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J Biomol NMR. 2016 Apr;64(4):281-9. doi: 10.1007/s10858-016-0027-z. Epub 2016 Mar 17.

Optimization of protein samples for NMR using thermal shift assays.

Author information

1
SPC Facility, European Molecular Biology Laboratory (EMBL), Hamburg Outstation, Notkestrasse 85, 22607, Hamburg, Germany.
2
SCB Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117, Heidelberg, Germany.
3
BMWZ and Institute of Organic Chemistry, Leibniz University Hannover, Schneiderberg 38, 30167, Hannover, Germany.
4
SCB Unit, European Molecular Biology Laboratory (EMBL), Meyerhofstrasse 1, 69117, Heidelberg, Germany. teresa.carlomagno@oci.uni-hannover.de.
5
BMWZ and Institute of Organic Chemistry, Leibniz University Hannover, Schneiderberg 38, 30167, Hannover, Germany. teresa.carlomagno@oci.uni-hannover.de.
6
Research Group of NMR-based Structural Chemistry, Helmholtz Centre for Infection Research, Inhoffenstrasse 7, 38124, Brunswick, Germany. teresa.carlomagno@oci.uni-hannover.de.
7
SPC Facility, European Molecular Biology Laboratory (EMBL), Hamburg Outstation, Notkestrasse 85, 22607, Hamburg, Germany. sboivin77@hotmail.com.

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.

KEYWORDS:

Differential scanning fluorimetry; Nuclear magnetic resonance; Protein thermal stability; Sample optimization; Thermal shift assay; ThermoFluor

PMID:
26984476
PMCID:
PMC4869703
DOI:
10.1007/s10858-016-0027-z
[Indexed for MEDLINE]
Free PMC Article

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