Deamidation of human βB1 alters the elongated structure of the dimer

Kirsten Lampi, Julie T. Oxford, Hans Peter Bachinger, Thomas (Tom) Shearer, Larry David, Deborah M. Kapfer

Research output: Contribution to journalArticle

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Abstract

The purpose of these experiments was to determine if truncation and deamidation alter the structure of a human lens protein, βB1-crystallin. Recombinant wild type and a deamidated form of recombinant βB1 were expressed in Escherichia coli. Wild type βB1 was also enzymatically cleaved to generate a physiologically-relevant truncated βB1. Purity and size of the expressed proteins were confirmed by SDS-PAGE and electrospray ionization mass spectrometry. Size exclusion chromatography and light scattering were used to determine aggregation states of βB1. Protein conformations were predicted from sedimentation velocity analysis. Molecular weights of 49 000 and 54 000 Da were obtained for wild type βB1 by sedimentation equilibrium and light scattering, respectively. A sedimentation coefficient of 2.7 S was determined for wild type βB1. Molecular weights of 54 000 and 60 000 Da were determined for deamidated βB1 by sedimentation equilibrium and light scattering, respectively. However, deamidated βB1 eluted earlier than wild type βB1 on size exclusion chromatography, with an estimated molecular weight between 78 000 and 116 000 Da. Loss of the extensions of βB1 caused abnormal association of the protein with the stationary phase during size exclusion chromatography. Wild type βB1 was predicted to form a dimer with an elongated structure. The earlier elution of the deamidated βB1 dimer on size exclusion chromatography suggested the dimer was less compact. Truncation caused abnormal column interactions suggesting an altered conformation. These changes are important because truncation and deamidation occur extensively in aging human lenses and may be important for senile cataract formation.

Original languageEnglish (US)
Pages (from-to)279-288
Number of pages10
JournalExperimental Eye Research
Volume72
Issue number3
DOIs
StatePublished - 2001

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Gel Chromatography
Crystallins
Molecular Weight
Light
Protein Conformation
Electrospray Ionization Mass Spectrometry
Cataract
Lenses
Polyacrylamide Gel Electrophoresis
Proteins
Escherichia coli

Keywords

  • Beta-crystallin
  • Deamidation
  • Protein conformation
  • Truncation

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems

Cite this

Deamidation of human βB1 alters the elongated structure of the dimer. / Lampi, Kirsten; Oxford, Julie T.; Bachinger, Hans Peter; Shearer, Thomas (Tom); David, Larry; Kapfer, Deborah M.

In: Experimental Eye Research, Vol. 72, No. 3, 2001, p. 279-288.

Research output: Contribution to journalArticle

Lampi, Kirsten ; Oxford, Julie T. ; Bachinger, Hans Peter ; Shearer, Thomas (Tom) ; David, Larry ; Kapfer, Deborah M. / Deamidation of human βB1 alters the elongated structure of the dimer. In: Experimental Eye Research. 2001 ; Vol. 72, No. 3. pp. 279-288.
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