Folding is coupled to dimerization of Tctex-1 dynein light chain.
Equilibrium analyses have been performed to elucidate the role of dimerization in folding and stability of dynein light chain Tctex-1. The equilibrium unfolding transition was monitored by intrinsic fluorescence intensity, fluorescence anisotropy, and circular dichroism and was modeled as a two-state mechanism where a folded dimer dissociates to two unfolded monomers without populating thermodynamically stable monomeric or dimeric intermediates. Sedimentation equilibrium and chemical cross-linking experiments performed at increasing concentrations of denaturants show no change in the association state before the unfolding transition and are consistent with the two-state model of dissociation coupled to unfolding. A linear dependence on denaturant concentration is observed by fluorescence intensity and anisotropy before unfolding in the 0-2 M GdnCl, and 0-4 M urea concentration range. This change is not protein concentration-dependent and possibly reflects relief of quenching associated with premelting conformational disorder in the vicinity of Trp 83. The data clearly show that the dissociation-coupled unfolding mechanism of Tctex-1 is different from the three-state denaturation mechanism of its structural homologue light chain LC8. The absence of a stable monomer in Tctex-1 offers insight into its functional differences from LC8.
Talbott
Matthew
M
Department of Chemistry and Biochemistry, Ohio University, Athens, Ohio 45701, USA.
Hare
Michael
M
Nyarko
Afua
A
Hays
Thomas S
TS
Barbar
Elisar
E
D000818:Animals; D056445:Cytoplasmic Dyneins; D019281:Dimerization; D004213:Dissociative Disorders; D029721:Drosophila Proteins / Q000737:chemistry*; D004398:Dyneins / Q000737:chemistry*; D011489:Protein Denaturation; D017510:Protein Folding
D029721:Drosophila Proteins; C430717:Dlc90F protein, Drosophila; D056445:Cytoplasmic Dyneins; D004398:Dyneins