To demonstrate this tool, we developed a prion detection assay. We used the yeast prion Sup35 as a model protein and incorporate two non‑canonical amino acids (p-acetophenylalanine and propargyllysine). After purification, the recombinant produced Sup35 could be labeled with two different fluorophores (Cyanin 3 and Cyanin 5). The emission spectra of the fluorophores depend on their distance. When this test protein gets in contact with prions, the prions conformational changes result in the change of the fluorophores spectra. Therefore, the test prion could be used to detect prions in medical samples.
Structural Analysis with Non-canonical Amino Acids
The first step is the incorporation of the non-canonical amino acids. In proteins which contain no cysteines naturally (cysteines are the only canonical amino acids, that could be labeled specific) or in which the exchanges of cysteines does not influence the structure, only one ncAA and one cysteine at specific points need to be incorporated to be labeled. In proteins that contain cysteines, two ncAAs need to be incorporated for the labeling (Kim et al., 2013).
Non-canonical amino acids could be incorporated by orthogonal tRNA/aaRS pairs using the amber stop codon. However, this allows only the incorporation of one non-canonical amino acid. To incorporate the second amino acid, another orthogonal amino acid has to be used for the incorporation. Another codon that could be repurposed is the rarely used leucine codon CUA. With the use of this and the amber codon, two different ncAAs could be used. For structural analysis, the amino acids are specific labeled with chromophores. This labeling (shown in figure 1) is possible due to the functional groups of the amino acids, which could form a covalent bond to the fluorophores in a chemical reaction. After the protein is labeled, the fluorescence of the chromophores could be measured to draw conclusions on the distance of the ncAAs from each other (Brustad et al., 2008, Kim et al., 2013).
Figure 1: Target protein labeled with fluorophores.
The ncAAs AcF and PrK are incorporated in the target protein. After bi-orthgonal chemical conjugation the ncAAs are coupled with the fluorescent dyes cyanin 3 (Cy3) and cyanin 5 (Cy5).
- Name: Propargyllysine
- Short: PrK
- CAS: 1428330-91-9
- MW: 228.25 g mol-1
- Storage: 4 °C
- Source: Sichem
- Prize: 1g - £300.00
- Function: Propargyl group for click-chemistry reaction
Figure 2: Structure of PrK
- Name: p‑Acetylphenylalanine
- Short: AcF
- CAS: 122555-04-8
- MW: 207,23 g mol-1
- Storage: -20 °C
- Source: abcr
- Prize: 1g - £509.00
- Function: Ketone group for hydrazide reaction
Figure 3: Structure of AcF
Foerster Resonance Energy Transfer (FRET)
Figure 4: Animations of a FRET fluorophore pair
Animation of the distance dependent energy transfer of two fluorophores.
r intermolecular distance
R0 Foerster distance for a given dye pair
Cyanin 3 and Cyanin 5
Figure 5: Spectra of the fluorophore pair
Extinction and emission spectra of Cy3 and Cy5.
Prion Detection Assay
Prions are proteins that could infect other proteins to change their conformation.
This is often causing a loss of function and aggregation of these proteins. Prions are
the cause for diseases like transmissible spongiform encephalopathies (TSEs),
neurodegenerative disorders that effect humans and animals (Wickner et al., 2015).
Sup35 is a yeast translation termination factor from Saccharomyces cerevisiae.
The prion form of Sup35 is known to form amyloids consisting of beta-sheet rich protein
aggregates with beta-strands perpendicular to the long axis of the filament. The domain
responsible for the conformational change is the NM region. This region of the protein
contains two different sections. The N‑section (amino acids 1‑124) forms the major part
of the amyloid core that that directs the protein into the prion form. The M-section
(amino acids 124‑250) is highly charged and provides the solubility to the native form
of Sup35. In the prion form the M region changes its conformation to a beta‑sheet rich
conformation, while the N‑section stays nearly unchanged in its conformation (Mukhopadhyay et al., 2007; Wickner et al., 2015).
ReferencesBrustad, E. M., Lembke, E. A., Schultz, P. G., Dentz, A. A.(2008). A General and Efficient Method for the Site-Specific Dual-Labeling of Proteins for Single Molecule Fluorescence Resonance Energy Transfer. American Chemical Society. 130: 17664-17665..
Kim, J., Seo, M., Lee, S., Cho, K., Yang, A., Woo, K., Kim, H., Park, H.(2012). Simple and Efficient Strategy for Site-Specific Dual Labeling of Proteins for Single-Molecule Fluorescence Resonance Energy Transfer Analysis. Analytical Chemistry.85: 1468-1474.
Lembke, E. a.(2011). Site-Specific Labeling of Proteins for Single-Molecule FRET Measurements Using Genetically Encoded Ketone Functionalities. Bioconjugation Proocols: Strategies and Methods in Molecular Biology. 751: 3-15.
Mukhopadhyay, S., Krishnan, R., Lembke, E. A., Lindquist, S., Deniz, A. A.(2007)A natively unfolded yeast prion monomer adopts an ensemble of collapsed and rapidly fluctuating structures.PNAS.104(8):2649-2654.
Wickner, R. B., Shewmaker, F. P., Bateman, D. A., Edskes, H. K., Gorkovsky, A., Dayani, Y., Bezsonov, E. E.82015) Yeast Prions: Structure, Biology, and Prion-Handling Systems. Microbiology and Molecular Reviews. 79(1):1-17.