Hierarchy of local structural and dynamics perturbations due to subdenaturing urea in the native state ensemble of DLC8 dimer

• Published in: Biophysical chemistry Vol. 153; no. 1; pp. 17 - 26
• Main Authors: Mohan, P.M. Krishna, Chakraborty, Swagata, Hosur, Ramakrishna V.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier B.V 01.12.2010
• Subjects: Circular Dichroism; Cytoplasmic Dyneins - chemistry; Dimerization; Dynein Light Chain protein (DLC8); Equilibrium states; Fluorescence spectroscopy; Humans; Hydrogen - chemistry; Nuclear magnetic resonance; Nuclear Magnetic Resonance, Biomolecular; Protein Structure, Tertiary; Protein Unfolding; Spectrometry, Fluorescence; Thermodynamics; Unfolding mechanism; Urea - chemistry; aa; Fluorescence spectroscopy; CD; NOE; DTT; TOCSY; Unfolding mechanism; Circular dichroism; HX; Dynein Light Chain protein (DLC8); Equilibrium states; CPMG; Nuclear magnetic resonance; HSQC; GdnHCl; TALOS; NHX
• ISSN: 0301-4622; 1873-4200
• DOI: 10.1016/j.bpc.2010.09.010

Local structural and dynamic modulations due to small environmental perturbations reflect the adaptability of the protein to different interactors. We have investigated here the preferential local perturbations in Dynein light chain protein (DLC8), a cargo adapter, by sub-denaturing urea concentrations. Equilibrium unfolding experiments by optical spectroscopic methods indicated a two state like unfolding of DLC8 dimer, with the transition mid-point occurring around 8.6 M urea. NMR studies identified the β3 and β4 strands, N-, C- terminal regions, loops connecting β1 to α1, α1 to α2 and β3 to β4 as the soft targets of urea perturbation and thus indicated potential unfolding initiation sites. Native-state hydrogen exchange studies suggested the unfolding to traverse from the edges towards the centre of the secondary structural elements. At 6 M urea the whole protein chain acts like a cooperative unit. These observations are expected to have important implications for the protein's multiple functions. [Display omitted] ►Investigation of preferential local perturbations in DLC8 by urea. ►Optical spectroscopy indicates a two state like unfolding ►Structural and motional characterizations by NMR reveal potential unfolding initiation sites. ►Native-state hydrogen exchange studies predict hierarchy of unfolding.