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Thermal Shift Analysis - Case Studies - October 2010
XTAL’s Thermal Shift Analysis Services
Thermal shift analysis has a broad utility to address important issues in the quality and treatment of proteins, and as a biophysical technique for ligand binding studies.
- Quality control: each lot of protein produced at XTAL has a Tm as part of the characterization package.
- ‘Footprint’ Screen: evaluate the pH & ionic strength profile of a protein for protein purification, solubility, and stabilization.
- ‘Broadcast’ Screen: evaluate parameters important for assay design, protein stabilization, and crystallization.
- Ligand binding: confirm binding, effect of binding (stabilizing vs destabilizing), screening ligand library’s or fragment-based panels, titrations, etc.
Thermal Shift Analysis:XTAL Footprint ScreenProtein Stability Characterization Profiling
A 24 condition screen to evaluate protein solubility/stability as a function of pH and ionic strength. The screen is a rapid and efficient method to profile the thermal stability of purified proteins. This provides insight into quality issues, storage, solubility, and stability.
Thermal Shift Analysis:XTAL Broad-Cast ScreenProtein Stability Characterization Profiling
A 96 condition screen to evaluate parameters relevant to general stability and solubility profile of a protein.
The screen assists in addressing key biochemical/biophysical paramaters that are a paramount when developing/optimizing purification protocols, quality controls, and high throughput enzyme assays:
1.
Urea unfolding concentration
2.
Solubility/stability in ammonium sulfate
3.
Effect of DMSO
4.
pH profile
5.
Buffer interaction (at pH 7.5)
6.
Hofmeister ion series
7.
Zn, Cu, Mn, Co, Ni
8.
Glycerol
9.
Misc. additives
Thermal Shift Analysis:NADP+Cofactor Binding to Purified Enzyme
• Experiment confirmed NADP+binding to purified protein, providing a +4.5 °C stability shift.
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This provide a batch-to-batch quality control measurement.
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System established for subsequent inhibitor binding screening studies.
Thermal Shift AnalysisInhibitor Binding Studies -Conformation & Classification
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Protein only (black)
• Tm = 48.5 °C
Protein + Inhibitor 1
• Tm = 59.0 °C
•+10.5 °C stabilization
•Binds at cofactor site (confirmed by X-ray structure)
Protein + Inhibitor 2
• Tm = 42.0 °C
•-4.5 °C destabilization, some aggregation
•Suggested to bind in secondary site (substrate binding site?)
Protein + Inhibitor 1 + Inhibitor 2
• Tm = 51.5 and 58.5 °C
•Bimodal, confirmation of simultaneous binding to independent sites
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Assay system established for subsequent inhibitor binding screening studies to provide:
(i) secondary conformation of binding, (ii) classification of binding site, and (iii) stabilization/destabilization mode.
Thermal Shift AnalysisInhibitor Binding Studies –Binding Titration
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