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Kirsten Lampi, Larry David; Probing structure of human β-crystallins by protein cross-linking. Invest. Ophthalmol. Vis. Sci. 2013;54(15):5748.
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To determine conformational changes upon heterodimer formation of β-cyrstallins that lead to stabilizing interactions using cross-linking and proteomic methods.
Human βB2 and βA3 homodimers, or βB2/βA3 heterodimers were treated DSP cross-linker, digested with trypsin, and cross-linked peptides identified by mass spectrometry following cleavage of the disulfide bond in the linker by electron transfer dissociation. Hydrogen/deuterium exchange with mass spectrometry was also performed.
Crosslinks in βB2 homodimers were found between lysines 11/101, 101/168, 108/168, and108/172; while βB2/βA3 heterodimers contained crosslinks between lysines βB2-108/βB2-121, βB2-108/βB2-168, and βB2-101/βA3-44. No cross-links could be identified in the βA3 homodimer.
Finding an identical intramolecular crosslink between βB2 K108 and 168 in both βB2 homodimer and βB2/βA3 heterodimer supports that the βB2 C-terminal domains in both structures are similar. The crosslinks between βB2 lysines 108/121 and lysine 101/44 in βB2 and βA3, respectively, in the heterodimer also suggest a βB2 homodimer like structure. This result is inconsistent with the H/D exchange data for the heterodimer that supports a structure similar to the βB1 homodimer with a bent linker. The difference in the results suggests that βB2/βA3 heterodimers may adopt both structures in solution. This finding would be consistent with their apparent polydispersity.
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