Differential scanning fluorimetry (DSF) is a rapid and inexpensive screening method to identify low-molecular-weight ligands that bind and stabilize purified proteins. The temperature at which a protein unfolds is measured by an increase in the fluorescence of a dye with affinity for hydrophobic parts of the protein, which are exposed as the protein unfolds. A simple fitting procedure allows quick calculation of the transition midpoint; the difference in the temperature of this midpoint in the presence and absence of ligand is related to the binding affinity of the small molecule, which can be a low-molecular-weight compound, a peptide or a nucleic acid. DSF is best performed using a conventional real-time PCR instrument. Ligand solutions from a storage plate are added to a solution of protein and dye, distributed into the wells of the PCR plate and fluorescence intensity measured as the temperature is raised gradually. Results can be obtained in a single day.

The use of differential scanning fluorimetry to detect ligand interactions that promote protein stability

Noncovalent, extrinsic fluorescent dyes are applied in various fields of protein analysis, e.g. to characterize folding intermediates, measure surface hydrophobicity, and detect aggregation or fibrillation. The main underlying mechanisms, which explain the fluorescence properties of many extrinsic dyes, are solvent relaxation processes and (twisted) intramolecular charge transfer reactions, which are affected by the environment and by interactions of the dyes with proteins. In recent time, the use of extrinsic fluorescent dyes such as ANS, Bis-ANS, Nile Red, Thioflavin T and others has increased, because of their versatility, sensitivity and suitability for high-throughput screening. The intention of this review is to give an overview of available extrinsic dyes, explain their spectral properties, and show illustrative examples of their various applications in protein characterization.

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Extrinsic fluorescent dyes as tools for protein characterization.

Metalloproteins are proteins capable of binding one or more metal ions, which may be required for their biological function, or for regulation of their activities or for structural purposes. Genome sequencing projects have provided a huge number of protein primary sequences, but, even though several different elaborate analyses and annotations have been enabled by a rich and ever-increasing portfolio of bioinformatic tools, metal-binding properties remain difficult to predict as well as to investigate experimentally. Consequently, the present knowledge about metalloproteins is only partial. The present bioinformatic research proposes a strategy to answer the question of how many and which proteins encoded in the human genome may require zinc for their physiological function. This is achieved by a combination of approaches, which include:  (i) searching in the proteome for the zinc-binding patterns that, on their turn, are obtained from all available X-ray data; (ii) using libraries of metal-binding protei...

Counting the zinc-proteins encoded in the human genome.

Thermofluor-based high-throughput stability optimization of proteins for structural studies.

Thermal shift assays are used to study thermal stabilization of proteins upon ligand binding. Such assays have been used extensively on purified proteins in the drug discovery industry and in academia to detect interactions. Recently, we published a proof-of-principle study describing the implementation of thermal shift assays in a cellular format, which we call the cellular thermal shift assay (CETSA). The method allows studies of target engagement of drug candidates in a cellular context, herein exemplified with experimental data on the human kinases p38α and ERK1/2. The assay involves treatment of cells with a compound of interest, heating to denature and precipitate proteins, cell lysis, and the separation of cell debris and aggregates from the soluble protein fraction. Whereas unbound proteins denature and precipitate at elevated temperatures, ligand-bound proteins remain in solution. We describe two procedures for detecting the stabilized protein in the soluble fraction of the samples. One approach involves sample workup and detection using quantitative western blotting, whereas the second is performed directly in solution and relies on the induced proximity of two target-directed antibodies upon binding to soluble protein. The latter protocol has been optimized to allow an increased throughput, as potential applications require large numbers of samples. Both approaches can be completed in a day.

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The cellular thermal shift assay for evaluating drug target interactions in cells

A High Throughput Method for the Detection of Metalloproteins on a Microgram Scale

https://febs.onlinelibrary.wiley.com/doi/pdf/10.1016/j.febslet.2004.03.085

X-ray structure of tRNA pseudouridine synthase TruD reveals an inserted domain with a novel fold.

Pseudouridine, the 5-ribosyl isomer of uridine, is the most common modification of structural RNA. The recently identified pseudouridine synthase TruD belongs to a widespread class of pseudouridine synthases without significant sequence homology to previously known families. TruD from Escherichia coli was overexpressed, purified and crystallized. The crystals diffract to a minimum Bragg spacing of 2.4 A and belong to space group P212121, with unit-cell parameters a = 63.4, b = 108.6, c = 111.7 A.

Ulrika B. Ericsson

Papers

Thermofluor-based high-throughput stability optimization of proteins for structural studies.

A High Throughput Method for the Detection of Metalloproteins on a Microgram Scale

X-ray structure of tRNA pseudouridine synthase TruD reveals an inserted domain with a novel fold.

Expression, purification, crystallization and preliminary diffraction studies of the tRNA pseudouridine synthase TruD from Escherichia coli.