Monitoring Protein-Folding Processes with Environment-Sensitive Dyes—Note 9.1

1,8-ANS and bis-ANS (A47, B153; Other Nonpolar and Amphiphilic Probes—Section 13.5) have proven to be sensitive probes for partially folded intermediates in protein-folding pathways. These applications take advantage of the strong fluorescence enhancement exhibited by these amphiphilic dyes when their exposure to water is lowered (Figure 1, Figure 2). Consequently, fluorescence of ANS increases substantially when proteins to which it is bound undergo transitions from unfolded to fully or partially folded states that provide shielding from water. Molten globule intermediates are characterized by particularly high ANS fluorescence intensities due to the exposure of hydrophobic core regions that are inaccessible to the dye in the native structure. Binding of 1,8-ANS and bis-ANS to proteins is noncovalent and involves a combination of electrostatic and hydrophobic modes. Some investigators have noted that the dye-binding event itself may induce protein conformational changes, indicating the advisability of correlating ANS fluorescence measurements with data obtained using other physical techniques. In particular, high-resolution structural analysis of an ANS–protein complex by X-ray crystallography has demonstrated the occurrence of local rearrangements of the protein structure to accommodate the dye.

Figure 1. Fluorescence emission spectra of equal concentrations of 1,8-ANS (A47) in ethanol:water mixtures. The labels adjacent to each curve indicate the percentage of ethanol in the solvent mixture.

Figure 2. Fluorescence enhancement of 1,8-ANS (1-anilinonaphthalene-8-sulfonic acid, A47) upon binding to protein. The image shows aqueous solutions of 1,8-ANS excited by ultraviolet light. Addition of protein (bovine serum albumin) to the solution in the cuvette on the left results in intense blue fluorescence. In comparison, the fluorescence of uncomplexed free dye in the cuvette on the right is negligible.

The basic mechanism of protein-folding detection by ANS has been developed as the basis of fluorescence thermal shift (a.k.a. differential scanning fluorometry) assays for high-throughput analysis of protein stability. The assay readout is a profile of protein–dye complex fluorescence intensity as a function of temperature. Profiles are obtained and compared for multiple samples in which environmental or structural factors influencing protein stability are systematically varied. Thermocycler instruments designed for real-time PCR monitoring provide a readily adaptable instrument platform for these measurements. Most high-throughput fluorescence thermal shift assays use SYPRO Orange dye (S6650, S6651; Protein Detection on Gels, Blots and Arrays—Section 9.3) instead of ANS. Other environment-sensitive dyes with demonstrated utility include SYPRO Red dye (S6653, S6654; Protein Detection on Gels, Blots and Arrays—Section 9.3), Dapoxyl sulfonic acid (D12800, Other Nonpolar and Amphiphilic Probes—Section 13.5), nile red (N1142, Other Nonpolar and Amphiphilic Probes—Section 13.5) and CPM (D346, Thiol-Reactive Probes Excited with Ultraviolet Light—Section 2.3). Within the broad context of protein stability optimization, fluorescence thermal shift assays have many applications including:

• Analysis of ligand binding to proteins of unknown function
• Identification of protein–protein interaction inhibitors
• Analysis of protein stabilization by peptide aptamers and amino acid ligands
• Characterization of engineered protein variants
• Optimization of protein crystallization conditions
• Enhancement of recombinant protein quality and yield