Download 364431 - IDEALS @ Illinois

70th ISMS at UIUC
Dynamics/Kinetics/Ultrafast
Tuesday, June 23, 2015
Multiscale Dynamics of Diffusing
Molecules in Crowded Environments
Ahmed A. Heikal
Department of Chemistry & Biochemistry
University of Minnesota-Duluth, Duluth, MN, 55812, USA
Living Cells are Crowded by Macromolecules
and Organelles
The cell is a crowded environment:
Medalia et al., 2003; Ellis 2001 ; Rivas et al.,
2004; Minton, 2001

Crowding influences the kinetics
of biochemical reactions:
Minton, 2001 and 2006; Somalinga & Roy,
2002; Ellis & Minton, 2003

Actin Filaments
Membranes

Crowding influences protein
folding and protein assembly:
Banks and Fradin, 2005; Minton 1977, 2000;
Tokuriki et al., 2004

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
Medalia, et al. Science (2002)
Random Walk and Stokes-Einstein Model
Diffusion coefficient (cm2/s) for
a molecular undergoing
a random walk:
Thermal Energy kBT
D=
=
Friction
6pha
www.google.com/image
Temperature: D 
T 
Viscosity:
D
η 
Size:
D
a 
éhcrowding é
Dbuffer
=é
é
Dcrowding é hbuffer éT, a
Excluded Volumes in Crowded Environments
Excluded
volume
Excluded
volume
Minton, 2001; Rivas et al., 2003
Diffusion in Crowded Environments: Hypothesis
Control
Homogeneous
& S-E Model
C
R
G
Scenario I
O
W
D
Scenario II
I
N
Scenario III
Confinement
Binding (Specific
Electrostatic
(Buffer-Like Cages) or Non-specific)
Interactions
(Transient/Long-Lived)
Biomimetic Crowding Agents and Methods
For Diffusion Measurements
(Rhodamine Green)
Continuum Solution:
- Glycerol-Enriched Solutions
Crowded Solutions:
- Ficoll-70 (70 kDa)
- Bovine Serum Albumin, BSA (66 kDa)
- Ovalbumin (45 kDa)
Bulk Viscosity Measurements:
- Ubbelohde Viscometer
Diffusion Measurements:
- Time-Resolved Anisotropy (ns)
- FCS (μs – tens of seconds)
Heikal. In “Advances in Planar Lipid Bilayers & Liposomes” Elsevier (2010)
r(x, y,t) =
I // (x, y,t) - I ^ (x, y,t)
I // (x, y,t) + 2I ^ (x, y,t)
Heikal. In “Reviews in Fluorescence” (2011)
 
Anisotropy
Yu et al., J. Biomed. Optics (2008)
V
k BT
DR 
k BT
8a 3
Fluorescein:
 = 140 ps
Time (ns)
TCSPC: 2P-FLIM & Time-Resolved Anisotropy
Intensity Image
FLIM Image
950 nm
Crowding Effect on Fluorescence Lifetime:
Refractive Index
BSA
Ovalbumin
Glycerol
Ficoll-70
Strickler-Berg Equation
Anisotropy, r(t)
Rotational Diffusion Reveals Binding
(A) RhG (Glycerol)
(C) RhG (BSA)
(B) RhG (Ficoll-70)
(D) RhG (Ovalbumin)
Time (ns)
Time (ns)
RhG Binding with BSA
fbound =
[BSA]
K d + [BSA]
Kd = 7.0 ± 0.6  M
Do/Dc
DR =
1 kBT
=
6j hV
D0 éhc é
=é é
Dc éh0 éT , a
ηc/ηo
ηc/ηo
FCS:
2wz
2wxy
G()

Ci (r , t )  Di  2Ci (r , t )   TijCi (r , t )
t
j
Rhodamine Green:
N
17.6 molecules
D
0.13 ms
D
2.8 ×10-6
cm2s-1
 xw
250 nm
Vobs
~1 × 10-15 L
G ( ) 
F
D
Watt W. Webb
(1972)
Lag Time (ms)
F (t ).F (t   )
2

2
wxy
4D
k BT
D 
6a
Hess, Huang, Heikal, Webb. Biochemistry, 2002.
Crowding Effects on Translational Diffusion
(C) RhG (BSA)
(B) RhG (Ficoll-70)
(D) RhG (Ovalbumin)
G()
G()
(A) RhG (Glycerol)
Correlation Time (ms)
Correlation Time (ms)
Do/Dc
ηc/ηo
kBT
DT =
6pha
D0 éhc é
=é é
Dc éh0 éT , a
ηc/ηo
ηc/ηo
Anisotropy, r(t)
Anisotropy, r(t)
Anisotropy, r(t)
G()
Correlation Time (ms)
RhG (BSA)
RhG (Glycerol)
Time (ns)
RhG (Ficoll-70)
Time (ns)
Viscosity, cP
Bulk Viscosity versus Microviscosity
[Glycerol], g/L
- In homogeneous environment, molecules experience the
same viscosity on ns & ms timescales as bulk viscosity
Diffusion in Crowded Environments: Conclusion
Control
Homogeneous
& S-E Model
C
R
G
Scenario I
O
W
D
Scenario II
I
N
Scenario III
Confinement
Binding (Specific
Electrostatic
(Buffer-Like Cages) or Non-specific)
Interactions
(Transient/Long-Lived)
BACE1 Cellular Trafficking
BACE1
BACE 1 activity is the rate limiting step for β-amyloid formation in AD
Heikal, Biomarkers in Medicine, (2010)
Heikal, SPIE (2014)
University of Minnesota Duluth: Graduates
D. Wickramasinghe
R. Welty
K. Hewawasam
J. Alfveby
S. Gardeen
University of Minnesota Duluth: Undergraduates
J. Bartusek
J. Bentley
M. Velasquez
C. Thao
(McNair Scholar)
H. Israelson
T. Ward
R. Timerman
B. Berry
T. Fransen
Megan Currie