Details

Autor(en) / Beteiligte

• Alexander, Crispin G.
• Wanner, Randy
• Johnson, Christopher M.
• Breitsprecher, Dennis
• Winter, Gerhard
• Duhr, Stefan
• Baaske, Philipp
• Ferguson, Neil

Titel

Novel microscale approaches for easy, rapid determination of protein stability in academic and commercial settings

Ist Teil von

• Biochimica et biophysica acta. Proteins and proteomics, 2014-12, Vol.1844 (12), p.2241-2250

Ort / Verlag

Elsevier B.V

Erscheinungsjahr

2014

Link zum Volltext

Quelle

Elsevier ScienceDirect Journals Complete

Beschreibungen/Notizen

• Chemical denaturant titrations can be used to accurately determine protein stability. However, data acquisition is typically labour intensive, has low throughput and is difficult to automate. These factors, combined with high protein consumption, have limited the adoption of chemical denaturant titrations in commercial settings. Thermal denaturation assays can be automated, sometimes with very high throughput. However, thermal denaturation assays are incompatible with proteins that aggregate at high temperatures and large extrapolation of stability parameters to physiological temperatures can introduce significant uncertainties. We used capillary-based instruments to measure chemical denaturant titrations by intrinsic fluorescence and microscale thermophoresis. This allowed higher throughput, consumed several hundred-fold less protein than conventional, cuvette-based methods yet maintained the high quality of the conventional approaches. We also established efficient strategies for automated, direct determination of protein stability at a range of temperatures via chemical denaturation, which has utility for characterising stability for proteins that are difficult to purify in high yield. This approach may also have merit for proteins that irreversibly denature or aggregate in classical thermal denaturation assays. We also developed procedures for affinity ranking of protein–ligand interactions from ligand-induced changes in chemical denaturation data, and proved the principle for this by correctly ranking the affinity of previously unreported peptide–PDZ domain interactions. The increased throughput, automation and low protein consumption of protein stability determinations afforded by using capillary-based methods to measure denaturant titrations, can help to revolutionise protein research. We believe that the strategies reported are likely to find wide applications in academia, biotherapeutic formulation and drug discovery programmes. [Display omitted] •Chemical denaturant titrations are slow, lengthy and consume lots of protein sample.•We developed very fast methods for measuring chemical denaturant titrations.•Automated titrations can be measured in minutes using only μl sample volumes.•Ligand interactions rapidly screened and ranked via changes in protein stability•These advances make chemical denaturation more suitable for commercial research.