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Detergent Screening Via Immobilized-Protein Stability Assay

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

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Detergent Screening Via Immobilized-Protein Stability Assay

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  1. Detergent Screening Via Immobilized-Protein Stability Assay James M. Vergis Laboratory of Michael Wiener

  2. 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

  3. Current Methods • A320nm/A280nm ratio • HPSEC profile • Ultracentrifugation/SDS-PAGE

  4. 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., Methods34, 364-372 (2004)

  5. 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., Structure14, 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)

  6. 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)

  7. 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

  8. 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

  9. False-Positive Example • 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 IMAC ─ FosCH12 ─ DM

  10. 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

  11. 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)

  12. Basis for Detergent AIPSA • Based on “old-school” detergent exchange method • Bind protein to column and wash extensively with new detergent • Incompatible detergents will cause protein to precipitate on resin while compatible detergents maintain the protein’s solubility

  13. 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)

  14. 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

  15. 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).

  16. 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

  17. 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

  18. Example #3 - KcsA

  19. 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

  20. 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

  21. Acknowledgements Michael Wiener Michael Purdy Peter Horanyi David Shultis Christian Banchs Supported by NIH Roadmap Grant 5R01 GM075931 (MW)

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