Premium
Guilt by association: functional annotation of TM0486 from Thermotoga maritima by identification of its bound ligands
Author(s) -
Palanski Brad A,
Snider Mark J
Publication year - 2010
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.24.1_supplement.518.3
Subject(s) - thermotoga maritima , mass spectrometry , chemistry , affinity chromatography , size exclusion chromatography , ligand (biochemistry) , function (biology) , chromatography , biochemistry , biology , escherichia coli , microbiology and biotechnology , enzyme , receptor , gene
As the result of recent structural genomic initiatives, the Protein Data Bank contains approximately 3000 protein structures classified as having unknown function. The crystal structures of over 80 of these proteins show that they bind at least one ligand of unknown chemical identity. We analyze one such protein (TM0486 from Thermotoga maritima ) and hypothesize that the isolation and determination of the identity of the unknown bound ligands will suggest a function for this protein. E. coli BL21 DE3 cells were transformed with expression plasmids, protein expression was optimized, and the His 6 ‐tagged protein was purified by affinity chromatography. The protein was denatured by ethanol to release the ligands, and the precipitated protein was removed by centrifugal filtration. The filtrate was analyzed by electrospray liquid chromatography‐mass spectrometry. Preliminary chromatograms show several peaks with promising m/z ions that suggest that the ligands may be isolated. Once verified, additional analyses by mass spectrometry and nuclear magnetic resonance should reveal their identity and allow a functional hypothesis for TM0486 to be proposed and examined.