title: The novel isotopically coded short-range photo-reactive crosslinker 2,4,6-triazido-1,3,5-triazine (TATA) for studying protein structures.
abstract: 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)-12C3/13C3, for MS-based protein crosslinking studies. Upon activation by 254nm UV light, TATA-12C3/13C3generates 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.
  
  
  
  The isotopically labeled short-range non-specific crosslinker TATA-12C3/13C3was 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-12C3/13C3had 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.
authors.affiliation: University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101 - 4464 Markham St., Victoria, BC V8Z 7X8, Canada.
authors.forename: Nicholas I
authors.initials: NI
authors.lastname: Brodie
authors.affiliation: University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101 - 4464 Markham St., Victoria, BC V8Z 7X8, Canada.
authors.forename: Evgeniy V
authors.initials: EV
authors.lastname: Petrotchenko
authors.affiliation: University of Victoria - Genome British Columbia Proteomics Centre, Vancouver Island Technology Park, #3101 - 4464 Markham St., Victoria, BC V8Z 7X8, Canada; Department of Biochemistry and Microbiology, University of Victoria, Petch Building Room 207, 3800 Finnerty Rd, Victoria, BC V8P 5C2, Canada. Electronic address: christoph@proteincentre.com.
authors.forename: Christoph H
authors.initials: CH
authors.lastname: Borchers
mesh_terms: D002853:Chromatography, Liquid; D003432:Cross-Linking Reagents / Q000138:chemical synthesis / Q000737:chemistry* / Q000528:radiation effects; D006801:Humans; D007553:Isotope Labeling / Q000379:methods*; D008958:Models, Molecular; D009211:Myoglobin / Q000737:chemistry*; D010455:Peptides / Q000737:chemistry; D055668:Photochemical Processes; D017433:Protein Structure, Secondary; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D014227:Triazines / Q000138:chemical synthesis / Q000737:chemistry* / Q000528:radiation effects; D014466:Ultraviolet Rays; D051844:alpha-Synuclein / Q000737:chemistry*
chemical_list: C000624745:2,4,6-triazido-1,3,5-triazine; D003432:Cross-Linking Reagents; D009211:Myoglobin; D010455:Peptides; D014227:Triazines; D051844:alpha-Synuclein
keywords: 2,4,6-Triazido-1,3,5-triazine (TATA); Crosslinking; Non-specific crosslinker; Short-range crosslinker; Structural proteomics