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

Nicholas I. Brodie, Evgeniy V. Petrotchenko, Christoph H. Borchers

Результат исследований: Вклад в журналСтатьярецензирование

15 Цитирования (Scopus)

Аннотация

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 (14 Å), 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)-12C3/13C3, for MS-based protein crosslinking studies. Upon activation by 254 nm UV light, TATA-12C3/13C3 generates up to three nitrene radicals capable of non-selective crosslinking at ~ 5 Å. 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-12C3/13C3 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-12C3/13C3 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.

Язык оригиналаАнглийский
Страницы (с-по)69-76
Число страниц8
ЖурналJournal of Proteomics
Том149
DOI
СостояниеОпубликовано - 21 окт. 2016
Опубликовано для внешнего пользованияДа

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