Team:TU-Eindhoven/ProteinSelection

Contents 1 CEST Based Marker Proteins 2 Scanning for Candidates 3 Molecular Dynamics 4 References

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 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 .

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
1IWQ	5.2279	5.6850	4.6562	5.3046
1PJN	8.1621	4.2782	4.5149	8.5247
2IGR	4.7848	4.7331	4.7331	4.7331
2KLW
2PCO
1G70	2.9608	2.2858	2.2338	2.7171
1BY0	5.5306	7.1884	6.7331	5.7483
1NWD	2.7855	2.2016	2.2016	2.2016
1PEH
2L9A	3.0711	2.1342	2.1342	2.1342
2L96
2L99
1LYP
1LQ7	0.0814	0.7923	0.3797	0.0810

References