Team:Bielefeld-CeBiTec/Project/toolbox/analysing

Analyzing

Short Summary

As part of our toolbox, structural analysis of a protein could be used to study distances between non-canonical amino acids with Foerster Resonance Energy Transfer (FRET). This provides measuring distances within specific incorporated amino acids in the target protein to gain insight into protein folding or structural changes under different conditions.
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 structure of proteins could be detected through protein crystallography. However, there are some problems when it comes to highly flexible proteins, or proteins which change their structure within different conditions. To analyze these proteins and the changes in their conformation we want to establish a tool, that allows to detect changes in protein conformation. For the detection of those changes, two amino acids are incorporated at specific positions into the protein. These amino acids could then be labeled with chromophores, enabling the measurement of the proteins distances with Foerster Resonance Energy Transfer (FRET)(Lembke, 2011, Kim et al., 2013).
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).

Propargyllysine (PrK)

One amino acid which provides a functional group different to the canonical amino acids is propargyllysine. The propargyl group of PrK could form a covalent bond to acidic groups in a click chemistry reaction. PrK is commercially available or could be synthesized chemically in two steps starting with Boc‑I‑Lys‑OH (Kim et al., 2013). The click chemistry reaction is performed at neutral pH, native buffers and temperatures of 4° C to 37 °C. However, for the click‑chemistry reaction copper is required which is toxic for living cells, so PrK could not be used for in vivo labeling (Kim et al., 2013).
  • 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
Propargyllysine (PrK).

p‑Acetylphenylalanine (AcF)

Another amino acid with an additional functional group to the canonical amino acids is p‑acetylphenylalanine. The ketone group of AcF is able to build a covalent bond to a hydroxylamine coupled dye in a hydrazide reaction. This reaction is carried out at low pH‑values which causes problems with certain proteins (Kim et al., 2013).
  • 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
p-Acetylphenylalanine (AcF).

Foerster Resonance Energy Transfer (FRET)

Foerster Resonance Energy Transfer or Fluorescence Energy Transfer, short FRET, describes an energy transfer between two chromophores. During this process the donor chromophore is excited by light of a certain wavelength and transfers the energy to the acceptor chromophore if they are within a certain distance to each other. In biochemistry, FRET is mostly used as measurement tool with the help of fluorescent dyes. Using FRET, the measuring of distances from 1 to 10 nm is possible (Brustad et al., 2006). The FRET process is shown in Figure 4.

Figure 4: Animations of a FRET fluorophore pair
Animation of the distance dependent energy transfer of two fluorophores.

The FRET efficiency (E) is used to estimate the intramolecular distance of molecules.
E=[1+(r/R0)6)]-1

E FRET efficiency
r intermolecular distance
R0 Foerster distance for a given dye pair

Cyanin 3 and Cyanin 5

Cyanin 3 (Cy3) in combination with Cyanin 5 (Cy5) is a chromophore pair which is suitable for FRET measurements. Cy3 operates as the donor dye and Cy5 as the acceptor dye. The extinction and emission spectra of both chromophores is shown in Figure 5 (Kim et al., 2013).

Figure 5: Spectra of the fluorophore pair
Extinction and emission spectra of Cy3 and Cy5.

For the labeling of the noncanonical amino acids with these chromophores a functional group is required to build a covalent bond in chemical conjugation. PrK could be labeled with Cy3‑azide and AcF with Cy5‑hydrazide. Both chromophores are relatively advantageously priced in comparison to other chromophore pairs and commercially available from Lumiprobe.

Prion Detection Assay

Prions

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

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).

References

Brustad, 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.