Identifying proteins that can form tyrosine-cysteine crosslinks

Area of Science:

• Biochemistry
• Structural Biology
• Post-translational Modifications

Background:

• Protein cofactors are redox-active posttranslational modifications essential for metalloprotein function.
• Current identification methods, primarily crystallography, likely underestimate the prevalence of protein cofactors.
• The known number of proteins with cofactors is limited, necessitating new discovery techniques.

Purpose of the Study:

• To develop and validate a computational method for identifying novel protein cofactors.
• To discover new protein cofactors by searching for specific chemical environments conducive to their formation.
• To experimentally confirm the formation of a protein cofactor in a candidate metalloprotein.

Main Methods:

• Computational screening of protein structures for tyrosine and cysteine side chain proximity and favorable chemical environments.
• Utilizing hydrogen bonding and metal center proximity to filter candidate proteins.
• Experimental validation using copper-dioxygen chemistry, liquid chromatography-MALDI mass spectrometry, and optical spectroscopy.

Main Results:

• A computational search identified 300 candidate proteins, narrowed down to four highly viable candidates.
• A novel tyrosine-cysteine crosslink was successfully formed and confirmed in the orphan metalloprotein BF4112.
• The findings validate the predictive methodology for identifying proteins capable of forming tyrosine-cysteine crosslinks.

Conclusions:

• The developed predictive method is effective in identifying proteins with the potential for cofactor formation.
• The discovery of a tyrosine-cysteine crosslink in BF4112 expands the known repertoire of protein cofactors.
• This approach, combined with functional studies, can uncover additional protein-derived cofactors.