Dimerization and folding of LC8, a highly conserved light chain of cytoplasmic dynein

Biochemistry. 2001 Feb 13;40(6):1596-605. doi: 10.1021/bi002278+.


Cytoplasmic dynein is a multisubunit ATPase that transforms chemical energy into motion along microtubules. LC8, a 10 kDa light chain subunit of the dynein complex, is highly conserved with 94% sequence identity between Drosophila and human. The precise function of this protein is unknown, but its ubiquitous expression and conservation suggest a critical role in the function of the dynein motor complex. We have overexpressed LC8 from Drosophila melanogaster and characterized its dimerization and folding using analytical ultracentrifugation, size-exclusion chromatography, circular dichroism, and fluorescence spectroscopy. Sedimentation equilibrium measurements of LC8 at pH 7 reveal a reversible monomer-dimer equilibrium with a dissociation constant of 12 microM at 4 degrees C. At lower pH, LC8 dissociates to a monomer, with a transition midpoint at pH 4.8. Far-UV CD and fluorescence spectra demonstrate that pH-dissociated LC8 retains native secondary and tertiary structures, while the diminished near-UV CD signal shows loss of quaternary structure. The observation that dimeric LC8 dissociates at low pH can be explained by titration of a histidine pair in the dimer interface. Equilibrium denaturation experiments with a protein concentration range spanning almost 2 orders of magnitude indicate that unfolding of LC8 dimer is a two-stage process, in which global unfolding is preceded by dissociation to a folded monomer. The nativelike tertiary structure of the monomer suggests a role for the monomer-dimer equilibrium of LC8 in dynein function.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Aspergillus nidulans / enzymology
  • Carrier Proteins / chemistry*
  • Carrier Proteins / metabolism*
  • Chromatography, Gel
  • Circular Dichroism
  • Conserved Sequence
  • Cytoplasm / enzymology
  • Dimerization
  • Drosophila Proteins*
  • Drosophila melanogaster / enzymology
  • Dyneins / chemistry*
  • Dyneins / metabolism*
  • Fungal Proteins / chemistry
  • Fungal Proteins / metabolism
  • Guanidine
  • Humans
  • Insect Proteins / chemistry
  • Insect Proteins / metabolism
  • Protein Conformation
  • Protein Denaturation
  • Protein Folding*
  • Spectrometry, Fluorescence
  • Thermodynamics
  • Ultracentrifugation


  • Carrier Proteins
  • Drosophila Proteins
  • Fungal Proteins
  • Insect Proteins
  • ctp protein, Drosophila
  • Dyneins
  • Guanidine