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Fluorescence- and Chromatography-Based Protein Thermostability Assay

SBKB [doi:10.1038/sbkb.2012.104]
Technical Highlight - October 2012
Short description: A new thermostability assay for medium- to high-throughput screening helps identify membrane protein crystallization conditions.

Workflow for FESC-TS assay. Reprinted with permission from Elsevier. 1

Structure determination by X-ray crystallography requires the growth of well-ordered, diffracting crystals. The identification of optimal isolation and crystallization conditions is often a long and laborious process, particularly in the case of membrane proteins. This typically involves testing the effects of salts and organic precipitants, and also of various additives and ligands that can affect protein stability. To aid in this process, Gouaux and colleagues have developed a new fluorescence-detection size-exclusion chromatography-based thermostability assay (FSEC-TS) that builds on the sensitivity of fluorescence detection and the ease of SEC.

The process begins with a small-scale heat treatment of an EGFP-fusion in a typical thermal cycler, followed by the injection of the material into a SEC column equipped with an in-line fluorescence detector. Monodisperse proteins will produce symmetric, single-Gaussian peaks during elution, while heterogeneous samples require multiple Gaussian curves to obtain a good fit to the peak data. By quantifying the fluorescence obtained at different denaturation temperatures, classical melting curves can also be constructed and used to evaluate the stabilizing effects of chemical additives. The authors employed this assay to the precrystallization screening of two membrane proteins, the P2X receptor and the glutamate-gated chloride channel in the presence of ligands, divalent cations and lipids. The FSEC-TS-derived melting temperatures were in agreement with the values derived from classical radioligand binding assays, and the addition of the identified stabilizing agents aided crystallization.

FSEC-TS can be performed using purified protein in nanogram-to-microgram amounts, but also with unpurified material from whole-cell extracts, greatly simplifying the process and making it particularly suitable for medium- to high-throughput precrystallization screening. Unlike other high-throughput membrane protein thermal stability assays employing thiol-reactive fluorophores, FSEC-TS does not require free cysteines embedded in the protein core. Moreover, while the presence of an EGFP tag confers distinct advantages in a whole-lysate format (notably a wide spectral separation between GFP fluorescence and intrinsic protein fluorescence), FSEC-TS is also applicable to purified proteins lacking a fluorescent tag. In this case, the readout is the intrinsic tryptophan fluorescence rather than the EGFP signal.

Alexandra M. Deaconescu

References

  1. M. Hattori et al. A fluorescence-detection size-exclusion chromatography-based thermostability assay for membrane protein precrystallization screening.
    Structure. 20, 1293-1299 (2012). doi:10.1016/j.str.2012.06.009

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