Deamidation of human βB1 alters the elongated structure of the dimer

Kirsten J. Lampi, Julie T. Oxford, Hans Peter Bachinger, Thomas R. Shearer, Larry L. David, Deborah M. Kapfer

Research output: Contribution to journalArticle

65 Scopus citations

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 - Jan 1 2001

Keywords

  • Beta-crystallin
  • Deamidation
  • Protein conformation
  • Truncation

ASJC Scopus subject areas

  • Ophthalmology
  • Sensory Systems
  • Cellular and Molecular Neuroscience

Fingerprint Dive into the research topics of 'Deamidation of human βB1 alters the elongated structure of the dimer'. Together they form a unique fingerprint.

  • Cite this