Equilibrium unfolding of DLC8 monomer by urea and guanidine hydrochloride: distinctive global and residue level features

Chatterjee, Amarnath ; Krishna Mohan, P. M. ; Prabhu, Arati ; Ghosh-Roy, Anindya ; Hosur, Ramakrishna V. (2007) Equilibrium unfolding of DLC8 monomer by urea and guanidine hydrochloride: distinctive global and residue level features Biochimie, 89 (1). pp. 117-134. ISSN 0300-9084

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Official URL: http://linkinghub.elsevier.com/retrieve/pii/S03009...

Related URL: http://dx.doi.org/10.1016/j.biochi.2006.09.007


We present circular dichroism (CD), steady state fluorescence and multidimensional NMR investigations on the equilibrium unfolding of monomeric dynein light chain protein (DLC8) by urea and guanidine hydrochloride (GdnHCl). Quantitative analysis of the CD and fluorescence denaturation curves reveals that urea unfolding is a two-state process, whereas guanidine unfolding is more complex. NMR investigations in the native state and in the near native states created by low denaturant concentrations enabled residue level characterization of the early structural and dynamic perturbations by the two denaturants. Firstly, 15N transverse relaxation rates in the native state indicate that the regions around N10, Q27, the loop between β2 and β4 strands, and K87 at the C-terminal are potential unfolding initiation sites in the protein. Amide and 15N chemical shift perturbations indicate different accessibilities of the residues along the chain and help identify locations of the early perturbations by the two denaturants. Guanidine and urea are seen to interact at several sites some of which are different in the two cases. Notable among the common interaction site is that around K87 which is in close proximity to W54 on the protein structure, but the interaction modes of the two denaturants are different. The secondary chemical shifts indicate that the structural perturbation by 1 M urea is small, compared to that by guanidine which is more encompassing over the length of the chain. The probable (Φ, ψ) changes at the individual residues have been calculated using the TALOS algorithm. It appears that the helices in the protein are significantly perturbed by guanidine. Further, comparison of the spectral density functions of the native and the two near native states in the two denaturants implicate greater loosening of the structure by guanidine as compared to that by urea, even though the structures are still in the native state ensemble. These differences in the early perturbations of the native state structure and dynamics by the two denaturants might direct the protein along different pathways, as the unfolding progresses on further increasing the denaturant concentration.

Item Type:Article
Source:Copyright of this article belongs to Elsevier Science.
Keywords:Protein Unfolding; near Native States; Secondary Chemical Shifts; NMR; N Relaxation; Denaturants; DLC8; Torsion Angle Predictions; Circular Dichroism; Fluorescence
ID Code:16601
Deposited On:15 Nov 2010 13:35
Last Modified:03 Jun 2011 08:46

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