Detergent Screening Via Immobilized-Protein Stability AssayJames M. VergisLaboratory of Michael Wiener

Background

• Membrane protein structural studies typically performed in detergent – Detergent preserves structure/function of protein by

mimicking the membrane environment

• Choosing the “right” detergent critically important

• Detergent-space is historically biased

Current Methods

• A320nm/A280nm ratio• HPSEC profile• Ultracentrifugation/SDS-PAGE

A320nm/A280nm Ratio

• Protocol:– Concentrate protein in current detergent– Dilute into new concentrated detergent solution– Compare A320nm/A280nm ratios

• A320nm increase indicative of aggregation (i.e. BAD detergent)

• Pros:– Combined with a microplate spectrophotometer, allows parallel detergent

screening– Fast and simple

• Cons:– Requires large amounts of protein– Detergent can concentrate with protein leading to false positives

Wiener, M.C., Methods 34, 364-372 (2004)

HPSEC Profile• Protocol #1:

– Inject protein onto HPSEC column equilibrated in new detergent– Assess quality of Abs280nm chromatogram

• Protocol #2 (FSEC) (Kawate, T. and Gouaux, E., Structure 14, 673-681 (2006)) :– GFP-label protein and monitor Fluorescence instead of Abs280nm

– Solubilize/dilute/exchange protein into new detergents– Inject onto HPSEC column using same detergent mobile phase for all samples– Assess quality of chromatogram

• Pros:– HPSEC provides readout on monodispersity– Nanogram quantities of unpurified protein necessary (FSEC)– Same detergent mobile phase speeds up HPSEC runs (FSEC)

• Cons:– Time consuming (detergents evaluated in series)– GFP-tag can have deleterious effect on protein (FSEC)

Ultracentrifugation/SDS-PAGE• Protocol:

– Purify the protein, dilute into new detergent, & spin-concentrate– Repeat dilution/concentration step three more times– Ultracentrifuge sample to pellet aggregated protein– Compare before and after ultracentrifuge samples by SDS-PAGE

• Pros:– Detergents evaluated in parallel– Relatively small amount of protein required

• Cons:– Original detergent may still be present due to concentration steps

Gutmann, D.A.P. et al., Protein Science 16, 1422-1428 (2007)

Shortcomings

• Dilutions NOT exchanges• HPSEC is too slow to test large

numbers of detergents• Concentration step can lead to false

positives– i.e. detergent looks good but in reality is not– Original detergent is not removed or diluted below its

CMC

Detergent Concentrates• Free micelle

concentration can be determined using RI and HPSEC– Protein:

• 0.1mg/ml to 3.3mg/ml– 33X concentration

– Detergent :• 2.5mM to 63mM

– 25X concentration

• Particularly problematic with small size difference between PDC and detergent micelle

False-Positive Example

20070102 2L wtPBR IMAC in DM001:10_UV1_280nm 20070117 2L wtPBR imac normal001:10_UV1_280nm

0

500

1000

1500

2000

2500

3000

3500

4000

mAU

0 20 40 60 80 100 120 ml

• Detergent Dilution– A320nm/A280nm ratio low– HPSEC looks good

• BUT do detergent exchange on IMAC and protein precipitates on column– Led to AIPSA idea!

HPSEC─ FosCH12─ DM

─ FosCH12─ DM

IMAC

20070301 wtPBR small GF in D2O001:10_UV1_280nm 20061117 PBR in DM small G200:10_UV1_280nm@01,NORM

0

500

1000

1500

2000

mAU

0.0 5.0 10.0 15.0 20.0 25.0 ml

Why do we a new method?

• False positives probable• Large amounts of protein may be

required• High-throughput testing not possible• HPSEC is time expensive

Affinity-Immobilized Protein Stability Assay (AIPSA)• Not just for detergent screening

– Can be used to screen buffers, pH, salts, etc.

• Protocol:– Bind protein to affinity matrix– Extensively wash bound protein with new buffer (20CV)– Elute protein in new buffer (3CV)– Analyze protein by SDS-PAGE and HPSEC

• Pros:– Requires small amounts of protein and materials– Screening easily performed in parallel– True exchange

• Cons:– Still uses HPSEC (for now)

Basis for Detergent AIPSA

1. Based on “old-school” detergent exchange method– Bind protein to column and wash extensively with new

detergent

2. Incompatible detergents will cause protein to precipitate on resin while compatible detergents maintain the protein’s solubility

Detergent Screening via AIPSA• Amounts of material used:

– 10µl resin/condition (1ml total)– 1-50µg protein/condition (0.1-5mg total)– 230µl detergent (20CV wash and 3CV elution)

• Special apparatus/consumables:– 96-well filter plates– Multi-channel pipette or fluid handling robot– Vacuum manifold or microplate centrifuge– E-PAGE gels (48 or 96 well) and E-Base power supply– iBLOT system and transfer stacks– Superdex™ 200 5/150 gel filtration column (a.k.a. short column)

Detergent Screening via AIPSA • 30µl elution volume

– 5-10µl typically used for gel– Remaining volume available for other experiments

• Gels blotted to nitrocellulose membranes– E-PAGE gels take too long to destain– Membranes stained with Thermo MemCode™ Stain

• HPSEC is final step to evaluate “goodness”• Panel of 94 detergents evaluated in parallel

– Resin to gel readout only 1hr!!!– Each HPSEC run 6min

• 10hrs worse case

• Selection criteria– CMC between 0.03mM and 40mM– Soluble in water– Commercially available

Ruman, P. et al., Cellular and Molecular Life Sciences 63, 36-51 (2006).

water solubilizing detergent

ANZERGENT®

3-12(12)

ANZERGENT®

3-14 n-Decyl-N,N-

dimethyl-glycine n-Dodecyl-N,N-dimethyl-glycine

n-Decyl-N,N-dimethyl-amine-

N-oxide (10)

n-Undecyl-N,N,-dimethyl-amine-

oxide

n-Dodecyl-N,N-dimethyl-

amine-N-oxide (108)

C-DODECAFOS CYCLOFOS-4 CYCLOFOS-5

CYCLOFOS-6 CYCLOFOS-7 FOS-CHOLINE®-10

FOS-CHOLINE®-11

FOS-CHOLINE®-12

(32)

FOS-CHOLINE®-13

FOS-CHOLINE®-14

(1)

FOS-CHOLINE®-

ISO-11

FOS-CHOLINE®-ISO-11-6U

FOS-CHOLINE®-

ISO-9

FOS-CHOLINE®-

UNSAT-11-10

DHPC(6)

LysoPC-10 LysoPC-12 FOSFEN-9 CHAPS(1)

CHAPSO(15) DDMAU DDMAB LAPAO

(2) TRIPAO ANAPOE®-20 ANAPOE®-35ANAPOE®-

X-100 (2)

ANAPOE®-X-114

ANAPOE®-X-305

ANAPOE®-X-405

(2)

ANAPOE®-NID-P40 APO8 APO9 APO10

(1) APO11 APO12 C6E3 C6E4 C6E5

C7E5 C8E4(52)

C8E5 (1) C8E6 C10E5

(5)C10E6

(1)ANAPOE®-

C10E9 C12E8

(19)ANAPOE®-C12E9 (19)

ANAPOE®-C12E10

ANAPOE®-C13E8 Big CHAP

Big CHAP, deoxy

Octyl-2-hydroxyethyl-

sulfoxide(17)

Rac-2,3-dihydroxy-propyl

octylsulfoxideGenapol X-100 n-Heptyl-β-D-

thioglucoside (3)

n-Octyl-β-D-glucoside

(130)

n-Nonyl-β-D-glucoside

(19)CYGLU®-3 ANAMEG®-7 Hega-9 C-Hega-10 C-Hega-11

CYMAL®-3(1)

CYMAL®-4(1)

CYMAL®-5(7)

CYMAL®-6(3) CYMAL®-7

2,6-Dimethyl-4-heptyl-β-D-maltoside

n-Octyl-β-D-maltoside

(2)

n-Nonyl-β-D-maltoside

(2)

n-Decyl-α-D-maltoside

n-Decyl-β-D-maltoside

(56)

n-Undecyl-α-D-maltoside

n-Undecyl-β-D-maltoside

(9)

ω-Undecylenyl-β-D-maltoside

n-Dodecyl-α-D-maltoside

(1)

n-Dodecyl-β-D-maltoside

(80)

n-Tridecyl-β-D-maltoside

(1)

n-Octyl-β-D-thiomaltoside

n-Nonyl-β-D-thiomaltoside

(1)

n-Decyl-β-D-thiomaltoside

n-Undecyl-β-D-thiomaltoside

n-Dodecyl-β-D-thiomaltoside

(1)Sucrose8 Sucrose10 Sucrose12

Example1 - AqpZ

20µl load, 0.5ml/min, Superdex 200 5/15020mM Tris pH 7.4, 500mM NaCl, 10% glycerol, 40mM OG

*

OGDDMABBig CHAP, deoxy

Example #1 - AqpZ

*

500µl load, 0.5ml/min, Superdex 200HR 10/3020mM Tris pH 7.4, 500mM NaCl, 5mM FosCH12 or 2.6mM C12E9

Example #2 – Human X

Example #3 - KcsA

Future Improvements• Use two 48-well gels instead of one 96-well gel

– Migration appears less affected by detergent in 48-well gels– Transfer seems more consistent for 48-well gels– Or skip the gel and use a dot-blot?

• In-line fluorescence detection for HPSEC– 100-1000x more sensitive than UV detection

• E-PAGE/iBLOT → 1–5µg/condition • E-PAGE/iBLOT + HPSEC (UV) → 50µg/condition• E-PAGE/iBLOT + HPSEC (Fluor) → 1–10µg/condition• HPSEC (Fluor) only → 0.5µg/condition

• Incorporate HPLC autosampler

Discussion/Conclusions• AIPSA detergent screening works• SDS-PAGE alone not sufficient for assessing the “goodness” of a

detergent; an HPSEC step is required• 1st running a protein gel eliminates negative samples from HPSEC• Despite HPSEC being the “slow step” for this method, using the short

column and a constant mobile phase are large timesavers• Fluorescence detector will reduce total amount of protein required• Once the best detergents are found, repeating HPSEC with those

detergents in the mobile phase is recommended and when used in conjunction with RI and SLS, the masses of bound detergent, protein, and the PDC can be estimated

• Other protein quality tests can be easily dialed in• AIPSA not limited to just detergent stability

Acknowledgements

Michael WienerMichael PurdyPeter HoranyiDavid ShultisChristian Banchs

Supported by NIH Roadmap Grant 5R01 GM075931 (MW)