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Ahmed A. Heikal Department of Chemistry & Biochemistry
University of Minnesota-Duluth, Duluth, MN, 55812, USA
PHYS 1021:
Exploring Current Topics in Physics
November 7, 2013
Biophysical Perspective of Bioenergetics
& Macromolecular Crowding
Some of the unpublished results
were removed from these slides.
Heikal
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1988 - 95
Caltech Cornell
1997 - 2003 2009 - Present
UMD
2003 - 09
PSU
1995 - 97
JPL
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Heikal, Biomarkers in Medicine, (2010)
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Huang, Heikal and Webb. Biophys. J. (2002)
Heikal, Biomarkers in Medicine, (April 2010)
Heikal, Annual Reviews in Fluorescence (2011)
Dr. Huang
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2. How to quantify the population fraction of free & enzyme-
bound NADH in live cells?
NADH concentration & conformations correlate with the physiological & redox
state of living cells
1. Cellular/tissue autofluorescence: A friend (diagnostics) or a
foe!
Intracellular coenzymes (e.g., NADH, FAD) as natural biomarkers for cell
physiology & pathology
3. What are the key factors that determine the nature of
anisotropy decay for a mixture of biomolecules?
Developing fluorescence anisotropy as a non-invasive & quantitative methods
Challenges and Opportunities
4. What is the role of molecular crowding in NADH-Enzyme
binding reactions?
Confinement in macromolecule-induced caging vs diffusion
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Ariola et al., PCCP (2006); Ariola et al., Biophys. J. (2009)
Heikal. In “Advances in Planar Lipid Bilayers &
Liposomes” Editors: Iglic & May. Elsevier (2010) Yu et al., J. Biomed. Optics (2008)
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C3H 10T1/2 fibroblast (RhG-123)
TCSPC:
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Time (ns)
[NADH]/[Enzyme]
<
fl>
, p
s
LDH (4) mMDH (2)
Flu
ore
scen
ce (
no
rmalize
d)
- Free NADH - HTB125
- HTB126
mMDH
LDH
Yu
an
d H
eik
al,
J. P
ho
toc
he
m. P
ho
tob
iol.
(B
), 9
5:
46–5
7 (
20
09
)
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FAD NADH
500 1000 1500 2000 2500 3000 35000
100
200
300
400
500
600
700
Lifetime (ps)
# o
f P
ixels
Qianru Yu
FAD
Heikal, Biomarkers in Medicine, (April 2010)
Heikal, Annual Reviews in Fluorescence (2011)
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0.1
ns
1.5
Heikal, Biomarkers in Medicine, (April 2010). Heikal, Annual Reviews in Fluorescence (2011)
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(A) Excitation Photoselectivity: Heterogeneous Population
Enzymei(NADH )nEnzymeNADHn 1k
2k
(B) Fluorescence Depolarization
I┴
E
x
z
x I//
q
P (0˚)
P (90˚)
r(x, y, t) = a1e(-t/t1)b1e
(-t/f1) +a2e(-t/t2 )b2e
(-t/f2 ){ } / a1e(-t/t1) +a2e
(-t/t2 )( )
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Time (ns)
An
iso
tro
py
Fluorescein:
= 130 ps
G = 1.66
NADH (Tris, pH 8.0):
1 = 63 ps (1=0.39)
2 = 350 ps (2=0.18)
Time-Resolved Anisotropy: Control Experiments
Tk
V
B
38 a
TkD
B
R Heikal, Biomarkers in Medicine, (April 2010)
Heikal, Annual Reviews in Fluorescence (2011)
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Normoxic
Hypoxic
Vishwasrao, Heikal, Kasischke, Webb, J. Biol. Chem. (2005); Press Release.
H. Vishwasrao
K. Kasischke
SR
SP
Hippocampus
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Qianru Yu
Hs578st H
eik
al,
An
nu
al R
ev
iew
s in
Flu
ore
sc
en
ce
, 2
011
Yu and Heikal, J. Photochem. Photobiol. (B), 95: 46–57 (2009)
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Heikal, Biomarkers in Medicine, (2010)
J. Alfveby
R. Timerman
H. Israelson
J. Bartusek
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The cell is a crowded environment: Medalia et al., 2003; Ellis 2001 ; Rivas et al.,
2004; Minton, 2001
Molecular Crowding Influences Biochemical
Reactions & Diffusion in Living Cells
Crowding influences the kinetics of
biochemical reactions:
Minton, 2001 and 2006; Somalinga & Roy,
2002; Ellis & Minton, 2003
Crowding influences protein folding &
protein assembly: Banks and Fradin, 2005; Minton 1977, 2000;
Tokuriki et al., 2004
Medalia, et al. Science (2002)
Me
mb
ran
es
Actin Filaments
Crowding influences diffusion:
Banks and Fradin, 2005; Verkman 2002;
Lavalette et al., 1999; Zorilla et al., 2007;
Sanabria et al., 2007; Dix and Verkman, 2008
Macromolecules
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Crowding: Challenges & Opportunities
Diffusion of biomolecules & substrates are essential to
biochemical reactions, molecule-molecule interactions &
signaling in living cells
How to differentiate between environmental restriction &
binding of biomolecules using diffusion-based methods?
Non-specific interactions (steric, electrostatic, hydrophobic)
between solute & crowding agents
Diffusion mechanisms in crowded environments: Sensitivity to
the spatial & temporal resolution of employed methods
How do biomimetic crowding agents compare? From polymers
“inert” macromolecules , proteins, to cell lysates
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Yosef et al. J. Am. Chem. Soc. 2005, 127, 15138-15144.
(A) An enzymatic reaction in homogeneous environment:
Biochemical Reactions in Crowded Environments
(B) An enzymatic reaction in a crowded environment:
RDDk BAAB )(4 Confinement (I) (II)
A
B
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Rhodamine Green: N 17.6 molecules D 0.13 ms D 2.8x10-6 cm2s-1
xw 250 nm
Vobs ~1x10-15 L
Lag Time (ms)
G(t
)
2wz
2wxy
2
)().()(
F
tFtFG
tt
a
TkD
B
6
DDxy
4
2wt
j
iijiii trCTtrCDtrCt
),(),(),( 2
Randi Timerman Chang Thao
D. Wickramasinghe R. Welty
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2. Biophysics of cellular autofluorescence provides a
non-invasive, quantitative approach for monitoring
physiological & pathological changes
1. Intrinsic coenzymes (e.g., NADH, FAD) are natural
biomarkers for cellular metabolism & the redox states in
living cells (i.e., potential diagnostics)
3. Macromolecular crowding affects both diffusion &
biochemical reactions in the complex milieu of living cells
or tissues
Closing Remarks
4. Formulating a mechanistic model for diffusion in a crowded,
heterogeneous environment is challenging due to
non-specific binding & the multiscale processes involved
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A. Davey
Dr. Y. Liu
R. Walvick
K. Krise
F. Ariola
C. Cornejo
D. Mudaliar Q. Yu
PSU: University of Minnesota Duluth: Graduates
D. Wickramasinghe R. Welty K. Hewawasam (Physics)
J. Alfveby
University of Minnesota Duluth: Undergraduates
M. Velasquez R. Timerman J. Bentley T. Fransen J. Bartusek
K. Paul C. Thao (McNair Scholar)
H. Israelson