Immunohistochemistry based approach to detect synucleinopathy conformations i...
JG_ResearchPoster-fe
1. Role of Conformational Transitions in Proteins related to Prion and
Parkinson’s Disease
The misfolding of the Human Prion Protein (HPP) plays a key role in
prion diseases such Creutzfeldt-Jakob disease, fatal familial
insomnia, and Gerstmann-Straussler-Scheinker (GSS) syndrome.
Similarly, the misfolding of the Alpha-Synuclein protein plays a key
role in Parkinson’s disease, an irreversible and progressive
neurodegenerative disorder that affects about 60,000 Americans per
year1
. The conformations of these proteins are of great interest
because the dynamic shape and structure of these molecules dictate
the protein’s functions and properties. These misfolds, or
conformational disorders, can also lead to protein aggregation, a
mechanism by which misfolded proteins, rich in β-sheet structure,
clump together and deter the function of the respective system that
they inhabit. Our simulations can shed light on the transitory
conformations that lead to misfolding of these proteins and can
inform targets for drug design to prevent unfavorable misfolding
events.
In analyzing the conformations of the HPP and Alpha-
Synuclein protein, Steered Molecular Dynamics Simulations
(SMD) were performed using GROMACS suite of MD programs.
Only specific peptides of each protein were used—residues 61-84
for the HPP and residues 57-83 for Alpha-Synuclein. The SMD
simulations involved non-equilibrium pulling methods in order to
apply an external force to specific atoms of both proteins and
analyze their trajectories as they become stretched. Umbrella
sampling methods were used for non-equilibrium pulling, where a
harmonic potential is applied between the center of mass of two
groups, making the force proportional to the displacement of the
protein.
GROMACS is a high-performance software package
designed to perform molecular dynamics on biochemical
molecules like proteins, lipids, and nucleic acids4
. Analyses of
conformational changes of these peptides upon quasi-static
pulling (SMD simulations) has been performed to obtain root-
mean-square deviations (RMSD), changes in hydrogen-bonding
and dihedral angles corresponding to transitions among various
conformational states.
Throughout all of the simulations, certain conformations were
preferred and maintained by the proteins at specific time frames
within the pulling. At the initial stages of the pulling where residues
were much closer to each other, hydrogen bonds remained intact
within both proteins, allowing for an initial conformation to
dominate. Since hydrogen bonds hold helices and sheets together,
any condition that interferes with their formation can disrupt or
destroy the protein’s structure, causing a misfold. The misfolding of
proteins into β-sheet aggregated structures characterize many of the
diseases that can be caused by these proteins.
The equilibrated conformations of the peptides bear strong structural
resemblance to each other and also to the NMR structure (1OEI).
This may be expected because both peptides are composed of
hydrophobic residues and multiple glycine residues. NAC peptide
retains a high degree of alpha-helical character even in this partially
folded state.
The NAC peptide transitions from an equilibrated partial fold to
conformations with variable amounts of alpha-helical structure and
eventually to a alpha-helical peptide (Figures 4,5,6).
The HPP peptide exhibits multiple conformations with high-degree
of hydrogen bonding (Figures 4,5).
• Run simulations using different pulling rates and determine
whether this has a significant effect on protein pulling and on
their conformations.
• Analyse the conformations of significant mutations of both
proteins and perform recoil simulations of the peptides going
from extended to folded state.
• Use Jarzynski’s Equality in order to obtain Free Energies between
the proteins’ native and folded states which will give us useful
information about the stability of the proteins and its misfolded
conformations.
Research supported by Louis Stokes Alliance for Minority Participation
(LSAMP), an NSF funded program under grant No. 06-552. Jerry
expresses gratitude to Prof. F. Escobedo of the Chemical &
Biomolecular Engineering Department at Cornell University and his
doctoral student Sai Pooja for their mentorship. Special thanks to
Diversity Programs in Engineering at Cornell University (DPE), and
the Collegiate Science and Technology Entry Program (CSTEP) at
Syracuse University.
Figure 3: Illustration of protein pulling through various stages– initial
(starting) conformation, equilibrated conformation, a conformation as it
is being pulled, and another displaying its final pulled(stretched)
conformation.
Introduction
Aim
Method Results Conclusion
Acknowledgements
The purpose of this research is to analyze the conformations and
physical changes of the HPP and Alpha-Synuclein in order to gain
insight on the events at the molecular level that lead to misfolding
and aggregation of these proteins.
Certain properties and variables such as hydrogen bonding and
root mean square deviation (RMSD) will be analyzed for further
understanding of protein misfolding. RMSD plots will help us gain
information on the different conformations that the proteins take as
they undergo Steered Molecular Dynamics (SMD). By
understanding the structural transitions of these proteins, we can
learn more about how protein folding can go astray and work on
ways to prevent these unfavorable misfoldings.
Figure 1: The primary
structure of the Human
Prion Protein5
.
Future workFigure 2: The
regions and primary
structure of Alpha-
Synuclein2
.
Figure 5: Evolution of the number of hydrogen bonds formed
along the peptide of the Human Prion Protein (Residues 61-84),
and Alpha-Synuclein (Residues 57-83).
Human prion protein peptide
sequence61 84
HGGGWGQPHGGGWGQPHGGGWGQP
Alpha-Synuclein peptide sequence
57 83
EKTKEQVTNVGGAVVTGVTAVAQKTVE
I. II.
III.
I. II.
III. IV.
Figure 4: RMSD of the HPP (Top) and NAC (Bottom) peptides with
conformations corresponding to each of the major transitions
observed during pulling simulations. Green sticks represent certain
Carbon atoms; Yellow represents the hydrogen bonds; Red, blue, and
white represent certain O, N, and H atoms, respectively. All atoms are
categorized with respect to their position along the peptide. i.e. C-
Alpha, C-Beta, etc.
References
1. "Parkinson's Disease." NIHSeniorHealth:. N.p., n.d. Web. 02 Aug.
2015
2. Gallegos S, et.al (2015) Neurosci. 9:59.
3. Nucl. Acids Res. (2000) 28 (1): 235-242.
4. van der Spoel, et al. (2005) J. Comput. Chem. 26: 1701-1718.
5. Zahn R, J. Mol. Biol. (2003) 334, 477-488.
Jerry Gomez
L.C.Smith College of Engineering & Computer Science, Syracuse University, Syracuse, NY, 13210, USA
The program GNUPLOT was utilized to generate analytical
graphs of the data obtained from the GROMACS simulations.
The program PYMOL was used to view the conformational
transitions.
Peptide Pulling Rate
[nm/ps]
Pulling Force Constant
[kJ/mol-nm]
# of SMD Simulations
HPP 0.0001 1000 50
NAC 0.001 1000 50
PDB:1OEI
PDB:1XQ8
Conformational Transitions observed during simulations
Figure 6: High-propensity for alpha-helical structure observed
for NAC peptide.
IV.