ABSTRACT
Chemical modification of proteins has been crucial in engineering protein-based therapies, targeted biopharmaceutics, molecular probes, and biomaterials. Here, we explore the use of a conjugation-based approach to sense alternative conformational states in proteins. Tyrosine has both hydrophobic and hydrophilic qualities, thus allowing it to be positioned at protein surfaces, or binding interfaces, or to be buried within a protein. Tyrosine can be conjugated with 4-phenyl-3H-1,2,4-triazole-3,5(4H)-dione (PTAD). We hypothesized that individual protein conformations could be distinguished by labeling tyrosine residues in the protein with PTAD. We conjugated tyrosine residues in a well-folded protein, bovine serum albumin (BSA), and quantified labeled tyrosine with LC-MS/MS. We applied this approach to alternative conformations of BSA produced in the presence of urea. The amount of PTAD labeling was found to relate to the depth of each tyrosine relative to the protein surface. This study demonstrates a new use of tyrosine conjugation using PTAD as an analytic tool able to distinguish the conformational states of a protein.
Competing Interest Statement
The authors have declared no competing interest.
Footnotes
Rewritten manuscript and new data in Supplemental Figure and Figure 4.
ABBREVIATIONS
- BSA
- bovine serum albumin
- CD
- circular dichroism
- DBCO
- dibenzocyclooctyne
- DBH
- 1,3-dibromo-5,5-dimethylhydantoin
- LC-MS/MS
- liquid chromatography and tandem mass spectrometry
- NH(urea)
- amine conjugated isocyanate
- PTAD
- 4-phenyl-3H-1,2,4-triazole-3,5(4H)-dione
- PTAD-N3
- 4-(4-(2-azidoethoxy)phenyl)-1,2,4-triazolidine-3,5-dione, N3-Ph-Ur for e-Y-CLICK
- red·PTAD-N3
- reduced PTAD-N3
- SEC
- size exclusion chromatography
- UPLC-MS
- ultra-high pressure liquid chromatography and mass spectrometry
- Y(1)
- tyrosine with single PTAD conjugation
- Y(2)
- tyrosine with two PTAD conjugations