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