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Detergent Screening

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					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

HPSEC
                        • Detergent Dilution
─ FosCH12
─ DM                      – A320nm/A280nm ratio low
                          – HPSEC looks good


                        • BUT do detergent
                          exchange on IMAC and
            IMAC          protein precipitates on
                          column
            ─ FosCH12
            ─ DM

                          – Led to AIPSA idea!
    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
                                 ANZERGENT®                                               n-Decyl-N,N- n-Undecyl-N,N,- n-Dodecyl-N,N-
                  solubilizing              ANZERGENT® n-Decyl-N,N- n-Dodecyl-N,N-                                                         C-
    water                            3-12                                               dimethyl-amine- dimethyl-amine- dimethyl-amine                              CYCLOFOS-4       CYCLOFOS-5
                   detergent                    3-14  dimethyl-glycine dimethyl-glycine                                                DODECAFOS
                                     (12)                                                 N-oxide (10)       oxide      -N-oxide (108)


                                                              FOS-                  FOS-CHOLINE® FOS-CHOLINE® FOS-CHOLINE® FOS-CHOLINE®
                                 FOS-CHOLINE® FOS-CHOLINE®             FOS-CHOLINE®                                                     FOS-CHOLINE®                                     DHPC
CYCLOFOS-6       CYCLOFOS-7                                CHOLINE®-12                   -14           -            -            -
                                      -10          -11                      -13                                                          -UNSAT-11-10                                     (6)
                                                               (32)                      (1)        ISO-11      ISO-11-6U      ISO-9


                                                                                                                                                                                      ANAPOE®-
                                                   CHAPS          CHAPSO                                              LAPAO
 LysoPC-10        LysoPC-12        FOSFEN-9                                         DDMAU            DDMAB                             TRIPAO        ANAPOE®-20     ANAPOE®-35          X-100
                                                     (1)            (15)                                                (2)
                                                                                                                                                                                         (2)


                                   ANAPOE®-
 ANAPOE®-         ANAPOE®-                     ANAPOE®-NID-                                          APO10
                                     X-405                          APO8             APO9                             APO11            APO12            C6E3            C6E4             C6E5
   X-114            X-305                          P40                                                 (1)
                                      (2)



                     C8E4            C8E5                           C10E5            C10E6         ANAPOE®-           C12E8          ANAPOE®-        ANAPOE®-        ANAPOE®-
    C7E5                                            C8E6                                                                                                                               Big CHAP
                      (52)            (1)                            (5)              (1)           C10E9              (19)          C12E9 (19)       C12E10          C13E8


                   Octyl-2-
                                                                                 n-Octyl-β-D-      n-Nonyl-β-D-
  Big CHAP,      hydroxyethyl- Rac-2,3-dihydroxy                 n-Heptyl-β-D-
                                    -propyl      Genapol X-100                    glucoside          glucoside      CYGLU®-3        ANAMEG®-7          Hega-9        C-Hega-10        C-Hega-11
    deoxy          sulfoxide     octylsulfoxide                thioglucoside (3)
                                                                                    (130)               (19)
                     (17)


                                                                                 2,6-Dimethyl-4-   n-Octyl-β-D-     n-Nonyl-β-D-                     n-Decyl-β-D-                    n-Undecyl-β-D-
  CYMAL®-3         CYMAL®-4        CYMAL®-5      CYMAL®-6                                                                            n-Decyl-α-D-                   n-Undecyl-α-D-
                                                                  CYMAL®-7         heptyl-β-D-      maltoside         maltoside                       maltoside                         maltoside
     (1)              (1)             (7)           (3)                                                                               maltoside                        maltoside
                                                                                    maltoside          (2)               (2)                             (56)                              (9)


                 n-Dodecyl-α-D- n-Dodecyl-β-D- n-Tridecyl-β-D-                    n-Nonyl-β-D-                                      n-Dodecyl-β-D-
ω-Undecylenyl-                                                    n-Octyl-β-D-                     n-Decyl-β-D-    n-Undecyl-β-D-
                    maltoside     maltoside       maltoside                       thiomaltoside                                      thiomaltoside    Sucrose8        Sucrose10        Sucrose12
β-D-maltoside                                                    thiomaltoside                     thiomaltoside    thiomaltoside
                       (1)           (80)            (1)                               (1)                                                (1)



                   • 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)

				
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