Effects of Detergents on the 

Crystallization of 

Bacteriorhodopsin 

Emily Blick

Mentor: Thomas Cleveland

Summer Undergraduate Research Fellowship
OUTLINE

• Protein crystallization

• Why?

• How?

• Membrane proteins

• How is approach to crystallization different?

• Lipidic cubic phase: what is it, and why use it?

• Project Aims

• Approach

• Results
PROTEIN CRYSTALLIZATION

• Protein crystals are necessary for structure 

determination by x-ray crystallography

• Protein crystal is exposed to x-ray beam

• Results in diffraction patterns from electron clouds

• Determines three dimensional structure of proteins 

and macromolecules

• Structures necessary for understanding protein 

function

• binding to other proteins

• binding to drugs

• enzyme mechanisms

Bacteriorhodopsin diffraction patterns and resulting 

structural determination.

X-ray

Bacteriorhodopsin 

crystal

X-ray diffraction

Detector
SOLUBLE PROTEINS

• Highly concentrate target protein

• Introduce precipitant to encourage 

crystal growth

• Hanging drop vapor diffusion

• Proteins are not soluble

• Need detergents!

• Highly concentrate target protein

• Introduce precipitant to encourage 

crystal growth

• Protein and detergent complexes may 

also be incorporated into the lipidic 

cubic phase

MEMBRANE PROTEINS

HOW IS IT USUALLY DONE?

H20

Slide cover

Protein, precipitant, and 

water

Precipitant solution
OVERVIEW

• In meso crystallization is important method for some classes of membrane 

proteins

• Small proteins with few crystal contacts in hydrophilic domains

• Better crystal packing can lead to higher-resolution crystal structures

• Detergents are used to solubilize proteins

• Importance of detergent identity unknown in the cubic phase

• Currently expensive and highly purified detergents in use

• Protein and detergent complexes are incorporated into lipidic cubic phase

• Compare success of wide span of detergents
THE ROLE OF DETERGENT

• Amphiphilic nature allows hydrophobic parts 

of membrane proteins to be solubilized 

• Forms lipid bilayer

• Large span of detergent properties

• Detergent characteristics affect chemical 

properties

• Detergent monomers

• Micelles have different shapes and sizes

• Prepares for incorporation into lipidic cubic 

phase

Hydrophilic head group

Hydrophobic tail
DETERGENTS IN SOLUTION

• Detergents form micelles in solution, and around membrane proteins

• Hydrophobic effect

• Micelles can be different shapes and sizes

• Some detergents are “better” than others for some proteins

• This varies from protein to protein
THE CUBIC PHASE

• Three dimensional bilayer with water, lipid, 

and protein

• Forms a bicontinuous phase

• Protein reconstituted into the lipid bilayer

• Protein remains in native and active conformation

• Protein mobility

• Precipitant added to induce phase separation

• Phase that has high levels of protein can 

encourage crystal growth

• Viscous and difficult handling
IN MESO CRYSTALLOGENESIS

• Lipidic cubic phase formed through lipid hydration

• 40% hydration for protein incorporation

• Cubic mesophase as environment for crystallization

• Hydrated monoolein to Pn3m stage
CRYSTALLIZATION 

OVERVIEW

Protein/ detergent 

solution Monoolein

Homogenized to 

create cubic phase

Dispense bolus onto 

slide

Add excess precipitant

Cover slide and 

store in the dark

Visualize crystals in 3 

days to a week No apparent crystals
MAKING LCP AND PERFORMING 

CRYSTALLIZATION TRIALS

Sandwich plates

Mix lipid:protein in correct ratio 

(60:40 for monoolein)

Dispenser for manual trays

glass slide

coverslip

double-stick spacer

precipitant

LCP

Side View:
OUTLINE

• Protein crystallization

• Why?

• How?

• Membrane proteins

• How is approach to crystallization different?

• Lipidic cubic phase: what is it, and why use it?

• Project Aims

• Approach

• Results
PROJECT AIMS

1. Determine whether a variety of detergents (other than the 

standard octyl glucoside) support crystallization of bR in LCP.

Other crystallization-related side issues:

• SANS is done in D2O. We need to verify that crystals can still be obtained.

• Crystallization is usually done in bR after size exclusion chromatography. For rapid 

detergent screening, we will only use centrifugation. 

2. Use scattering to measure shape/size of micelles for common 

detergents

3. Determine what happens to these detergents upon incorporation 

into the LCP, and after addition of precipitant.
PROJECT AIMS

“Does the detergent 

matter?”

1. Determine whether a variety of detergents (other than the 

standard octyl glucoside) support crystallization of bR in LCP.

Other crystallization-related side issues:

• SANS is done in D2O. We need to verify that crystals can still be obtained.

• Crystallization is usually done in bR after size exclusion chromatography. For rapid 

detergent screening, we will only use centrifugation. 

2. Use scattering to measure shape/size of micelles for common 

detergents

3. Determine what happens to these detergents upon incorporation 

into the LCP, and after addition of precipitant.

Why does it 

(not) matter?
MOTIVATION

 In solution, detergents that are good for protein stability can be bad for crystallization:

• Large micelle 

• Heterogeneous

 Detergents that are good for crystallization can be bad for stability, or can present 

other practical difficulties:

• Can be extremely costly

• Other poor properties (e.g., low solubility, complex pH/temperature behavior, etc.)

 Hypothesis: micelles dissociate upon incorporation into the lipidic cubic 

phase, so the detergent identity becomes less important than in solution.

 It would be nice to be able to work with any detergent that your protein is 

stable in without having to separately consider whether that detergent will 

allow crystallization.
OUTLINE

• Protein crystallization

• Why?

• How?

• Membrane proteins

• How is approach to crystallization different?

• Lipidic cubic phase: what is it, and why use it?

• Project Aims

• Approach

• Results
MODEL SYSTEM: 

BACTERIORHODOPSIN (BR)

• Photosynthetic transmembrane protein in 

Halobacterium salinarum

• Converts light energy into proton gradient

• Naturally present in “purple membrane:” twodimensional crystals embedded in cell membrane

• Structure and function studied in detail

• Stable

• Crystallization propensity
BR CAN BE EXPRESSED AND PURIFIED 

IN LARGE AMOUNTS

Cell Pellet Low-Salt

Lysis

Washed 

Membranes

Sucrose

Gradient

“Purple 

Membrane”

bR

bacterioruberin

debris
FURTHER PURIFICATION BY SIZE EXCLUSION 

CHROMATOGRAPHY (SEC)

Addition of OG to 

purple membrane:

t = 0 1 day

Size exclusion 

chromatography column
DETERGENT CLASSES SELECTED FOR STUDY

Maltoside Detergents 

5-Cyclohexylpentyl β-Dmaltoside

Neopentyl Glycol Detergents 

n-Octyl-β-D-Glucopyranoside $20.12

Elugent $1.48

n-Dodecyl-N,N-Dimethylamine-N-Oxide 

Fos-Choline 

Tween Detergents

Polyoxyethylene Ether 

Detergents

Triton Detergent
SMALL-ANGLE NEUTRON SCATTERING

• Scattering of neutrons through interaction with nuclei

• Scattering is result of inhomogeneities in sample (scattering length density)

• Detector can move to reach a range of scattering angles

Velocity Selector Sample

Collimation Plates

Neutron Guide

Scattered Beam

Incident Beam

q

Detector

Ki

Ks

q

QQ = 2KKKK sin θθ =

4ππ

λ sin θθ
WHY NEUTRONS?

• Contrast matching

• Exchanging hydrogen for deuterium in order to match 

your solvent to a component of sample

• D2O has scattering length density of 6.3×10-6 Å-2

• H2O has scattering length density of -0.56×10-6 Å-2

• H2O/D2O mixture can match any sample 

component with SLD between those values

• Can be used to silence some features and examine 

others
SANS WITH CONTRAST-MATCHED LIPIDS

Solvent 

Channels

Adjust [H2O]/[D2O] of solvent so that its 

scattering length density matches the lipid

Without contrast match: 

Scattering will be mostly 

from the lipid (which makes 

up most of the sample)

With contrast matching:

Scattering will be mostly 

from structures embedded 

in the (now-invisible) lipid
OUTLINE

• Protein crystallization

• Why?

• How?

• Membrane proteins

• How is approach to crystallization different?

• Lipidic cubic phase: what is it, and why use it?

• Project Aims

• Approach

• Results
RESULTS

1. Crystallization trials

• Different detergents?

• H2O vs D2O?

• Size-exclusion chromatography necessary or not?

2. Scattering Measurements

• Detergent micelles

• Detergents in the lipidic cubic phase

 After initial mixing

 Immediately after precipitant addition
PROTEIN SOLUBILIZATION

Maltoside Detergents 

5-Cyclohexylpentyl β-Dmaltoside

Neopentyl Glycol Detergents

n-Octyl-β-D-Glucopyranoside $20.12

Elugent $1.48

n-Dodecyl-N,N-Dimethylamine-N-Oxide 

Fos-Choline 

Tween Detergents 

Polyoxyethylene Ether 

Detergents

Triton Detergent
PROTEIN SOLUBLIZATION
CRYSTALLIZATION TRIALS

1 M

2.5 M 2.75 M 3 M

1.75 M 2 M 2.25 M

1.25 M 1.5 M

H2O D2O

Concentration of precipitant (Na/K Pi pH 5.6) Typical plate layout

LCP 

(with bR)

Surrounding 

precipitant 

droplet

Well
CRYSTALLIZATION TRIALS

1 M

2.5 M 2.75 M 3 M

1.75 M 2 M 2.25 M

1.25 M 1.5 M

H2O D2O

Concentration of precipitant (Na/K Pi pH 5.6) Typical plate layout

LCP 

(with bR)

Surrounding 

precipitant 

droplet

Well
CRYSTALLIZATION TRIALS

Octyl Glucoside Elugent Triton X-100 No hits yet:

OGNG

LMNG

C12E9

DDM

LDAO

Crystals after ~5 days:

D2O H2O H2O D2O H2O D2O

2.5

1.75

1.0

3.0

2.25

1.5

2.75

2.0

1.25

Key

(Na/K Pi precipitant 

concentration in M)
CRYSTALLIZATION TRIALS

Octyl Glucoside Elugent* Anapoe X-100** No hits yet:

OGNG

LMNG

C12E9

DDM

LDAO

Crystals after ~5 days:

*Essentially a less-pure (far less expensive) 

form of octyl glucoside that has a distribution 

of different carbon chain-lengths. 

**The same compound as Triton X-100, but this specific 

product is supplied with low peroxide content and 

packaged under inert gas. 
CRYSTALLIZATION TRIALS

Octyl Glucoside Elugent* Anapoe X-100** No hits yet:

OGNG

LMNG

C12E9

DDM

LDAO

Crystals after ~5 days:

Neither Elugent nor Triton X-100 are 

typically used for solution crystallization 

of membrane proteins!

• Both of them are heterogeneous, low-purity 

detergent mixtures.

• Both are extremely inexpensive
RESULTS

1. Crystallization trials

• Different detergents?

• H2O vs D2O?

• Size-exclusion chromatography necessary or not?

2. Scattering Measurements

• Detergent micelles

• Detergents in the lipidic cubic phase

 After initial mixing

 Immediately after precipitant addition
DETERGENTS IN SOLUTION

Detergent micelles can have a variety of shapes/sizes.

Example scattering curves:

Shapes:

(cartoon only)

LDAO OG LMNG
ELLIPTICAL CYLINDER

Detergent Radius

LDAO 20.271

n-Decyl 21.648

CYMAL 5 21.885

n-Dodecyl 25.192

C12E8 29.102

SPHERE

MICELLE SHAPES AND SIZES

Detergent Length (A) Radius (A)

n-Tridecyl 17.998 17.998

n-Tetradecyl 18.089 18.089

LMNG 27.256 18.507

Fos-Choline 31.147 14.006

Triton 35.261 22.887

Anapoe-40 41.354 18.898

Anapoe-100 42.076 18.773

Anapoe-58 43.043 33.713

C10E9 43.589 15.863

C12E10 50.307 18.717

Detergent Length (A) Radius (A)

C12E9 54.262 22.839

C10E6 55.898 16.142

Anapoe-35 56.375 20.153

Anapoe-305 58.193 18.127

Anapoe-20 62.894 21.363

C13E8 64.197 19.776

Anapoe-80 73.597 27.083

OG 139.471 10.454

ELUGENT 187.012 13.901

OG Neo 224.900 9.3717
WHAT HAPPENS TO THE 

DETERGENT IN THE LIPIDIC 

CUBIC PHASE?
Solution Micelles Dispersed?

Phase-separated 

structures?

WHAT HAPPENS TO THE DETERGENTS 

IN THE LIPIDIC CUBIC PHASE?

H2O

D2O

Scattering 

Length Density

Without contrast matching: most observed scattering will just be from the LCP

solvent
WHAT HAPPENS TO THE DETERGENTS 

IN THE LIPIDIC CUBIC PHASE?

H2O

D2O

Scattering 

Length Density

Without contrast matching: most observed scattering will just be from the LCP

solvent
WHAT HAPPENS TO THE DETERGENTS 

IN THE LIPIDIC CUBIC PHASE?

H2O

D2O

Scattering 

Length Density

With contrast-matched lipids: scattering from any structures formed by detergents will be observed

solvent
DETERGENTS IN THE LCP – SANS

Compare cubic phases with/without detergent:

1. Without detergent

2. With detergent

3. With detergent + 2 M Na/K Pi Precipitant

1.

2.

3.

Very little change
DETERGENTS IN THE LCP – SANS

Curves

1. Detergent solution micelle

2. Similar amount of detergent in LCP

3. LCP/detergent after addition of precipitant

1.

2.

3.

Compare cubic phase scattering to 

what we would see if there were 

aggregated structures such as micelles

Detergent aggregates are not seen.

Detergent is dispersed in the cubic phase.
DETERGENTS IN THE LCP – SANS

Anapoe X-100 DDM OG

LMNG C12E9 LDAO

Similar results were found for all detergents tested.

DETERGENTS IN THE LCP – X-RAY

• Detergent solutions (3%) were mixed with the lipid 

to form cubic phases

• Bragg peak positions can be used to determine the 

lattice spacing of the cubic phase

• Detergents cause swelling in the cubic phase

• Crystals were obtained from Triton X-100 and 

Octyl Glucoside

• No direct correlation between lattice parameter vs. 

crystallization hits.

Detergent aggregates were not seen; but what effects do 

they have on the cubic phase lattice? Used x-ray diffraction:

Detergent Lattice Par. (Å)

OG 113.8

DDM 111.8

LDAO 108.1

C12E9 108.1

TX100 106.4

LMNG 106.4

None 101.4
CONCLUSIONS

• In LCP crystals of bR can be obtained in additional detergents aside from the traditionally-used octyl glucoside

• include atypical (for crystallization studies) detergents such as Triton X-100 and Elugent

• Less-refined detergents in advantageous due to:

• Low cost

• May be most suitable for protein stability in certain proteins

• Absence of LCP crystals other detergents may not be due to “incompatibility” with crystallization but instead:

• Poor solution stability before LCP incorporation (LDAO which showed color changes indicative of bR denaturation)

• Detergents matter less in the cubic phase because they disaggregate

• Properties depend more on the lipid then dispersed detergent
ACKNOWLEDGEMENTS

• My mentor: Thomas Cleveland

• SURF Coordinators: Julie Borchers, Joseph Dura, and Brandi Toliver

• Center for High Resolution Neutron Scattering

• NIST Center for Neutron Research

• My fellow NCNR SURFers.
REFERENCES

• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1186895/

• https://phys.org/news/2015-12-bacteriorhodopsin-crystals-consume-smallercounterparts.html

• http://www.chem.ucla.edu/~harding/ec_tutorials/tutorial60.pdf

• https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3182643/

• http://www.chem.uwec.edu/Chem455/expressbR.pdf