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Some of the unpublished results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