June 2015
Volume 56, Issue 7
ARVO Annual Meeting Abstract  |   June 2015
Interactions within β-crystallin complexes revealed by crosslinking and mass spectrometry
Author Affiliations & Notes
  • Kirsten J Lampi
    Integrative Biosciences, Oregon Health and Science University, Portland, OR
  • Eileen Yue
    Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR
  • Larry L David
    Biochemistry and Molecular Biology, Oregon Health and Science University, Portland, OR
  • Footnotes
    Commercial Relationships Kirsten Lampi, None; Eileen Yue, None; Larry David, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 5879. doi:
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      Kirsten J Lampi, Eileen Yue, Larry L David; Interactions within β-crystallin complexes revealed by crosslinking and mass spectrometry. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):5879.

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      © ARVO (1962-2015); The Authors (2016-present)

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Purpose: Lens transparency is accounted for by the short-range order of the major structural proteins, the crystallins. In order to elucidate this order, interaction sites between crystallins were identified by chemical crosslinking βB2 and βA3 heterodimers.

Methods: Homodimers of recombinant βB2 and βA3 were mixed at equal molar concentrations overnight at 37 °C to form heterodimers that were confirmed by Blue Native-PAGE. The respective homodimers and heterodimer were then treated with equal amounts of the light and heavy isotope labeled chemical cross linker CyanurBiotinDimercaptoProprionylSuccinimide (CBDPS) (Creative Molecules, Inc. Vancouver, CAN) capable of crosslinking lysine residues 14 Å or less apart. Crosslinked peptides in tryptic digests were purified using an avidin column and analyzed by mass spectrometry using an Orbitrap Fusion (Thermo Scientific, Inc.) to generate accurate parent and daughter ion masses for the crosslinked peptides. The data was analyzed using StavroX software (version: 3.4.9 , University of Halle-Wittenberg) to identify crosslinked peptides by comparison to theoretical crosslinked peptides calculated from a protein database containing βA3 and βB2 sequences.

Results: There were 9 and 7 confidently assigned crosslinks in βB2 homodimer and βB2/βA3 heterodimer, respectively. Crosslinks at Ser, Thr, and Tyr residues, as well as Lys were observed, as previously reported by others (J. Mass Spectrom. 2009, 44, 694-706). A crosslink within βB2 occurred between T90 in the N-terminal domain and K107 at the C-terminal end of the connecting peptide, suggesting these residues were within 14 Å of each other in the heterodimer. The predicted distance between these two residues in the extended βB2 homodimer is 39 Å. While many dead end reactions were observed in βA3 peptides, no crosslinks were detected that contained βA3 peptides in either the βA3 homodimer or βA3/βB2 heterodimer.

Conclusions: Chemical crosslinking of βB2 peptides within the βB2/βA3 heterodimer suggested that the linker in βB2 is bent in the βB2/βA3 oligomer and may adopt a structure analogous to the closed βB1 structure. The difficulty in finding crosslinks in βA3 are likely due to the smaller number of lysines in the protein, compared to βB2, and their lack of proximity to one another in the structure. Future experiments probing βA3 structure should use crosslinkers that are not entirely based on NHS ester chemistry.


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