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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Some of the unpublished results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
1988 - 95
Caltech Cornell
1997 - 2003 2009 - Present
UMD
2003 - 09
PSU
1995 - 97
JPL
Heikal, Biomarkers in Medicine, (2010)
Huang, Heikal and Webb. Biophys. J. (2002)
Heikal, Biomarkers in Medicine, (April 2010)
Heikal, Annual Reviews in Fluorescence (2011)
Dr. Huang
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
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)
C3H 10T1/2 fibroblast (RhG-123)
TCSPC:
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
)
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)
0.1
ns
1.5
Heikal, Biomarkers in Medicine, (April 2010). Heikal, Annual Reviews in Fluorescence (2011)
(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 )( )
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)
Normoxic
Hypoxic
Vishwasrao, Heikal, Kasischke, Webb, J. Biol. Chem. (2005); Press Release.
H. Vishwasrao
K. Kasischke
SR
SP
Hippocampus
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)
Heikal, Biomarkers in Medicine, (2010)
J. Alfveby
R. Timerman
H. Israelson
J. Bartusek
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
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
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
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
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
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