Optimization of protein samples for NMR using thermal shift assays

Area of Science:

• Biochemistry
• Structural Biology
• Biophysics

Background:

• Maintaining protein stability is crucial for biochemical and structural studies, particularly at higher temperatures and concentrations.
• Recombinant proteins often present folding challenges, necessitating optimization of buffer conditions for stability.

Purpose of the Study:

• To describe a thermal shift assay optimized for nuclear magnetic resonance (NMR) studies.
• To identify buffer components that enhance the stability of challenging recombinant proteins.

Main Methods:

• Utilized thermal shift assays (ThermoFluor®) for high-throughput screening of buffer components.
• Applied the assay to assess thermal stability of protein samples under various conditions simultaneously.
• Evaluated the impact of identified stabilizing ingredients on NMR spectral quality.

Main Results:

• The multicomponent screen successfully identified ingredients that significantly increased the thermal stability of two challenging proteins.
• Optimized conditions led to marked improvements in the quality of NMR spectra.
• Demonstrated the effectiveness of thermal shift assays in finding suitable conditions for NMR studies.

Conclusions:

• Thermal shift assays are an effective, economic, and time-efficient method for optimizing protein sample conditions for NMR studies.
• This approach enhances protein stability, leading to better data quality for structural investigations.
• The described method provides a valuable tool for researchers working with difficult-to-stabilize proteins.