Abstract
We have studied the conformational stability of the two homologous membrane skeletal proteins, the erythroid and non-erythroid spectrins, in their dimeric and tetrameric forms respectively during unfolding in the presence of urea and guanidine hydrochloride (GuHCl). Fluorescence and circular dichroism (CD) spectroscopy have been used to study the changes of intrinsic tryptophan fluorescence, anisotropy, far UV-CD and extrinsic fluorescence of bound 1-anilinonapthalene-8-sulfonic acid (ANS). Chemical unfolding of both proteins were reversible and could be described as a two state transition. The folded erythroid spectrin and non-erythroid spectrin were directly converted to unfolded monomer without formation of any intermediate. Fluorescence quenching, anisotropy, ANS binding and dynamic light scattering data suggest that in presence of low concentrations of the denaturants (up-to 1M) hydrogen bonding network and van der Waals interaction play a role inducing changes in quaternary as well as tertiary structures without complete dissociation of the subunits. This is the first report of two large worm like, multi-domain proteins obeying twofold rule which is commonly found in small globular proteins. The free energy of stabilization (ΔGu H 2 0) for the dimeric spectrin has been 20 kcal/mol lesser than the tetrameric from.
Highlights
Understanding of the stability of a protein under different chemical conditions is crucial for protein chemists and biologists
For monitoring denaturant induced structural and dynamical properties of spectrin, all experiments are performed at 25°C after incubation with the denaturants for 1 hour, during which no proteolytic cleavage of the protein occurred, shown in S1 Fig. We have studied the change in the ratio of intensities at 337 nm and 350 nm and emission maxima of Trp fluorescence in presence and absence of urea and guanidine hydrochloride (GuHCl) in erythroid and non-erythroid spectrin (Fig. 1)
The λmax appeared at 338nm and 338.5nm respectively for both erythroid and non-erythroid spectrin indicating buried tryptophan residues in the folded protein [51]
Summary
Understanding of the stability of a protein under different chemical conditions is crucial for protein chemists and biologists. Urea and guanidine hychloride are commonly used as strong denaturants of proteins and difference in stability of protein in the native and unfolded states are studied in the presence and absence of these chaotropic agents their mechanism of action is still obscure [6, 7]. It is still an open question whether they exert their effect by direct binding to the protein or they act indirectly by altering the 3D network of water [8, 9]. Recent studies indicate that in a multi-domain protein, an individual domain could fold in a co-operative manner, i.e. the folding of one domain is influenced by the other, whereas in others, the individual domain might fold independently [13, 14, 15, 16]
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