D2O AND THE LIPIDIC CUBIC PHASE

Stacy Young

Mentor: Tom Cleveland
INTRODUCTION

2 components to project: 

–Protein preparation + crystallization

–Determining the phase diagram of monoolein in 

D2O
BACKGROUND

Why do we crystallize proteins?

 Analysis via xray scattering

structural information revealed

2 requirements of crystallization:

1) Protein stability 

2) Diffusible environment 

2 types of proteins

 Soluble vs. membrane
BACKGROUND

Membrane proteins:

 Embedded within lipid bilayer

Lose structural + functional stability when 

removed from membrane

 Critical role in cell processes

 26+% of proteins coded by human genome = 

membrane proteins*

 Over 50% of drugs target membrane proteins +

 As of 2014, 406 out 36,000 identified proteins 

are membrane proteins*

*Source: 2014 - Kynde et al. - Small-angle scattering gives direct structural information about a 

membrane protein inside a lipid environment.pdf

+http://www.irbbarcelona.org/en/news/more-than-50-of-drugs-target-membrane-proteins

Source: http://goo.gl/kAFA0C
BACKGROUND

Problems with methodology

Protein isolated, mixed with buffers + precipitants

Mostly only works with soluble proteins

Membrane proteins ....? 

Not in solution

Need to be purified but protected at the same time
METHODS: BR

Bacteriorhodopsin (bR) isolated from Halobacteria

1) Grow organism

2) Harvest

2) Lyse

3) Isolate membranes

4) Solubilize with octyl glucoside 

(OG)

Image from experiment: Halobacteria culturing 
Cell Pellet Low-Salt

Lysis

Washed 

Membranes

Sucrose

Gradient

“Purple Membrane”

bR

bacterioruberin

debris

BR EXPRESSION AND PURIFICATION
METHODS: BR

OG 

 Detergent

 Solublizes protein, prevents large 

masses from aggregating 

 Used to remove bR from membrane

 Formation of detergent micelles 

Protein purified...but still 

cannot crystallize

 Protein placed in lipidic cubic phase

Source: 

http://goo.gl/KVfuEQ

Standard 

phospholipid

Source: [Caf03] Caffrey, M. (2003) Membrane protein 

crystallization. Journal of Structural Biology 142:108-132. 
LIPIDIC CUBIC PHASE

Special lipid environment

Satisfies conditions for 

crystallization

Variations of cubic phases

Source: http://goo.gl/4SUIus
CRYSTALLIZED PROTEIN 

Placed 0.2 μL of cubic phase 

containing protein on slide

Layered 1 μL of phosphate precipitant 

onto each cubic phase drop

Crystals form over about 1.5 weeks

Crucial experimental checkpoint 

1.8 M Na/K Phosphate pH 5.6

2.6 M Na/K Phosphate pH 5.6
EXPERIMENTAL OBJECTIVE

2 knowns:

bR placed in lipidic cubic phase

bR WILL crystallize 

How?

Small Angle Neutron Scattering (SANS)

Examine early intermediates of protein crystal formation
METHODS: PHASE DIAGRAMS

Want to observe protein

contrast match out lipid

Creation of a phase diagram of monoolein in D2O

determine which conditions yield which cubic phase

Want to understand exactly where we are in the phase 

diagram
METHODS: PHASE DIAGRAMS

Capillaries

 filled with monoolein and varying concentrations of D2O or H2O

analyzed via Small Angle X-ray Scattering (SAXS) in different temperatures (17°C-

30 °C)

Fit2d program used to generate I vs. Q graph from scattering image
CAPILLARIES: SAXS IMAGE + CORRESPONDING GRAPH

25% D2O concentration

1.00

10.00

100.00

1,000.00

0.00 1.00 2.00 3.00 4.00 5.00

Q

I

I vs. Q 25% D2O 26°C
CAPILLARIES: SAXS IMAGE + FITTED GRAPH

first two graph peaks fit using a 

Gaussian function 

 determine position

ratio of peak position indicated 

the given cubic phase

Q

I

I vs. Q 25% D2O 26°C

1.00

10.00

100.00

1,000.00

0.00 1.00 2.00 3.00 4.00 5.00
PHASE DIAGRAM

10.00

15.00

20.00

25.00

30.00

35.00

20.00 25.00 30.00 35.00 40.00 45.00 50.00

Temperature

% aqueous

D2O Phase Diagram: Temperature vs. % 

Aqueous

Ia3d pn3m

0.00

5.00

10.00

15.00

20.00

25.00

30.00

0.00 10.00 20.00 30.00 40.00 50.00

Temperature

% aqueous

H2O Phase Diagram: Temperature vs. % 

Aqueous

Ia3d pn3m

LATTICE PARAMETER VS. % AQUEOUS

0.00

20.00

40.00

60.00

80.00

100.00

120.00

140.00

160.00

180.00

20.00 30.00 40.00 50.00

Lattice parameter

% aqueous

Lattice Parameter vs. % Aqueous 

(26 °C) 

D2O H2O

0.00

20.00

40.00

60.00

80.00

100.00

120.00

0.00 10.00 20.00 30.00 40.00

Lattice Parameter

Temperature

Lattice Parameter Vs. 

Temperature (°C) 

40% D2O

0.00

20.00

40.00

60.00

80.00

100.00

120.00

140.00

160.00

180.00

0.00 10.00 20.00 30.00 40.00 50.00

Lattice Parameter (Å)

% aqueous

Lattice Parameter vs. % 

Aqueous (17°C)

D2O H2O
BIG PICTURE...WHO CARES?

bR “easy” membrane protein, structure has already been identified

Why spend so much time with this protein?

Advantageous to study exact process of protein crystal formation

 bR excellent sample group

 Process applied to more difficult membrane proteins

Pharmaceutical applications

 More advanced drugs 
FINAL THOUGHTS

Structure, Structure, Structure! 

Entirety of life dependent on the interaction and workings of 

proteins

Small step...but large stride
ACKNOWLEDGEMENTS

Dr. Thomas Cleveland

Dr. Zvi Kellman

Dr. Paul Butler

Dr. Julie Borchers & Dr. Yamali Hernandez 

NIST SHIP Director

NIST Center for Neutron Research

Institute for Bioscience and Biotechnology Research

National Science Foundation + CHRNS