Thermal shift assay
DSF methodology includes techniques such as nanoDSF,[1][2] which relies on the intrinsic fluorescence from native tryptophan or tyrosine residues, and Thermofluor, which utilizes extrinsic fluorogenic dyes.
[3] The binding of low molecular weight ligands can increase the thermal stability of a protein, as described by Daniel Koshland (1958)[4] and Kaj Ulrik Linderstrøm-Lang and Schellman (1959).
nano-Differential scanning fluorimetry, or nanoDSF, is a biophysical characterization technique used for assessing the conformational stability of a biological sample, typically a protein.
[2] Samples are subjected to either temperature ramps or gradients of chemical denaturant, and the intrinsic fluorescence is measured and fit to determine the melting point (Tm).
Applications include formulation ranking, protein engineering (comparing mutants to wild type), and ligand binding (quantification of affinity constants).
Benefits include tag-free analysis, avoidance of extrinsic fluorophores, low sample consumption, easy of use, amenity to automation, and high screening throughput.
Drawbacks include a propensity for false positives and negatives, usually necessitating follow-up screening with a potentially lower-throughout orthogonal technique to confirm.
3 Dimensional Pharmaceuticals were the first to describe a high-throughput version using a plate reader[15] and Wyeth Research published a variation of the method with SYPRO Orange instead of 1,8-ANS.
[16] SYPRO Orange has an excitation/emission wavelength profile compatible with qPCR machines which are almost ubiquitous in institutions that perform molecular biology research.
When the protein unfolds, the exposed hydrophobic surfaces bind the dye, resulting in an increase in fluorescence by excluding water.
Knowing this effect can be very useful as a high relative fluorescence increase suggests a significant fraction of folded protein in the starting material.
Alexandrov et al. (2008)[20] published a variation on the Thermofluor assay where SYPRO Orange was replaced by N-[4-(7-diethylamino-4-methyl-3-coumarinyl)phenyl]maleimide (CPM), a compound that only fluoresces after reacting with a nucleophile.
The excitation and emission wavelengths for reacted CPM are 387 nm/ 463 nm so a fluorescence plate reader or a qPCR machine with specialized filters is required.
Alexandrov et al. used the technique successfully on the membrane proteins Apelin GPCR and FAAH as well as β-lactoglobin which fibrillates on heating rather than going to a molten globule.
The DSF-GTP technique was developed by a team led by Patrick Schaeffer at James Cook University and published in Moreau et al.
This technology is based on the principle that a change in the proximal environment of GFP, such as unfolding and aggregation of the protein of interest, is measurable through its effect on the fluorescence of the fluorophore.
The protein samples are simply mixed with the test conditions in a 96-well plate and subjected to a melt-curve protocol using a real-time thermal cycler.
Intrinsic fluorescence lifetime works with membrane proteins and detergent micelles but a powerful UV fluorophore (e.g. auto-fluorescent small molecule) in the buffer could drown out the signal.
The emission wavelengths of tryptophan residues are dependent on the surrounding chemical environment, notably solvation (see solvatochromism) and therefore differ between folded and unfolded protein, just as with the fluorescence lifetime.
To reduce the workload, western blots could be replaced by SDS-PAGE gel polyhistidine-tag staining, provided that the protein has such a tag and is expressed in adequate amounts.
Analogous to Thermofluor binding assays, a small volume of protein solution is heated up and the fluorescence increase is followed as function of temperature.
[43] Thermofluor pre-screens can be performed that sample a wide range of pH, ionic strength, and additives such as added metal ions and cofactors.
The generation of a protein response surface is useful for establishing optimal assay conditions and can frequently lead to improved purification scheme as required to support HTS campaigns and biophysical studies.
Thermofluor evaluation of conditions that stabilize proteins is consequently a useful strategy for finding optimal crystallization conditions[9][47][8] Since Thermofluor is a label-free assay that detects small molecule binding to high affinity binding sites on a target protein, it is well suited to finding small molecule inhibitors of protein-protein interactions or allosteric modulation sites.
[50][51] Recent developments have extended thermal shift approaches to the analysis of ligand interactions in complex mixtures, including intact cells.
Initial observations of individual proteins using fast parallel proteolysis (FastPP) showed that stabilization by ligand binding could impart resistance to proteolytic digestion with thermolysin.
In CETSA, aliquots of cell lysate are transiently heated to different temperatures, following which samples are centrifuged to separate soluble fractions from precipitated proteins.
Newer developments seek to merge aspects of FastPP and CETSA approaches, by assessing the ligand-dependent dependent proteolytic protection of targets in cells using mass spectroscopy (MS) to detect shifts in proteolysis patterns associated with protein stabilization.
[52] Present implementations still require a priori knowledge of expected targets to facilitate data analysis, but improvements in MS data collection strategies, together with the use of improved computational tools and database structures can potentially allow the approach to be used for de novo target decryption on the total cell proteome scale.
