Transcript Detergent Screening

Detergent Screening Via
Immobilized-Protein
Stability Assay
James M. Vergis
Laboratory 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
20070301 wtPBR small GF in D2O001:10_UV1_280nm
• Detergent Dilution
20061117 PBR in DM small G200:10_UV1_280nm@01,NORM
mAU
HPSEC
2000
– A320nm/A280nm ratio low
– HPSEC looks good
─ FosCH12
─ DM
1500
1000
500
0
0.0
5.0
20070102 2L wtPBR IMAC in DM001:10_UV1_280nm
10.0
15.0
20070117 2L wtPBR imac normal001:10_UV1_280nm
20.0
25.0
ml
mAU
4000
3500
IMAC
3000
─ FosCH12
─ DM
2500
2000
• BUT do detergent
exchange on IMAC and
protein precipitates on
column
– Led to AIPSA idea!
1500
1000
500
0
0
20
40
60
80
100
120
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
n-Dodecyl-N,NANZERGENT®
n-Decyl-N,N- n-Undecyl-N,N,ANZERGENT® n-Decyl-N,N- n-Dodecyl-N,NdimethylC3-12
dimethyl-amine- dimethyl-amine3-14
dimethyl-glycine dimethyl-glycine
amine-N-oxide DODECAFOS
(12)
N-oxide (10)
oxide
(108)
water
solubilizing
detergent
CYCLOFOS-6
CYCLOFOS-7
FOSCHOLINE®-10
FOSCHOLINE®-11
FOSCHOLINE®-12
(32)
FOSCHOLINE®-13
FOSCHOLINE®-14
(1)
FOSCHOLINE®ISO-11
FOSCHOLINE®ISO-11-6U
LysoPC-10
LysoPC-12
FOSFEN-9
CHAPS
(1)
CHAPSO
(15)
DDMAU
DDMAB
LAPAO
(2)
ANAPOE®X-114
ANAPOE®X-305
ANAPOE®X-405
(2)
ANAPOE®-NIDP40
APO8
APO9
APO10
(1)
C7E5
C8E4
(52)
C8E5
(1)
C8E6
C10E5
(5)
C10E6
(1)
Big CHAP,
deoxy
Octyl-2hydroxyethylsulfoxide
(17)
Rac-2,3dihydroxy-propyl
octylsulfoxide
Genapol X-100
n-Heptyl-β-Dthioglucoside
(3)
CYMAL®-3
(1)
CYMAL®-4
(1)
CYMAL®-5
(7)
CYMAL®-6
(3)
ω-Undecylenylβ-D-maltoside
n-Dodecyl-α-D- n-Dodecyl-β-D- n-Tridecyl-β-Dmaltoside
maltoside
maltoside
(1)
(80)
(1)
CYCLOFOS-4
CYCLOFOS-5
FOSCHOLINE®ISO-9
FOSCHOLINE®UNSAT-11-10
DHPC
(6)
TRIPAO
ANAPOE®-20
ANAPOE®-35
ANAPOE®X-100
(2)
APO11
APO12
C6E3
C6E4
C6E5
ANAPOE®C10E9
C12E8
(19)
ANAPOE®C12E9 (19)
ANAPOE®C12E10
ANAPOE®C13E8
Big CHAP
n-Octyl-β-Dglucoside
(130)
n-Nonyl-β-Dglucoside
(19)
CYGLU®-3
ANAMEG®-7
Hega-9
C-Hega-10
C-Hega-11
CYMAL®-7
2,6-Dimethyl-4heptyl-β-Dmaltoside
n-Octyl-β-Dmaltoside
(2)
n-Nonyl-β-Dmaltoside
(2)
n-Decyl-α-Dmaltoside
n-Decyl-β-Dmaltoside
(56)
n-Undecyl-α-Dmaltoside
n-Undecyl-β-Dmaltoside
(9)
n-Octyl-β-Dthiomaltoside
n-Nonyl-β-Dthiomaltoside
(1)
n-Decyl-β-Dthiomaltoside
n-Undecyl-β-Dthiomaltoside
n-Dodecyl-β-Dthiomaltoside
(1)
Sucrose8
Sucrose10
Sucrose12
• 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).
Example #1 - AqpZ
Example1 - AqpZ
OG
DDMAB
Big CHAP, deoxy
*
20µl load, 0.5ml/min, Superdex 200 5/150
20mM Tris pH 7.4, 500mM NaCl, 10% glycerol, 40mM OG
Example #2 – Human X
*
500µl load, 0.5ml/min, Superdex 200HR 10/30
20mM Tris pH 7.4, 500mM NaCl, 5mM FosCH12 or 2.6mM C12E9
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 Wiener
Michael Purdy
Peter Horanyi
David Shultis
Christian Banchs
Supported by NIH Roadmap Grant 5R01 GM075931 (MW)