December 2002
Volume 43, Issue 13
ARVO Annual Meeting Abstract  |   December 2002
Structural Analysis of ßB1-Crystallins in Solution
Author Affiliations & Notes
  • KJ Lampi
    Oral Molecular Biology Oregon Health Science Univ Portland OR
  • JA Carver
    University of Wollongong Wollongong Australia
  • R Lindner
    University of Wollongong Wollongong Australia
  • DM Kapfer
    Oral Molecular Biology Oregon Health Science Univ Portland OR
  • YH Kim
    Oral Molecular Biology Oregon Health Science Univ Portland OR
  • JB Feix
    Medical College of WI Milwaukee WI
  • Footnotes
    Commercial Relationships   K.J. Lampi, None; J.A. Carver, None; R. Lindner, None; D.M. Kapfer, None; Y.H. Kim, None; J.B. Feix, None. Grant Identification: Support: NIH Grant EY12239, RR01008, National Health of Australia
Investigative Ophthalmology & Visual Science December 2002, Vol.43, 4654. doi:
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    • Get Citation

      KJ Lampi, JA Carver, R Lindner, DM Kapfer, YH Kim, JB Feix; Structural Analysis of ßB1-Crystallins in Solution . Invest. Ophthalmol. Vis. Sci. 2002;43(13):4654.

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

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Abstract: : Purpose: To determine how the post-translational modification, deamidation, alters the structure of ßB1-crystallin in solution. Methods: Recombinant wildtype ßB1 (WT) and deamidated ßB1 (Q204E) were cloned and expressed. Oligomer state of the proteins at increasing concentrations were predicted from the molar mass determined by multi-angle laser light scattering. Structure at the single cysteine, residue 79, was examined by electron spin resonance spectroscopy after incubating with a methanethiosulfunate spin label. Regions of flexibility at 20 mg/ml of protein were determined by 2D 1H NMR spectroscopy. Results: ßB1 oligomerizes between 0.3 and 0.5 mg/ml with higher-ordered aggregates starting to form above 1 mg/ml. There were minimal differences in the EPR signal between 0.1 and 15 mg/ml. Fine differences in the spectra at the higher concentrations indicated restricted movement at the cysteine and suggested a more compact protein. Minimal differences were also seen between WT and Q204E. However, upon denaturation with urea, changes in the EPR signal indicated unfolding at the labeled site at a lower urea concentration for Q204E than for WT, suggesting a less stable protein. Both the N- and C- terminus of WT ßB1 and the C-terminus of Q204E were clearly visible by NMR indicating these regions were flexible in the proteins. Fewer cross-peaks from the N-terminal extension were observed in the NMR spectra of Q204E. The first 10 amino acids had significantly reduced flexibility. Conclusion: The flexibility of the extensions of native ßB1 in solution suggest these extensions are not involved in formation of homo-oligomers. Introduction of a negative charge at residue 204 may have resulted in electrostatic interactions of the N-terminal extension with the C-terminal domain. How this specifically decreases stability of the protein is unclear.

Keywords: 378 crystallins • 525 protein modifications-post translational • 527 protein structure/function 

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