Protein Stability Analysis Using the Optim 1000 Patrick Celie NKI Protein Facility, Amsterdam
NKI Protein Facility Fundamental and translation cancer research ~ 650 scientists + supporting personnel Connected to Antoni van Leeuwenhoek Hospital Provides infrastructure for protein research Personnel: Coordinator PostDoc (Biophysics) 2x Technician Facility shares equipment/lab with Structural Biology groups National Facility: Open to both NKI- and external users
Facility Services Construct Design (NKI LIC vectors) Protein expression and purification - E.coli - Insect cells (baculovirus) - HEK293 and derivatives Biophysical characterisation - Interaction: SPR (Biacore), ITC, Fluorescence Polarisation - Thermal shift assay - MALLS - Mass spec HTP crystallisation in 96-well format at 4 and 20 Provision of common reagents (Enzymes, Vectors, Antibodies)
Protein Quality Control Depending on the application, different criteria are used to assess protein quality % of Purified proteins Purity: SDS-PAGE Coomassie staining 100% Homogeneity: Size exclusion chromatography 80% Oligomeric state: SEC/MALLS 20% Protein integrity: Mass spec 5% Protein stability: Thermal Shift Assay 5% Tag (GST, Trigger Factor) removal 10%
Protein Thermal Stability Thermal Shift Assay : Monitor thermal unfolding of protein Advantages - Small amount of protein (2 5 µg / measurement) - Applicable in HTP fashion - relatively cheap Disadvantages -Does not work for all proteins - Sometimes difficult to interpret results (noise, multiple phases/transitions)
Optim 1000 Optim 1000 Simultaneous measurement of: Intrinsic protein fluorescence Static light scattering Extrinsic fluorescence from a range of probe dyes. Dimensions W x D x H: 85 cm x 69 cm x 76 cm 75 kg Two laser sources 266 nm laser to excite intrinsic protein fluorescence and for sensitive light scattering. 473 nm laser for light scattering and excitation of some dyes.
Preparing an Experiment Samples (9 µl) are loaded into a micro-cuvette array (MCA) One MCA contains 16 slots Optim 1000 contains space for 3 MCA s (48 samples) Proteins don t not have to be labeled Protein concentration: 0.1 2 mg/ml (preferably test more concentrations)
Measurement Results Protein 0.5 mg/ml Scattering 266 nm, small aggregates Fluorescence Trp, tyr, phe intrinsic fluorescence Scattering 473 nm, large aggregates
Fluorescence Data (1) Upon protein unfolding Intrinsic (tryptophan) fluorescence peak shifts from ~330 to ~340/350) nm. Tyr and Phe may contribute to a small extent Typically, fluorescent signal (300 400 nm) decreases with increasing temperature due to increased quenching (by water molecules) (IgG s show the reverse..) Protein 0.5 mg/ml
Fluorescence Data (2) Protein 0.5 mg/ml Transition Total Fluorescence intensity (SIl ;280 450 nm) vs temperature peak intensity (between 280 and 450 nm) vs temperature Similar trend: Decrease in fluorescent signal
Examples of Fluorescense data Unfolding Rapid aggregation Unfolding Aggregation No change in fluorescence aggregation
Analysis Fluorescence measurements Protein 0.5 mg/ml Plot changes in Fluorescense detection 1) Plot ratio of Fluorescence Intensity at 330 and 350 nm vs temperature 2) Plot peak height (nm) vs temperature 3) Calculate Barycentric mean fluorescence (280 450 nm): Preferred method: Less susceptible to noise Uses information of whole spectrum
Analysis Fluorescence measurements (2) Protein 0.5 mg/ml Tm Calculate 1 st derivative to get Tm value
Scattering Measurement Protein 0.5 mg/ml Onset of aggregation: ~50 C Small aggregates (scatter at 266 nm) form slightly earlier then larger aggregates And coincides with unfolding: Tm
Similar Onset Aggregation and Unfolding At two different protein concentrations, aggregation and unfolding occurs simultaneously
Comparison of TSA and Optim 1000 (1) TSA: Nice signal Optim: Nice signal TSA: Nice Optim: Formation of aggregates Unfolding and aggregation occur simultaneously and correspons to TSA
Comparison of TSA and Optim 1000 (2) TSA: Nice signal Optim: No obvious Tm from fluorescense TSA: Nice signal Optim: Formation of aggregates Conclusion: Aggregation is detected without clear Tm for unfolding
Comparison of TSA and Optim 1000 (3) TSA: No signal (hydrophobic protein) Optim: Nice signal Tm from fluorescense TSA: No signal (hydrophobic protein) Optim: Formation of aggregates
Aggregation is Concentration dependent 0.2 mg/ml Tm = 46.8 C 0.5 mg/ml Tm = 47.0 C 1.0 mg/ml Tm = 51.0 C Aggregation is protein concentration-dependent At high protein concentration aggregation occurs before unfolding
Stability of different Protein-Peptide complexes Tm = 46.5 Tm 54 C TSA Tm = 39.5 32 C Tm = 30.5
Fluorescense Optim vs CD Tm 70.8 C 37.0 C 67.8 C A-A CD Tm ~68 C ~30 C ~62 C A-B Thermal unfolding measured by CD and Optim shows comparable results
Conclusions Positive Both aggregation (scattering) and unfolding (intrinsic fluorescence) are measured No labeling or fluorescent probes are required Sypro orange can be used as in TSA (not tested by us) MCA s can be washed and re-used (not recommended by Avacta..) May work in occassions where TSA does not give proper data (and vice versa..) Relatively easy to set up (for medium throughput) About 1 to 20 µg protein per sample Negative Machine is expensive (~100k ) MCA s are expensive (~96 euro for 16 slots; 6 Euro per sample) It is big (newer version Optim 2 is much smaller) Is the scientific result worth the investment (time + money)? Does sample quality /stability correlates with crystallisability?
Acknowledgements Alex Fish! Structural Biology labs of Tassos Perrakis and Titia Sixma