Analysing

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 measuringdistances between 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 are developinga prion detection assay. We use the yeast prion Sup35 as a model protein and incorporate to non-canonical amino acids (p-acetophenylalanine and propargyllysine). After the purification of 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 between each other. 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 noncanonical amino acids

The structure of proteins could be detected through protein crystallography. However, there are a lot of problems when it comes to highly flexible proteins or proteins which change their structure under 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 these changes two amino acids are incorporated at specific positions in the protein. These amino acids could then be labeled with chromophores, enabeling the measurement of the proteins distances with Foerster Resonance Energy Transfer (FRET)[Aramburu 2017, Kim 2013].
The first step is the incorporation of the non-canonical amino acids. In proteins naturally containing no cysteins (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 cysteine two ncAAs need to be incorporated for the labeling [Kim 2013].
Noncanonical amino acids could be incorporated by orthogonal tRNA/aaRS synthetases in response to 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 could be used for the incorporation in response to a rarely used leucine codon. For structural analysis the amino acids are specific labeled with chromophores. This labeling 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 ncAA from each other. [Brustad 2008, Kim 2013]

Figure 1:
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 [Lemke5]. 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 2013].

Figure 2:
Propargyllysine (PrK).

p-Acetylphenylalanine

Another amino acid with an additional functional group to the canonical amino acids is p-acetylphenylalanine. The ketone 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 2013].

Figure 3:
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 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 2006].

Figure 4:
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 efficency
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 4[Kim 2013].

Figure 5:
Extinction and emission spectra of Cy3 and Cy5.

For the labeling of the non-canonical 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 inexpensible in comparison to other chromophore pairs and above commercially available (Lumiprobe).

Prion detection assembly

Prions

Prions are proteins that could infect other proteins to change their conformation. Often this causes 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 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 2007,Wickner 2015, ]

Team:Bielefeld-CeBiTec/Project/toolbox/analysing