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Isolation and Identification of
Chroococcidiopsis

Photosystems

Brian
Moran
1
,
Stephen
Richardson
2
,
Li
Meng
3
,
Brendan
Williams
3
,
Barry
D. Bruce
3

1.
Department
of Chemical and Biomolecular
Engineering,
Vanderbilt
University; 2. Department
of Biochemistry,Maryville
College; 3.
Department of Biochemistry, Cellular and Molecular
Biology, The University of Tennessee, Knoxville


Photosystem Isolation By Sucrose Ultracentrifugation

Sucrose Gradients

Chroococcidiopsis

T. Elongatus

3A)

3B)

Method
:

Thylakoid

membranes

of

Chroococcidiopsis

and

T
.

elongatus

(
0
.
2

ug/uL)

were

solubilized

with

1
%

DM

for

1
.
5

hours

at

25
°
C

before

loading

on

to

sucrose

gradients

(
10
%
--
30
%
)

with

0
.
05
%

DM

and

ultra
-
centrifuging

for

16

hours

at

20000

RPM

.



3
A)

Two

bands

(
1
-
2
)

were

taken

from

the

T
.

elongatus

sucrose

gradient

to

run

on

a

Blue

NativePAGE

gel

(
3
C)
.


3
B)

Five

bands

(
3
-
7
)

were

taken

from

the

Chroococcidiopsis

sucrose

gradient

(
3
C)
.



Blue NativePAGE of Sucrose Gradient Samples

Results
:

3
C)

The

labels,

TE

and

Ch,

correspond

to

the

non
-
centrifuged

thylakoid

membranes

of

T
.

Elongatus

and

Chroococcidiopsis
,

respectively
.

The

labels,

1
-
2

and

3
-
7
,

correspond

to

3
A

and

3
B

(the

same

in

Figure

4
A)
.

The

upper

gel

has

been

stained
.

Lane

1

has

no

PSI

trimer

(
1068

kDa)

while

lane

2

has

a

dark

band

corresponding

to

PSI

trimer
.

Lane

3

does

not

have

the

middle

large

green

band

(~
890

kDa)

while

6

and

7

have

little

of

the

lowest

large

green

band

(~
420

kDa)
.

None

of

the

Chroococcidiopsis

sucrose

samples

contain

a

high

degree

of

the

top

large

green

band

(~
1180

kDa)
.

This

contrasts

the

results

from

2
A,

which

had

the

top

large

green

band

(solubilized

with

1
.
0
%

DM)
.


3
D)

A

separate

BN
-
PAGE

gel

tests

the

same

variables

as

the

sucrose

gradient

prep

(Method

on

left)

except

the

membrane

samples

were

not

ultracentrifuged
.

The

labels,

10
%
,

20
%
,

30
%
,

correspond

to

the

sucrose

percentage

in

the

sample
.

These

lanes

all

have

the

top

large

green

band
.

It

is

likely

that

the

act

of

centrifugation

and

sedimentation

results

in

the

loss

of

this

band
.

Absorption Spectrum of Isolated Photosystems

Identifying Photosystems by 2
-
D SDS
-
PAGE

Conclusions and Future Directions

Acknowledgements

Abstract

Introduction

Photosystem

I

(PS

I)

is

a

protein

complex

that

transfers

electrons

from

plastocyanin

or

cytochrome

c

to

ferredoxin

in

cyanobacteria

in

response

to

photo
-
excitation
.

Understanding

the

organization

and

structure

of

PS

I

will

lay

the

ground

work

for

utilizing

it

as

a

part

of

solar

energy

devices
.

Different

from

plant

PS

I,

cyanobacterial

PS

I

was

believed

to

be

exclusively

trimer,

until

a

recent

report

of

a

tetramer

form

of

PS

I

in

Anabaena
.

Chroococcidiopsis

photosystems

are

studied

here

considering

their

vitality

in

various

extreme

environments
.

Blue
-
Native

PAGE

and

sucrose

gradient

ultra
-
centrifugation

are

used

as

the

main

methods

to

analyze

and

isolate

different

photosystems
.

The

isolated

Photosystems

from

the

cells

are

further

identified

by

SDS
-
PAGE

and

spectral

analyses
.

Thermosynechococcus

elongatus

(TE)

is

used

as

a

control

and

to

identify

the

photosystems

in

Chroococcidiopsis
.

Our

results

indicate

a

photosystem

complex

that

may

be

a

PS

I

tetramer/trimer

with

a

higher

molecular

weight

than

TE

PS

I

trimer
.

This

new

organization

of

PS

I

will

shed

light

on

the

understanding

of

the

evolutionary

significance

of

PS

I

monomerization

and

oligomerization
.

Hopefully,

with

a

better

understanding

of

Chroococcidiopsis

photosystems,

these

Photosystems

can

be

utilized

to

create

better

solar

energy

apparatus
.

Conclusions
:

1
.
Chroococcidiopsis

has

a

Photosystem

I

complex,

potentially

a

trimer

or

tetramer,

that

is

larger

than

TE

PS

I

trimer
;

2
.
The

majority

of

Chroococcidiopsis

photosystems

are

Photosystem

I
;

3
.
The

Chroococcidiopsis

trimer/tetramer

is

sensitive

to

high

concentrations

of

detergent

(DM)

and

ultra
-
centrifugation

force
;

4
.
Sucrose

gradient

can

separate

different

photosystem

mixtures

with

phycocyanin

attached

but

not

for

intact

higher

order

tetramer/trimer

complexes
.

5
.
Phycocyanin

is

attached

to

the

Chroococcidiopsis

photosystem

samples
.

6
.
Chroococcidiopsis

may

have

more

than

three

states

(monomer,

dimer,

trimer/tetramer)

of

PS

I
.


Future

directions

and

study
:


Chroococcidiopsis

PS

I

trimer/tetramer,

dimer

and

monomer

need

to

be

isolated

and

purified

further

to

study

the

properties

of

the

molecular

complex
.

1
.
Chroococcidiopsis

PS

I

trimer/tetramer

isolation

using

moderate

methods

such

as

Chromatography
;

2
.
Componental

analysis

of

the

different

forms

of

PS

I

using

Mass

Spectrometry
;

3
.
Fluorescence

spectra

analysis

of

different

PS

I

to

characterize

the

pigment

components
;

4
.
Dynamic

equilibrium

assay

of

phycocyanin

PS

I

association
;

5
.
Stability

assay

of

Chroococcidiopsis


PS

I
;

6
.
Electron

transport

rates

assay

of

Chroococcidiopsis

PS

I
.

Effect of Varying DM Concentration


Result
:

2
A)

Three

large

green

bands

(PSI

or

PSII)

and

at

least

six

other

faint

bands

were

separated

(ref
.

Fig

7
)

for

the

0
.
6
,

0
.
8
,

and

1
.
0
%

DM
.

Yet,

for

the

2
.
0
,

3
.
0
,

and

4
.
0
%

the

top

large

green

band

mostly

disappears
.

Also,

for

those

higher

DM

concentrations,

the

middle

large

green

band

becomes

larger
.

This

suggests

that

with

higher

DM,

the

multicomplex

photosystem

disassociates

into

smaller

photosystem

complexes
.


2
B)

Three

large

green

bands

and

one

blue

band

were

detected
.

The

concentration

of

DM

did

not

have

a

drastic

effect

on

the

T
.

Elongatus
.

The

large

green

bands

do

not

match

the

size

of

the

large

green

bands

in

Chroococcidiopsis
.


1 2
3
4 5 6 7

TE Ch

kDa

356

1068

700

356

1068

700

3D) Ch 10% 20% 30%

DM% .6

.
8

1 2 3

4 .
6 .8
1 2 3
4

Method
:

The

thylakoid

membranes

(prepared

from

fresh

cells)

of

Chroococcidiopsis

and

T
.

elongatus

were

each

solubilized

for

1
.
5

hours

at

25
°
C

in

six

concentrations

(
0
.
6
,

0
.
8
,

1
.
0
,

2
.
0
,

3
.
0
,

4
.
0

%
)

of

the

detergent

n
-
dodecyl
-
β
-

-
maltopyranocide

(DM
)
.

The

samples

were

loaded

on

a

4
-
16
%

acrylamide

Blue

NativePAGE

gel
.

As

the

consumption

of

energy

continues

to

increase

worldwide

(now

greater

than

425

X

10
18

J),

the

demand

for

novel

and

renewable

energy

capture

devises

has

never

been

higher
.

One

rapidly

developing

area

of

energy

research

is

in

solar

energy

utilization

and

bioelectric

hybrid

systems
.

These

systems

involve

the

union

of

biological

photosynthetic

protein

complexes

(photosystems)

and

electrically

charged

metal

scaffolding,

which,

when

organized

in

a

particular

orientation,

allows

them

to

together

create

the

movement

of

electrons

and

thus

electric

current
.



Photosystems

from

a

cyanobacterial

species,

Thermosynechococcus

elongates
,

have

previously

been

studied

for

their

potential

in

bioelectric

devices

because

the

species

is

well

characterized

and

has

an

available

crystal

structure
.

Typically
,

T
.

elongates

photosystem

I

(PS

I)

exists

as

a

trimer

(depicted

in

Figure

1
A)
.


Until

recent

report

of

a

tetramer

in

Anabaena
,

it

was

believed

that

all

cyanobacterial

photosystems

existed

exclusively

as

trimers
.




1A

1B

1A)T.E. Trimer with PsaL shown. 1B)
Chroococcidiopsis

microscopic image

2A)
Chroococcidiopsis
2B)
T. elongatus

3C)

j

b

c

d

e

g

h

i

k

m

a

f

l

5A) TE Ch

4
B
) PSI Trimer

4A)
Isolated Sucrose Gradient
Samples

4C
)
Phycocyanin

*

*

*

*

*

*

Another

genus

of

cyanobacteria

that

may

exist

as

a

tetramer

and

has

recently

become

of

interest

in

the

science

of

bioelectrics

is

Chroococcidiopsis

(Fig

1
B)
.

This

organism

is

of

particular

interest

because

of

its

ability

to

survive

a

wide

range

of

extreme

environments

that

are

uninhabitable

to

most

other

life

forms
.

The

goal

of

this

study

is

to

isolate

photosystem

I

of

Chroococcidiopsis

and

characterize

the

complex

through

various

experimental

techniques

such

as

Blue

NativePAGE

and

SDS

Page

electrophoresis,

sucrose

gradient

ultracentrifugation,

and

Absorbance

spectrum

analysis
.

Using

these

methods

to

better

understand

PS

I

of

Chroococcidiopsis,

hopefully

will

determine

whether

this

protein

complex

could

be

used

in

future

bioelectric

device

research

and,

ultimately,

expedite

the

introduction

of

similar

devices

as

viable

sources

of

clean,

renewable

energy

in

the

future
.


1
.

Phillips,

Tony
.

Greening

of

the

Red

Planet
.

NASA

Science

News
.

[Online]
.

http
:
//science
.
nasa
.
gov/science
-
news/science
-
at
-
nasa/
2001
/ast
26
jan_
1
/

2
.

Lavan,

David

and

Jennifer

N
.

Cha
.

Approaches

for

biological

and

biomimetic

energy

conversion
.

Proceedings

of

the

National

Academy

of

Sciences
.

2006
,

103
(
14
)
:

5251
-
5255
.

3
.

Watanabe,

Mai

et

al
.

Novel

Supercomplex

Organization

of

Photosystem

I

in

Anabaena

and

Cyanophora

paradoxa
.

Plant

and

Cell

Physiology
.

2010
,

52
(
1
)
:

162
-
168
.

4
.

Lewis,

Nathan

S

and

Daniel

G
.

Nocera
.

Powering

the

planet
:

Chemical

Challenges

in

Solar

Energy

Utilization
.

Proceedings

of

the

National

Academy

of

Science
.

2006
,

103
(
43
)
:

15729
-

15735
.

5
.

Mai

Watanabe

,

Masako

Iwai,

Rei

Narikawa

and

Masahiko

Ikeuchi
.

Plant

Cell

Physiology
.

2009
,

50
(
9
)
:

1674

1680



Special

thanks

to

the

National

Science

Foundation

and

the

funding

of

the

TN
-
SCORE

Research

Experience

for

Undergraduates

(
NSF

EPS
-
1004083
)
.






Method:

To further characterize the photosystem components of
Chroococcidiopsis
, bands from Blue
NativePAGE separation (Fig 5A) were excised and subjected to separation via SDS
-
PAGE Gel
electrophoresis and subsequent silver staining.

Results:

Figure 5B shows the sample protein separations compared to the
T
.
elongatus

purified trimer
control. The banding patterns in samples “a,” “e,” “f,” “g,” “h,” “k,” and “l” are consistent with the
protein fragments in PS I (noted by blue asterisks), while the banding in samples “c,” “d,” “i,” j,” and
“m” are consistent with the expected fragment sizes of PS II (noted by red asterisks).

1A