Discriminating changes in protein structure using tyrosine conjugation

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..

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“…On the other hand, when NIC molecules get into the drug sockets of BSA, its total conformation would have changed. This protein-disrupted structure of BSA suggests that the conformation of BSA might have changed through a strong interaction between BSA and NIC [ 55 ].…”

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“…It is known that BSA possesses 21 tyrosine residues, [26] and HSA possesses 18 tyrosine residues [27] . Although, in the case of BSA at least, access to all the residues is limited for conjugation purposes as 10 Y residues are present in the hydrophobic core of the protein as demonstrated by 4‐phenyl‐3 H ‐1,2,4‐triazole‐3,5(4 H )‐dione (PTAD) labelling studies [28] . Tyrosine residues show decreasing reactivity towards PTAD labelling as they retreat into the protein core and distance from the surface increases, with Y424 ( ca .…”

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