magnet. NMR
spectrometry uses a spectrometer that
sends radio waves via wires to a magnet.
Our magnet generates 11.74 Tesla; the
magnetic field of the earth ranges from
0.3-0.6 Gauss and 10,000 Gauss = 1
Tesla. The solid sample spins 25,000
times per second to create a magic angle
spinning environment. This allows for the
most accurate data collection as samples
oscillate between ground and excited
states. During spinning Nitrogen gas or
compressed air is used to cool the
magnet. The protein’s nuclei and
electrons emit different radio waves that
are recorded through Fourier’s
Transform.
At this time there is a computerized molecular model of MAX8
in the process of being built and confirmed. Future NMR
experiments with RADA16 must continue to be conducted and
data must be collected and analyzed to learn more about how
the molecules in the protein interact at different temperatures
and in different solvents. This will help scientists learn more
about the nature of the protein, which will aid in determining
the molecular structure.
Megan and Kathy can stand near
the NMR magnet because it has an
“ultrashielded,” tight magnetic field.
Classroom Applications
The most important part of this experience was finding a way to
take what we learned back to our elementary classrooms. We
used beads to represent amino acids that self-assembled into
proteins. This is a hands-on way to show students functions
that occur for cell and tissue growth. The strands must have
the correct components, be in a specific order, and be close
enough to each other for self-assembly to occur. The same
process is followed in our bodies and with designer proteins.
A
NMR uses radio waves; the
lower frequency allows for
detailed information on molecular
structure and environment.
Fourier
Original
Real and
Real and
transform
function
imaginary
imaginary
with 3 and 5
showing
parts of
parts of
hertz
oscillation 3
integrand
integrand
labeled (now
hertz (this
for Fourier
for Fourier
the waves
is the “real” transform at transform at have been
transformed
wave, but
3 hertz.
5 hertz.
into data
you can’t
that we can
see
analyze).
it or get
data
from it).
C
D
E
Strand A did not self-assemble; B-E
did. B is folded, C has coils and folds,
D is coiled, and E has large folds.
References
Fourier’s Transform uses
mathematical equations to
turn radio waves into usable data.
Cell phones and computers use it
everyday to process information.
Megan tunes the probe to find
signals for specific elements in
RADA16.
Challenges with MAX8 and RADA16
Determining the molecular structure of the proteins is difficult
because many variables are involved. The NMR graph below shows
RADA16 emits different signals when it is heated. There are also
concerns about exposure to water vapor in the air and the protein
breaking down over time. MAX8 will currently dissolve in a solvent
but RADA16 will not, which interferes with the HPLC purification
process for proteins. Both proteins seem to form β-sheets and it
is possible that RADA16’s β-sheets are keeping it from dissolving.
Yellow-2.5 mm Probe; 25 kHz MAS; RADA16
4/24/10 no HPLC at room temperature.
Red-2.5 mm Probe; 25 kHz MAS; RADA16
4/24/10 no HPLC, CPMAS at 80 C, spinning
a) 
with N2 gas, a few hours later.
Blue-2.5 mm Probe; 25 kHz MAS; RADA16
4/24/10, no HPLC, CPMAS at room temperature, b) 
spinning with N2 gas, the next morning.
B
These beads represent amino
acids. Some will self-assemble to
form proteins, some will not.
MAX8 is made with 20 amino acids.
The peptide synthesizer is ready to go!
Kathy Gibbons-Adams
Lakeview Elementary School
Miami, FL 33167
RADA16 self-assembles and forms
β-sheets that seem to make it insoluble.
MAX8 self-assembles and forms
hairpin β-sheets but it will still
dissolve in different solvents.
1. Cormier, A. Paravastu, “Solid state NMR structural analysis of designer
self assembling proteins,” MRS Conference Poster, Spring (2010).
2. A. Paravastu, “Optical polarization of nuclear spins in gallium arsenide,”
University of California, Berkeley (2004).
3. A. Paravastu, R. Leapman, W. Yau, R. Tycko, “Molecular structural basis
for polymorphism in Alzeimer’s β-amyloid fibrils,” Proceedings of the National
Academy of Sciences of the United States Journal (2008).
4. H. Yokoi, T. Kinoshita, S. Zhang, “Dynamic reassembly of peptide RADA16
nanofiber scaffold,” Proceedings of the National Academy of Sciences of the
United States Journal (2004).
Acknowledgements
Because Dr. Paravastu collaborates with teams at FSU, FAMU, the College
of Engineering and the Magnet Lab, we had the unique opportunity to observe
many different and diverse scientists throughout the summer. We would like
to thank Umesh Goli, Dr. Rufina Alamo, Dr. Derek Lovingood, Dr. Ongi
Englander, Dr. Karunya Kandimalla, Dr. Subramanian Ramakrishnan, Dr.
Geoffrey Strouse and Dr. Penny Gilmer for helping us experience scientific
research on a daily basis. In addition, we appreciated the willingness of Dr.
Paravastu’s students Serena Huang, Stefanny Cortes, Alexa Buchannan, and
Andres Gutierrez as they allowed us to spend the summer learning with them.
Thank you to Dr. Pat Dixon, Jose Sanchez, Carlos Villa, Patrick Enderle,
Nguyen Nguyen and the RET participants for their support at the Mag Lab
education department; our days with the scientists became more meaningful as
we worked together to figure out how to connect authentic research to
inquiry-based learning in our classrooms. Our level of understanding would not
have been possible without graduate
student Ashley Cormier patiently
explaining data to us as she worked
through the scientific method
each day.
Finally, we are grateful for
everything
that Dr. Anant Paravastu
allowed us to
experience as we shared the
summer
with a true scientist.
We will take his
undying curiosity about
the world back
to our classrooms as we
try to inspire
future generations of scientists.