Oligomerization Interface of RAGE Receptor Revealed by MS-Monitored Hydrogen Deuterium Exchange
Figure 1
YexRAGE dimer formation via a dityrosine cross-link.
(A) PAGE and (B, C) mass spectra of exRAGE (A, lanes 1-4, B, C) and YexRAGE (A, lanes 5-7) incubated with horseradish peroxidase in the presence of hydrogen peroxide for 15 or 30 minutes (lanes 2 and 6 and lanes 3 and 7, respectively). The oligomeric forms visible in lanes 6 and 7 with a mass >45 kDa correspond to YexRAGE dimers and trimers covalently linked by dityrosine and trityrosine bonds, respectively. Such oligomeric forms are absent if the substrate of the reaction is exRAGE instead of YexRAGE (lanes 2 and 3). Lanes 1 and 5 represent the sample before reaction, lane M is a protein ladder, and lane 4 shows the monomeric fraction after SEC. Gels were overloaded on purpose to show the lack of oligomeric forms in the reaction with exRAGE. (B) MALDI-ToF mass spectra of exRAGE before and (C) after HRP/H 2O2 incubation. (D) Fluorescence emission spectra in the range 350-550 nm obtained during incubation of YexRAGE in the presence of hydrogen peroxide and horseradish peroxidase for a specified period of time after excitation at 315 nm. The band with a maximum at 402 nm is expected for dityrosine species, whereas the band at 419 nm represents trityrosine or tetratyrosine species. In the course of the reaction, the fraction of species yielding a signal at 419 nm compared to a band at 402 nm increases, indicating facile formation of multityrosine species in YexRAGE. See also Scheme S1.