protein stability analysis using the optim 1000 · optim 1000 optim 1000 simultaneous measurement...
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Protein Stability Analysis Using the Optim 1000
Patrick CelieNKI Protein Facility, Amsterdam
• 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
• Fundamental and translation cancer research
• ~ 650 scientists + supporting personnel
• Connected to Antoni van Leeuwenhoek Hospital
NKI Protein Facility
• 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)
Facility Services
Protein Quality Control
Depending on the application, different criteria are used to assess protein quality
% of Purified proteinsPurity: 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 1000Simultaneous measurement of:
• Intrinsic protein fluorescence • Static light scattering• Extrinsic fluorescence from a range of probe dyes.
Two laser sources266 nm laser to excite intrinsic protein fluorescence and for sensitive light scattering.473 nm laser for light scattering and excitation of some dyes.
Dimensions W x D x H: 85 cm x 69 cm x 76 cm75 kg
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
Scattering 266 nm, small aggregates
Protein 0.5 mg/ml
Scattering473 nm, large aggregates
FluorescenceTrp, tyr, phe intrinsic fluorescence
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
Total Fluorescence intensity (SIl ;280 – 450 nm) vs temperaturepeak intensity (between 280 and 450 nm) vs temperature
Similar trend: Decrease in fluorescent signal
Transition
Examples of Fluorescense data
UnfoldingAggregation
Unfolding Rapid aggregation
No change in fluorescenceaggregation
Analysis Fluorescence measurements
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):
Plot changes in Fluorescense detection
Preferred method: Less susceptible to noiseUses information of whole spectrum
Protein 0.5 mg/ml
Analysis Fluorescence measurements (2)
Protein 0.5 mg/ml
Tm
Calculate 1st derivative to get Tm value
Scattering Measurement
Onset of aggregation: ~50 °CSmall aggregates (scatter at 266 nm) form slightly earlier then larger aggregates
And coincides with unfolding:Tm
Protein 0.5 mg/ml
Similar Onset Aggregation and Unfolding
At two different protein concentrations, aggregation and unfolding occurs simultaneously
Comparison of TSA and Optim 1000 (1)
TSA: Nice signalOptim: Nice signal
TSA: NiceOptim: Formation of aggregates
Unfolding and aggregation occur simultaneously and correspons to TSA
Comparison of TSA and Optim 1000 (2)
TSA: Nice signalOptim: No obvious Tm from fluorescense
TSA: Nice signalOptim: 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
Aggregation is protein concentration-dependentAt ‘high’ protein concentration aggregation occurs before unfolding
0.2 mg/mlTm = 46.8 °C
0.5 mg/mlTm = 47.0 °C
1.0 mg/mlTm = 51.0 °C
Stability of different Protein-Peptide complexes
Tm = 46.5
Tm = 39.5
Tm = 30.5
Tm 54 °C
32 °C
TSA
Optim vs CD
Fluorescense
CD
A-A
A-B
Tm70.8 °C37.0 °C67.8 °C
Tm~68 °C~30 °C~62 °C
Thermal unfolding measured by CDand 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