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Jakub Kostal &
Steve Sontum
Thesis Presentation ‘06
Courtesy of
www.mcsrr.org
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Binding of CO/O2 to Fe in Heme
CO ligand
O2/CO
N
N
N
Fe
N
Process of Ligand binding in Heme
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Bound vs. free Heme:
The Ultimate Puzzle
• Myoglobin’s ability to bind oxygen is readily poisoned
by its stronger affinity for carbon monoxide
• The affinity for CO is greatly reduced compared to
free heme
How does ligand surroundings in myoglobin’s
Heme pocket influence ligand binding?
1. Sterics
2. Electrostatic interactions
Heme Pocket for Dummies
Studying Electrostatic Effects:
Vibration of CO bond
•
•
Triple bond character causes high
vibration stretching frequencies
(CO) used to characterize different
conformers of the bound state
•Equilibrium IR absorbance
spectrum of bound CO shows the
major sub states A0, A1 and A 3 are
associated with CO stretching
bands at 1966, 1945, and 1927cm-1
•Dispersion of A sub states thought
to be caused by electrostatic
interactions between the CO dipole
and the imidazole chain of the
distal His64, which assumes
different dynamic conformations
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Studying Electrostatic Effects:
Vibration of CO bond
•
•
Triple bond character causes high
vibration stretching frequencies
(CO) used to characterize different
conformers of the bound state
•Equilibrium IR absorbance
spectrum of bound CO shows the
major sub states A0, A1 and A 3 are
associated with CO stretching
bands at 1966, 1945, and 1927cm-1
•Dispersion of A sub states thought
to be caused by electrostatic
interactions between the CO dipole
and the imidazole chain of the
distal His64, which assumes
different dynamic conformations
Models for electrostatic interactions in the
CO complexes - amino acid mutations
A (A3): Asn68 Mb
B (A1, A2): Wild-Type Mb
C (A0): Val64/Thr68 Mb
(CO = 1938cm-1 ; Fe-C = 527cm-1)
(CO = 1945cm-1 ; Fe-C = 507cm-1)
(CO = 1984cm-1 ; Fe-C = 477cm-1)
Preliminary studies on our project:
Building a simple Theoretical Model
•
•
•
•
Generation of a vibrational force field
using RESP method for various heme
analogs with bound CO ligand
Classical MD model built
Dynamic Simulation of an out-of-plane
electric field using Li ions to predict
changes in CO vibrations based on
experimental observations
Hypothesis:
Li (+/-)
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Li (-/+)
(CO) < (CO)
Molecular Dynamics Trajectory
Observed Vibrational Shifts
Expected trends somewhat preserved only at high e. field intensities
1958
1956
Vib. frequency (1/cm)
•
1954
1952
1950
1948
1946
1944
1942
-100
-50
0
50
Charge (au)
100
150
“Torquing” motion of the CO ligand
• Fe-C-O bond locked in
one “torquing” mode
throughout the dynamic
trajectory at higher E.
field intensities
• Dominant mode at
higher E. field
• Torquing motion
accounts for additional
centripetal stretching of
the CO bond
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Analysis of the
torquing mode
Normal direction
•
•
•
•
Fe-O angle to the normal
() is greater than Fe-C
angle to the normal of the
heme plane (). This
differences increases with
reversed e. field
Direction of the electric field
changes  and 
As the intensity of e. field
increases,  and  increase
as well
As the temperature
increases, both angles
increase
Reversed direction
X-axis: time (0.01ps) Y-axis: angle (degree)
MD Trajectory of Full Myoglobin
100
Inside heme pocket
90
N
N
% of MD trajectory time
80
70
60
50
40
30
20
Out of heme pocket
10
0
A
B
Conclusions and Future Work
• We have successfully generated RESP force field for CO heme
model to study the effect of electrostatic fields on the vibration
of CO.
• We have observed a toquing motion of the CO ligand induced by
electrostatic fields of high intensities.
• We have analyzed 2ns MD trajectory of full myoglobin and
observed that distal His64 spends 88% of the time inside and
12% outside of the heme pocket.
• Generate force fields for similar O-O and NO bound heme
models (in progress)
Acknowledgments
Steve
Meghan
Judy
References
•Spiro T. G., Kozlowski P. M. 1998. Discordant results on FeCO deformability in heme
proteins reconciled by density functional theory. J. Am. Chem. Soc. 120: 4524-4525
•Phillips G. N., Teodoro M. L., Tiansheng L., Smith B., Olson J. S.. 1999. Bound CO Is a
Molecular Probe of Electrostatic Potential in the Distal Pocket of Myoglobin. J. Phys. Chem.
B. 103: 8817-8829
•Nienhaus K., Pengchi D., Kriegl J. M., Nienhaus G. U. 2003. Structural Dynamics of
Myoglobin: Effect of Internal Cavities on Ligand Migration and Binding. Biochemistry. 42:
9647-9658
•Ray, G. B., X.-Y. Li, J. A. Ibers, J. L. Sessler, and T. G. Spiro. 1994. How far proteins bend
the FeCO unit? Distal polar and steric effects in heme proteins and models. J. Am. Chem.
Soc. 116: 162-176.
•Rovira, C., K. Kunc, J. Hutter, P. Ballone, and M. Parrinello. 1997. Equilibrium geometries
and electronic structure of iron-porphyrin complexes: a density functional study. J. Phys.
Chem. A. 101:8914-8925.
Solvated WT Trajectory