Identifying proteins that can form tyrosine-cysteine crosslinks.
Protein cofactors represent a unique class of redox active posttranslational protein modifications formed in or by metalloproteins. Once formed, protein cofactors provide a one-electron oxidant, which is tethered to the protein backbone. Twenty-five proteins are known to contain protein cofactors, but this number is likely limited by the use of crystallography as the identification technique. In order to address this limitation, a search of all reported protein structures for chemical environments conducive to forming a protein cofactor through tyrosine and cysteine side chain crosslinking yielded three hundred candidate proteins. Using hydrogen bonding and metal center proximity, the three hundred proteins were narrowed to four highly viable candidates. An orphan metalloprotein (BF4112) was examined to validate this methodology, which identifies proteins capable of crosslinking tyrosine and cysteine sidechains. A tyrosine-cysteine crosslink was formed in BF4112 using copper-dioxygen chemistry, as in galactose oxidase. Liquid chromatography-MALDI mass spectrometry and optical spectroscopy confirmed tyrosine-cysteine crosslink formation in BF4112. This finding demonstrates the efficacy of these predictive methods and the minimal constraints, provided by the BF4112 protein structure, in tyrosine-cysteine crosslink formation. This search method, when coupled with physiological evidence for crosslink formation and function as a cofactor, could identify additional protein-derived cofactors.
Department of Chemistry & Biochemistry, Calvin College, 1726 Knollcrest Circle SE, Grand Rapids, MI 49546, USA.
Ryan J
RJ
Martinie
Pahan I
PI
Godakumbura
Elizabeth G
EG
Porter
Anand
A
Divakaran
Brandon J
BJ
Burkhart
John T
JT
Wertz
David E
DE
Benson
D001426:Bacterial Proteins / Q000737:chemistry / Q000378:metabolism; D002853:Chromatography, Liquid; D003545:Cysteine / Q000737:chemistry / Q000378:metabolism*; D030562:Databases, Protein; D008667:Metalloproteins / Q000737:chemistry / Q000378:metabolism*; D008958:Models, Molecular; D010084:Oxidation-Reduction; D011487:Protein Conformation; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014443:Tyrosine / Q000737:chemistry / Q000378:metabolism*
D001426:Bacterial Proteins; D008667:Metalloproteins; D014443:Tyrosine; D003545:Cysteine