Dimerization and folding of LC8, a highly conserved light chain of cytoplasmic dynein.
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.
Barbar
E
E
Department of Chemistry and Biochemistry, Ohio University, Athens, OH 45701, USA. barbar@ohio.edu
Kleinman
B
B
Imhoff
D
D
Li
M
M
Hays
T S
TS
Hare
M
M
D000818:Animals; D001233:Aspergillus nidulans / Q000201:enzymology; D002352:Carrier Proteins / Q000737:chemistry* / Q000378:metabolism*; D002850:Chromatography, Gel; D002942:Circular Dichroism; D017124:Conserved Sequence; D003593:Cytoplasm / Q000201:enzymology; D019281:Dimerization; D029721:Drosophila Proteins; D004331:Drosophila melanogaster / Q000201:enzymology; D004398:Dyneins / Q000737:chemistry* / Q000378:metabolism*; D005656:Fungal Proteins / Q000737:chemistry / Q000378:metabolism; D019791:Guanidine; D006801:Humans; D019476:Insect Proteins / Q000737:chemistry / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017510:Protein Folding; D013050:Spectrometry, Fluorescence; D013816:Thermodynamics; D014461:Ultracentrifugation
D002352:Carrier Proteins; D029721:Drosophila Proteins; D005656:Fungal Proteins; D019476:Insect Proteins; C542608:ctp protein, Drosophila; D004398:Dyneins; D019791:Guanidine