PSI Structural Biology Knowledgebase

PSI | Structural Biology Knowledgebase
Header Icons
E-Collection

Related Articles
Families in Gene Neighborhoods
June 2015
Expanding the Reach of SAD
April 2015
Greasing the Path for SFX
January 2015
Time-Resolved Crystallography with HATRX
December 2014
Structures Without Damage
August 2014
Error Prevention
July 2014
A Refined Refinement Strategy
May 2014
Membrane Proteome: Microcrystals Yield Big Data
April 2014
Optimizing Damage
February 2014
Getting Better at Low Resolution
January 2014
Building a Structural Library
November 2013
Drug Discovery: Identifying Dynamic Networks by CONTACT
October 2013
Microbiome: Solid-State NMR, Crystallized
September 2013
Fluorescence- and Chromatography-Based Protein Thermostability Assay
October 2012
Insert Here
October 2012
Native phasing
August 2012
Smaller may be better
April 2012
Metal mates
February 2012
Not so cool
December 2011
One from many
August 2011
Rosetta hone
July 2011
Solutions in the solution
June 2011
Beyond crystals, solutions, and powders
May 2011
Snapshot crystallography
March 2011
FERM-ly bound
February 2011
A new amphiphile for crystallizing membrane proteins
January 2011
'Super-resolution' large complexes
December 2010
Proteinase K and Digalacturonic Acid
September 2010
Some crystals like it hot
May 2010
Tips for crystallizing membrane proteins in lipidic mesophases
February 2010
Tackling the phase problem
November 2009
Crystallizing glycoproteins
September 2009
Crystals from recalcitrant proteins
August 2009
Tips for crystallizing membrane proteins
June 2009
Chaperone-assisted crystallography
March 2009
An “X-ray” ruler
January 2009
Methylation boosts protein crystallization
December 2008

Technology Topics Crystallography

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

Structural Biology Knowledgebase ISSN: 1758-1338
Funded by a grant from the National Institute of General Medical Sciences of the National Institutes of Health