title: Hierarchy in guanidine unfolding of DLC8 dimer: regulatory functional implications.
abstract: Folding-unfolding caused by environmental changes play crucial regulatory roles in protein functions. To gain an insight into these for DLC8, a cargo adaptor in dynein motor complex, we investigated here the unfolding of homodimeric DLC8 by GdnHCl, a standard unfolding agent. Fluorescence spectroscopy revealed a three-state unfolding transition with midpoints at 1.5 and 4.0 M GdnHCl. The HSQC spectrum at 1.5 M GdnHCl displayed peaks belonging to a folded monomer. NMR chemical shift perturbations, line broadening effects and (15)N relaxation measurements at low GdnHCl concentrations identified a hierarchy in the unfolding process, with the dimer interface--the cargo binding site--being the most susceptible followed by the helices in the interior. Similar observations were made earlier for small pH perturbations and thus the early unfolding events appear to be intrinsic to the protein. These, by virtue of their location, influence target binding efficacies and thus have important regulatory implications.
authors.affiliation: Department of Chemical Sciences, Tata Institute of Fundamental Research, Homi Bhabha Road, Mumbai 400 005, Maharashtra, India.
authors.forename: P M Krishna
authors.initials: PM
authors.lastname: Mohan
authors.forename: Mamata V
authors.initials: MV
authors.lastname: Joshi
authors.forename: Ramakrishna V
authors.initials: RV
authors.lastname: Hosur
mesh_terms: D020816:Amino Acid Motifs; D000595:Amino Acid Sequence; D001665:Binding Sites; D002942:Circular Dichroism; D056445:Cytoplasmic Dyneins; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004398:Dyneins / Q000737:chemistry* / Q000378:metabolism; D019791:Guanidine / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D011489:Protein Denaturation; D017510:Protein Folding; D020836:Protein Structure, Quaternary; D017433:Protein Structure, Secondary; D013050:Spectrometry, Fluorescence
chemical_list: C427758:DYNLL1 protein, human; D056445:Cytoplasmic Dyneins; D004398:Dyneins; D019791:Guanidine