Optimization of protein samples for NMR using thermal shift assays

Abstract

Maintaining a stable fold for recombinant proteins is challenging, especially when working with highly purified and concentrated samples at temperatures >20 °C. Therefore, it is worthwhile to screen for different buffer components that can stabilize protein samples. Thermal shift assays or ThermoFluor® provide a high-throughput screening method to assess the thermal stability of a sample under several conditions simultaneously. Here, we describe a thermal shift assay that is designed to optimize conditions for nuclear magnetic resonance studies, which typically require stable samples at high concentration and ambient (or higher) temperature. We demonstrate that for two challenging proteins, the multicomponent screen helped to identify ingredients that increased protein stability, leading to clear improvements in the quality of the spectra. Thermal shift assays provide an economic and time-efficient method to find optimal conditions for NMR structural studies. Electronic supplementary materialThe online version of this article (doi:10.1007/s10858-016-0027-z) contains supplementary material, which is available to authorized users.

Structural characterization of the Asf1–Rtt109 interaction and its role in histone acetylation

“…Rtt109 is prone to aggregation and precipitation in commonly used buffers. In previous work, we used a thermal-shift assay to optimize Rtt109 stability and found optimal conditions in a buffer containing citric acid at low pH (5.5–6.5) ( 31 ). Using this buffer, we titrated 15 N-labeled Asf1 with increasing amounts of unlabeled full-length Rtt109 and observed significant changes in the Asf1 15 N-HSQC spectrum ( 33–35 ) (Figure 1A and Supplementary Figure S2A ).…”

“…Codon-optimized full-length Saccharomyces cerevisiae Rtt109 was cloned in a pETM-11 vector containing a N-terminal hexa-histidine tag, followed by a TEV protease cleavage site. Rtt109 was expressed in E. coli BL21(DE3) and purified as previously described ( 31 ). Xenopus laevis histones were expressed and purified as reported by Luger et al.…”

Measuring Interactions of Globular and Filamentous Proteins by Nuclear Magnetic Resonance Spectroscopy (NMR) and Microscale Thermophoresis (MST)

“…It also serves as an opportunity to change conditions such as salt concentration or pH, which may impact the efficacy of certain downstream techniques or assays. Protein thermal shift (PTS) can be used to identify optimal buffers for downstream assays, especially for those requiring stable protein for prolonged periods of time at room temperature 8,9 .…”

How to Stabilize Protein: Stability Screens for Thermal Shift Assays and Nano Differential Scanning Fluorimetry in the Virus-X Project

“…Despite the development in the technology available to crystallographers, protein crystallization remains a time-consuming and labor-intensive empirical process 5 . Preliminary biophysical experiments to improve the protein stability in solution give clearly a better starting point for protein crystallization and consume usually only a comparatively small amount of protein sample 6,7,8,9 . The large number of target proteins to be studied in this project also necessitates scalable, high-throughput stability assays.…”