Team:TU-Eindhoven/ProteinSelection

From 2013.igem.org

Revision as of 21:16, 12 August 2013 by Pascalaldo (Talk | contribs)

Contents

CEST Based Marker Proteins

To create bacteria with the ability to generate contrast on a CEST MRI scan, polypeptides that have that ability had to be found. Various solutions based on short Lysine, Arganine, Threonine or Serine rich sequences were proposed.McMahonDIACESTM.T. McMahon, New "Multicolor" Polypeptide Diamagnetic Chemical Exchange Saturation Transfer (DIACEST) Contrast Agents for MRI. Magnetic Resonance in Medicine 60, 803-812 (2008) However, it is hard to predict how well these sequences will express in bacteria and whether they are sufficiently stable in vivo. To avoid these problems a new approach was taken. The suitability as a CEST based marker was estimated for proteins of which the structure is already clarified.

Scanning for Candidates

For good CEST contrast a protein should have a high Lysine or Arganine content.McMahonDIACESTGiladArtificialGeneA.A. Gilad, Artificial reporter gene providing MRI contrast based on proton exchange.. Nature biotechnology 25.2, 217-219 (2007) To find these suitable proteins the [http://www.pdb.org RCSB Protein Data Bank] was queried for all entries containing proteins. This was done using the SEARCH Web Service of PDB.org, for the XML query used see query.xml. A Python program was written to analyse the obtained amino acid sequences and calculate the ratio of Lysine or Arginine to the total chain length (see PDB.py and queryPDB.py). The results are visualized in and .

TU-Eindhoven Images LysineRatio.png
lysineRatioPlot Plot of the Lysine ratio and count of a broad range of proteins from PDB.org. Red dots represent potentially interesting proteins.
TU-Eindhoven Images ArginineRatio.png
arginineRatioPlot Plot of the Arginine ratio and count of a broad range of proteins from PDB.org. Red dots represent potentially interesting proteins.
Using this data a selection of interesting proteins was made for further analysis. The protein list was also filtered by chain length and general practicality of the sequences. The selection is shown below:

PDB ID Frequent Amino Acid Amount of Frequent Amino Acids (#) Chain Length (#) Ratio (-)
1ETF Arginine 11 23 0.48
1IWQ Lysine 7 19 0.37
1PJN Lysine 8 21 0.38
2IGR Lysine 15 34 0.44
2KLW Lysine 10 32 0.31
2PCO Lysine 8 26 0.30
1G70 Arginine 10 22 0.46
1BY0 Lysine 8 27 0.30
1NWD Lysine 8 28 0.29
1PEH Lysine 10 35 0.29
2L9A Lysine 8 24 0.33
2L96 Lysine 8 24 0.33
2L99 Lysine 8 24 0.33
1LYP Lysine 9 32 0.28
1LQ7 Lysine 17 67 0.25

Molecular Dynamics

To refine the selection the accessibility of the various exchangable hydrogen atoms of the Lysines and Arginines was taken into account. Hereto Molecular Dynamics simulations of the proteins in water were carried out.

PDB ID Backbone Secondary Amine Arginine Guanidine Arginine Secondary Amine Secondary Amine Lysine Primary Amine
1ETF 0.9935 1.0525 0.7954 0.8028 0.7953
1IWQ 5.2279 5.6850 4.6562 5.3046 5.1866
1PJN 8.1621 4.2782 4.5149 8.5247 7.6893
2IGR 4.7848 4.7331 4.7331 4.7331 3.2901
2KLW
2PCO
1G70 2.9608 2.2858 2.2338 2.7171 2.8389
1BY0 5.5306 7.1884 6.7331 5.7483 1.9289
1NWD 2.7855 2.2016 2.2016 2.2016 3.6024
1PEH
2L9A 3.0711 2.1342 2.1342 2.1342 3.4460
2L96
2L99
1LYP
1LQ7 0.0814 0.7923 0.3797 0.0810 1.7040

References