The novel isotopically coded short-range photo-reactive crosslinker 2,4,6-triazido-1,3,5-triazine (TATA) for studying protein structures.

Unlabelled: Short-distance molecular-modeling constraints are advantageous for elucidating the structures of individual proteins and protein conformational changes. Commonly used amine-reactive crosslinks are relatively long (14Å), partly due to the length of the lysine side-chain, and are sparsely distributed throughout a protein. Short-distance non-specific crosslinkers can provide a larger number of tighter molecular-modeling constraints. Here we describe the use of a short-range homo-trifunctional isotopically-coded non-specific photo-reactive crosslinking reagent, 2,4,6-triazido-1,3,5-triazine (TATA)-C/C, for MS-based protein crosslinking studies. Upon activation by 254nm UV light, TATA-C/C generates up to three nitrene radicals capable of non-selective crosslinking at ~5Å. This reagent was validated using cyclohexane, several test peptides, and myoglobin, and was found to react with a large number of amino acids, forming multiple crosslinked products. The myoglobin crosslinks detected by MS agreed with the known structure of myoglobin; arranging the protein's secondary-structure motifs into their correct fold was possible based solely on the constraints imposed by the crosslinks. Finally, TATA was used to crosslink the α-synuclein monomer. The 10 short-distance constraints provided by TATA crosslinking led to an initial model of the molten-globule form of the native α-synuclein monomer; this provides a suggested structure for the precursor of the misfolded α-synuclein proteoforms involved in synucleopathies.

Biological Significance: The isotopically labeled short-range non-specific crosslinker TATA-C/C was characterized for use in crosslinking-based protein structural studies. The crosslinking products of TATA can provide a distance constraint of merely 5Ǻ between crosslinked residues. TATA-C/C had broad reactivity, crosslinking a wide variety of amino acids, including lysine, glutamic and aspartic acid, asparagine, glutamine, glycine, alanine, valine, proline, methionine, serine, cysteine, tyrosine, and the N-terminus. The short-distance crosslinking constraints provided by TATA allowed us to predict the fold of myoglobin using a combination of these distance constraints with a prediction of myoglobin's secondary structure motifs. TATA was also used to crosslink α-synuclein in its native, molten globule form, which has not been characterized using other structural biology techniques. The distance constraints provided by the crosslinks allowed for the manual modeling of a rudimentary structure for the α-synuclein monomer.