Differential scanning flourimetry for identification of protein ligands and stabilizing conditions at TMPC

Technology

Annotation & Function

Summary

Differential Scanning Fluorimetry (DSF) was used to demonstrate binding by the mitochondrial protein CPT1a of its physiological ligand carnitine, complex formation by trypsin and trypsin inhibitor, and metal ion binding by mitochondrial protein C20orf7.

Description

In Differential Scanning Fluorimetry (DSF, or Thermofluor®), effects of environment on a protein are monitored by observing shifts in protein denaturation temperature as reported by fluorescent dyes that interact with residues exposed by heat denaturation. DSF was first described, as Thermofluor®, by researchers at 3D-Pharmaceuticals in 2001 (1). The original workers and many other researchers in academia and industry have greatly extended the applications of this technique in the intervening years (2). In addition to screens for small organic and metal ligands, protocols for pH, buffer component, and ionic strength optimization have been described, ligand binding constants have been determined and shown to correlate well to those obtained by more traditional methods, and membrane proteins have been successfully studied. Here we use DSF to study proteins produced by two Protein Structure Initiative centers, the Mitochondrial Protein Partnership (MPP) and the Transmembrane Protein Center (TMPC), as well as control proteins.

Many proteins are expressed with large purification/solubility tags like Maltose Binding Protein (MBP). The data in Figure 1 demonstrates that such fusion proteins can be studied by DSF; both the target protein and the tag are detected and can be identified by stability shifts imposed by their specific ligands.

Protein:protein interactions may be detected by the formation of a more stable complex from less stable components. In Figure 2, increasing quantities of trypsin inhibitor (TI) are titrated into trypsin, resulting in the disappearance of free trypsin and the appearance of a very stable complex of the two proteins. The complex itself becomes increasingly stable with increasing TI concentration; Tms of 85.1 degrees Celsius, 85.5 degrees Celsius, 85.9 degrees Celsius, 87.0 degrees Celsius, and 87.8 degrees Celsius were observed for TI:trypsin ratios of 0.5:1, 1:1, 2:1, 4:1, and 6:1, respectively.

In Figure 3, the human mitochondrial methyltransferase C20orf7 was subjected to DSF in the presence of various metals. Many methyltransferases require or are stimulated by divalent cations, Mg2+ being perhaps the most common. We tested binding of C20orf7 to a range of metals as well as the chelator EDTA, finding that it binds Zn2+, not Mg2+ or any of the other metals tried. Zn2+ stabilized the protein by more than 15oC.

Authors: Aceti DJ, Moyer KL, Wells SB

Figure


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Figure 1. Response to ligands of tag and target components of a fusion protein. Purified fusion protein consisting of residues 166-756 of the mitochondrial protein CPT1a connected by a 68 residue linker to a His8/Maltose Binding Protein(MBP)affinity/solubility tags, was subjected to DSF with Sypro Orange dye. Shown is the first derivative of the melt curves, where peaks identify the inflection point (equivalent to the halfway point or “Tm”) of denaturation. Two distinct phases were apparent: (A) The left peak shifted to higher melting temperatures with increasing quantities of CPT1a substrate carnitine (Tms were 38.4 degrees Celsius, 38.8 degrees Celsius, 39.1 degrees Celsius, and 40.5 degrees Celsius); (B) The right peak shifted to higher melting temperatures with addition of MBP substrate maltose (Tm’s were 56.7 degrees Celsius, 57.6 degrees Celsius, 59.0 degrees Celsius, and 61.2 degrees Celsius).

Figure 2. Formation of a Trypsin/Trypsin Inhibitor complex. Bovine pancreatic trypsin and and increasing concentrations of bovine trypsin inhibitor (Sigma) were subjected to DSF with Sypro Orange dye. Shown are the melt curve (A) and the first derivative (B). Melting curve baselines in (A) were normalized.

Figure 3. Metal binding by the C20orf7 protein. Purified C20orf7 residues 63-345 (supplied by Amelia Still) were subjected to DSF with Sypro Orange in the presence 10-fold molar excess of metals or EDTA. Error bars indicate the standard deviation of three replicate experiments.

Publication

Pantoliano MW, Petrella EC, Kwasnoski JD, Lobanov VS, Myslik J, Graf E, Carver T, Asel E, Springer BA, Lane P, Salemme FR. High-density miniaturized thermal shift assays as a general strategy for drug discovery. J Biomol Screen, 2001 6(6): 429-40. PubMed ID:11788061

Vedadi M, Niesen FH, Allali-Hassani A, Fedorov OY, Finerty PJ Jr, Wasney GA, Yeung R, Arrowsmith C, Ball LJ, Berglund H, Hui R, Marsden BD, Nordlund P, Sundstrom M, Weigelt J, Edwards AM. Chemical screening methods to identify ligands that promote protein stability, protein crystallization, and structure determination. Proc Natl Acad Sci USA, 2006 103(43): 15835-40. PubMed ID:17035505 | PMC Link

Contact

Brian G. Fox (bgfox@biochem.wisc.edu)

Transmembrane Protein Center

Availability

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Last edited:Mon 10 Mar 2014 - 3 years, 3 months ago