Towards Single-Molecule Protein Sequencing

Speaker: Chirlmin Joo
Department: Bionanoscience Department TU Delft
Location: TU Delft 
Date: January 12, 2017
Author: Teun Huijben

Chirlmin Joo starts his talk with a graph showing the costs for whole genome sequencing over the years. The costs were once around 100 million dollars, where sequencing is nowadays possible for less then 1000 dollars. This decrease is enormous, but to improve medical care even more, the need for cheaper and easier methods is still high. New techniques like single-molecule fluorescence sequencing methods and nanopore detection have the potential to fulfill these needs.

It is clear that DNA sequencing is important, but Chirlmin rises the question why not sequencing proteins? Proteins are the working machinery of the cell, they perform all kind of functions. To name a few: they replicate our DNA, generate ATP, transport all kind of molecules and take care of muscle contractions. Having a fast and cheap method to sequence proteins will therefore be very interesting for both medical care as fundamental research. The technique that is used nowadays to sequence proteins is mass spectrometry. However, it is quite difficult to use and a lot of sample is needed for a proper measurement.

Chirlmin and his group are searching for a single-molecule protein sequencing technique. Sequencing proteins in stead of DNA gives rise to a problem, where DNA consists of only 4 subunits (the 4 nucleotides), are proteins built of 20 different amino acids. The idea was to label all different amino acids with a different fluorophore, but the spectrum of visible light is too small to distinguish 20 different colors. The brilliant idea they came up with was protein fingerprinting (figure 1). This means that of the protein only 2 types of amino acids are labeled. Computationally was determined that if by sequencing the order of only these two amino acids (they chose lysine and cysteine) was known, the right protein could be determined from the database with some error. However, if also the relative distance between the amino acids of interest was known, the right protein could be determined with high precision. This means that in theory the technique of protein fingerprinting was able to sequence proteins. Now they had to find a method to sequence the labeled proteins.

protein-fingerprinting

figure 1: Protein fingerprinting. The idea of protein fingerprinting is labelling only two amino acids, in this case cysteine and lysine. When the order and relative distance of only these two amino acids is known in the unknown protein, comparison with the protein database will results in determining the correct protein.[1] 

Chirlmin came up with the idea to linearize the protein and let it be pulled through a chaperon protein. A chaperon is a cylindrical protein that grabs the C-terminus of a protein and pulls it through its cavity. In this way the protein is sort of scanned. This idea was combined with FRET (Förster resonance energy transfer). The lysines and cysteines are labeled with different fluorophores (red and green), which have the same excitation wavelength but a different emission wavelength. At the end of the chaperon protein an extra fluorophore is attached, called the donor dye. The emitted light of this donor dye has the exact wavelength that excites the fluorophores that are attached to the lysines and cysteins on the protein. So while the protein is translocating through the chaperon the labeled amino acids pass the donor dye and will be excited. This results in a series of red and green light flashes, and these indicate the number and order of lysines and cysteines in the protein. When this is compared with the database, the right protein can be determined.

This technique has many advantages, it is relatively simple and only one copy of the protein is needed to sequence it. It has therefore the potential to be optimized to a very useful technique. Nowadays Chirlmins group is working on adding an extra fluorophore to the system which will enable the sequencing of three different amino acids, this will make the protein determination more reliable.

[1] Yao Yao et al. Single-molecule protein sequencing through fingerprinting: computational assessment. Physics. Biol. 12 (2015).

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