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Fluorescence-Based Analytical Crystallization Technologies: A Paradigm Shift

Learn about the FACTs approach for fast protein crystallization screening, improving success rates. Discover the benefits of a quantitative method with fluorescence anisotropy measurements. Contact info@mi-research.com for more.

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Fluorescence-Based Analytical Crystallization Technologies: A Paradigm Shift

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  1. QUANTITATIVE SCREENING FOR CRYSTALLIZATION CONDITIONS FACTs Fluorescence-based Analytical Crystallization Technologies A paradigm shift in how macromolecule crystallization conditions are determined. Marc L. Pusey and Takahisa Minamitani MI Research, Inc., Suite 109, 515 Sparkman Drive, Huntsville, AL, 35816

  2. MI Research, Inc. September 2006 – MI Research, Inc. started by TM & ELM. Goals – development of biomedical instrumentation. October 2006 – MLP quits day job and joins company. Task – develop the FACTs (Fluorescence-based Analytical Crystallization Technologies*) for crystallization screening. March 2007 – Proof of concept instrument up and running Data collection from assay volumes of 25-30 µL. July 2007 – Improved ‘Phase 0’ instrument Assay volumes of 2.5 – 3.5 µL. (1.73 mg / 96 conditions) Started collecting and testing data on model proteins. (this talk). September 2007 – Additional improvements to control software Data collection time reduced to ~3-4 hrs. Now open for business… * patent pending

  3. Problems with the Crystallization Screening Process (my perspective) • Current methods are highly ‘digital’ – yes (crystal) / no (no crystal). Days to months to obtain results. • Data interpretation (visual image analysis) is highly subjective. machine-based methods even less subtle led to idea of trace fluorescent labeling (intensity an easier search parameter than straight lines). • Many clear or precipitated solution outcomes may be near misses, which are not evident upon visual analysis. AN IMPROVED SCREENING APPROACH • Not dependent upon appearance of crystals • Will provide feedback & let you know you are close – near misses. • Will be amenable to automation (data collection and analysis). • Will rapidly (< 2 days, purified protein to data) give screen results. info@mi-research.com

  4. Goal of the FACTs Approach A means of rapidly acquiring data about protein behavior in the presence of precipitant solutions. To use the FACTs data to find where this can become this B6 Optimized results after a single 2D screen. Scale bars =200 µm. Screen outcome

  5. Crystallization Variable B22 attractive 0 “Crystallization Slot” defined by B22. Expanded slot How Far? Basis for Our Approach Builds upon B22 work of Wilson and others, which shows that there is a range of attractive interactions which favors (but does not necessarily guarantee) protein crystallization. We propose that a quantitative approach to crystallization screening would result in more successes by indicating the proximity to likely crystallization conditions. info@mi-research.com

  6. Add together protein Precipitant A NEW APPROACH TO PROTEIN CRYSTALLIZATION Current Practice Observe Harvest and Proceed Incubate Days Weeks Months YES! Crystal? NO minimum feedback!! Optimize Measure Protein Response to Solution Conditions Our Approach Data we can learn from Add together Pick likely Crystallization Conditions. Time to this point < 2 DAYS! diluted protein with fluorescent label Precipitant Plot and Analyze Data – the quantitative component Advantages – Speed and Increased Success Rate

  7. IVV – IVH r0 τ r = = 1 + IVV + 2*IVH r θ ηV θ = RT F F F F F F F F Fluorescence Anisotropy Ex F V H Em SOLUBLE When exciting with vertically polarized light, if the rotation time of the fluorophore is ≥ than the fluorescent lifetime, τ, then the vertical and horizontal emission will be ~ equal and r ≈ 0 PRECIPITATION Aggregated protein mass rapidly builds at low concentrations, rapidly slowing down the rotational rate. Anisotropy  r0 at low concentrations. r = measured anisotropy r0 = fundamental anisotropy a property of the fluorophore τ = probe lifetime θ = rotational correlation time η = viscosity R = gas constant T = temperature V = volume of rotator Note dependencies!! These will affect data collection. CRYSTALLIZATION Slow self association as a function of protein concentration, as with crystallization, leads to a progressive increase in anisotropy with concentration. info@mi-research.com

  8. Anticipated Results What we expect to see in the data and how it will be interpreted •  Clear Solution outcomes – will have anisotropy vs. protein • concentration curves that are flat (or with a slightly • negative slope). • Precipitated Solution outcomes – will have high (and/or level) anisotropy values in anisotropy vs. concentration curves. • Crystallization conditions – will have curves showing a progressive increase in anisotropy with increasing protein concentration. info@mi-research.com

  9. Clear Solution Outcomes - Xylanase - Clear solution conditions typically have a flat or slightly negative slope in a plot of concentration vs. anisotropy. info@mi-research.com

  10. Precipitated Solution Outcomes - Xylanase - More variable, but generally higher anisotropy values and/or no positive slope in the anisotropy vs. concentration data. info@mi-research.com

  11. Crystallization Conditions Glucose Isomerase Initial testing is being carried out using model proteins, for which we know the crystallization outcomes for the solutions being tested and which are available in high purity and relatively large amounts. These tests are also used to develop the methodology for making the measurements. info@mi-research.com

  12. Glucose Isomerase - continued In a ‘standard’ crytallization screen, a number of wells resulted in clear solutions. Some of these gave anisotropy data suggesting that they could be crystallization conditions. These gave crystals with higher protein concentration A total of 8 conditions were tested, with protein concentrations of 50, 100, and 150 mg/mL, of which 6 produced crystals. info@mi-research.com

  13. UNCUT CANAVALIN Starting with this protein we decided to aggressively pursue all leads found using anisotropy measurements. “Standard” crystallization conditions are 0.1-0.2 M MgCl2, 15-20% MPD, pH 6.5 to 8.0 (cacodylate, bis-tris, hepes, tris). P212121 a=80.32, b=85.2, c=211.93 Screen results, condition B6 Divalent cations facilitate UCAN crystallization 1:1 2:1 4:1 Anisotropy data 0.2 M MgAcetate, 20% PEG 8K, .1 M NaCacodylate, pH 6.5 5% PEG 8K 0.05 M MgAc 0.1 M NaCac pH 6.5 scale bar = 500 µm. info@mi-research.com

  14. 1:1 2:1 4:1 UNCUT CANAVALIN In some cases it only took a simple 2D screen to convert precipitation conditions to crystallization conditions. HSHT Condition H3 0.2 M MgCl2 0.1 M Tris, pH 8.5 3.4M 1,6 Hexanediol MgAc .05 .2 .85 hex-diol 3.4 0.05 M Mg Acetate 2.55 M Hex diol 0.1 M Hepes, pH 7.5 Scale bar = 200 µm info@mi-research.com

  15. UNCUT CANAVALIN For other conditions it took several screens to convert precipitation conditions to crystallization conditions. HSHT condition C4 0.2 M Na Acetate 0.1 M Na Cacodylate, pH 6.5 30% PEG 8K 1:1 2:1 4:1 Second screen – all wells made 0.1 M Mg Acetate Na Acetate .05 .2 5 PEG 8K 30 0.05 M NaAcetate 13.3% PEG 8K 0.1 M NaCac, pH 6.5 0.1 M MgAc Scale bar = 100 µm info@mi-research.com

  16. UNCUT CANAVALIN A number of anisotropy-derived leads had citrate as a component. Condition A9 0.2 M Ammonium acetate 30% PEG 4K 0.1 M Na Citrate, pH 5.6 Screen outcomes 1:1 2:1 4:1 Replace the Citrate & added Mg2+ PEG 4K Am Acetate 15% PEG 4K 0.2 M Ammonium Ac 0.1 M NaCacodylate, pH 6.5 0.1 M Mg Acetate Scale bar = 100 µm 10 % PEG 4K 0.2 M Ammonium Ac. 0.1 M NaCitrate, pH 5.6 Scale bar = 500 µm info@mi-research.com

  17. 1:1 2:1 4:1 BmimCl 0.1 0.4 2 PEG 4K 8 UNCUT CANAVALIN Some anisotropy leads were at pH values < UCAN pI (~5.2) HSHT condition D1 0.1 M Na Acetate, pH 4.6 8% PEG 4K 0.1 M BmimCl1 6% PEG 4K 0.1M Na Acetate, pH 6.5 0.1 M Mg Acetate scale bar = 200 µm info@mi-research.com 1Pusey et al., Crystal Growth & Design (2007) 7;787-792

  18. PEG MME2K 30 15 0.05 AmmSulf 0.2 UNCUT CANAVALIN Crystals at pH < UCAN pI, continued HSHT screen condition F1 – screen outcome was a clear solution. Increasing the protein concentration (1x40 mg/mL, 2x40 mg/mL) gave precipitate. 1st screen around pcpt. conditions, w/BmimCl1 to reduce pcptn, no xtls. 2nd screen, replacing BmimCl w/ 0.1 M MgAc  crystals. (protein @ 40 mg/mL) HSHT condition F1 0.2 M Ammonium Sulfate 0.1 M Na Acetate, pH 4.6 30 % PEG MME 2K 15 % PEG MME2K 0.15, 0.2 M AmSulf 0.1 M NaAc, pH 4.6 0.1 M MgAcetate scale bar = 200 µm info@mi-research.com 1Pusey et al., Crystal Growth & Design (2007) 7;787-792

  19. UCAN vs. CCAN Hits CCAN UCAN p = by crystallization plate A = by anisotropy assay A B C D E F G H A A p A A p A p A p p p p p p A p A A A A p A A p A A A p A A A A A A A A p p A A p A p A A A A A A p p A p A A 1 2 3 4 5 6 7 8 9 10 11 12 info@mi-research.com

  20. Data Summary for Model* Proteins New leads found by FACTs # of FACTs Leads tested % Increase over plate Crystals obtained % Success 1 = clear solution 2-5 = precipitate 1 / 2-5 1 / 2-5 1 / 2-5 1 / 2-5 Plate score Protein (plate xtls 1:1 only) GI (33 20) 11 / 10 8 / 6 6 / 3 75 / 50 27.3 XLN (12 3) 01 / 28 01 / 24 01 / 17 0 / 70.8 141.67 CCAN (15 8) 27 / 12 27 / 8 8 / 7 29.6 / 87.5 100 UCAN (6 2) 22 / 18 22 / 14 10 / 11 45.5 / 78.6 350 Overall (66 33) 60 / 68 57 / 52 24 / 38 42.1 / 73.1 93.9 Values based on outcomes at 1:1, 2:1, & 4:1 conditions 1 All clear solution FACTs leads @ 1:1 had xtls at 2:1 &/or 4:1 * model = any protein for which we have plate screening data.

  21. Current Requirements Protein ~2 mg protein required per 96 condition screen (using 2.5 µL assay solution volumes). Goals – reduce this to ~0.8 mg (1.0 µL assay volume) within 6 months and ~8 µg (10 nL assay volume) within 2 years.  4 mg protein for screen + 5-10 mg for subsequent optimization. Time To prepare derivatized protein ~ 2 hrs. To set up Assay ~1 hr. To carry out 96 condition screen ~3-4 hrs. Less than 1 day, protein to screen results. NOTE: Labeled protein is only used for the assay, not subsequent optimizations. info@mi-research.com

  22. FUTURE GOALS • Assay volume reduction – to 1.0 µL solution/assay – manual solution dispensing methods - within 6 months (691 µg protein/96 condition screen), to ≤ 10 nL – robotic solution dispensing methods - within 2 years. • Faster measurements – reduce the time to collect a 96 condition screen data set to < 1 - 2 hrs. • Temperature control – temperature sensitive crystallizations, membrane proteins. • Automated data analysis – a rational (vs. artsy) approach to protein crystallization. • Rational screen design – Incomplete factorial approach with two (or more) levels of screening.

  23. SUMMARY What this approach WILL NOT do  Anisotropy leads are not automatically crystallization conditions. However – Knowing where to look - analysis of the data - may indicate those factors that are most important to obtaining crystals.  No indication or guarantee of crystal quality. However – An expanded set of crystallization conditions may help in finding better conditions.  Not a method for getting crystals from bad protein. Bad protein may give good looking data, but see above.

  24. SUMMARY • Faster – The fluorescence anisotropy screen approach can be used to carry out the initial crystallization screen in ~8 hrs. • More Successes – The anisotropy screen finds more lead conditions – outcomes that would be clear or precipitated solutions in ‘normal’ crystallization screens. • Quantitative – Anisotropy screen data is highly amenable to machine analysis.

  25. finis Contact Information: MI Research, Inc. Suite 109 515 Sparkman Drive Huntsville, AL 35816 info@mi-research.com

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