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Probing conformational stability and dynamics of erythroid and nonerythroid spectrin: effects of urea and guanidine hydrochloride.

Patra M, Mukhopadhyay C, Chakrabarti A - PLoS ONE (2015)

Bottom Line: The folded erythroid spectrin and non-erythroid spectrin were directly converted to unfolded monomer without formation of any intermediate.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 (ΔGuH20) for the dimeric spectrin has been 20 kcal/mol lesser than the tetrameric from.

View Article: PubMed Central - PubMed

Affiliation: Chemistry Department, University of Calcutta, Kolkata, West Bengal, India.

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 (ΔGuH20) for the dimeric spectrin has been 20 kcal/mol lesser than the tetrameric from.

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Compactness of erythroid and nonerythroid spectrin under different conditions.Histograms show size distribution of spectrin species obtained by DLS measurements of erythroid spectrin in (A) native; (B) unfolded in 8M urea and (C) unfolded in 6M GuHCl and of non-erythroid spectrin in (D) native; (E) unfolded in 8M urea and (F) unfolded in 6M GuHCl.
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pone.0116991.g005: Compactness of erythroid and nonerythroid spectrin under different conditions.Histograms show size distribution of spectrin species obtained by DLS measurements of erythroid spectrin in (A) native; (B) unfolded in 8M urea and (C) unfolded in 6M GuHCl and of non-erythroid spectrin in (D) native; (E) unfolded in 8M urea and (F) unfolded in 6M GuHCl.

Mentions: DLS measurements were carried out to monitor the changes in size of the erythroid and nonerythroid spectrin in presence and absence of urea or GuHCl. The changes in the apparent hydrodynamic radii (Rh) of spectrin against increasing concentrations of urea and GuHCl, are shown in Fig. 4. Surprisingly, the apparent hydrodynamic radii of dimeric erythroid and tetrameric nonerythroid spectrin were estimated to be 60±10 nm and 40±5 nm respectively, which are in good agreement with Budzynski and co-workers [55, 56]. The hydrodynamic radius of both spectrin remained almost same up to 1M urea and beyond that the particle size significantly increased with denaturant concentration up to 4M. At a concentration of 8M urea, the hydrodynamic radii of completely unfolded erythroid and nonerythroid spectrin were 95±10 nm and 85±7 nm respectively, shown in Fig. 5. The hydrodynamic radii increased by the factor f = Rh unfold / Rh nat of 1.6 and 2.0 respectively for erythroid and nonerythroid spectrin in 6M GuHCl. Previous reports showed that the hydrodynamic stokes radius of lysozyme and RNase A increased by a factor 1.45 and 1.37 respectively in GuHCl induced denatured state [72, 73, 74]. The hydrodynamic radius (Rh ) measured for BSA in urea and GuHCl are shown in S6 Fig., used as control. The DLS profile of native BSA shows two peaks with hydrodynamic radii of 3.8±0.2 nm and 80 nm respectively, corresponding to the monomeric BSA and that of oligomeric BSA. On the other hand, the completely unfolded BSA showed a hydrodynamic radius of 8.5±0.5 nm in higher than 7M urea and 9.5±0.5 nm in higher than 3M GuHCl which is comparable to the previously obtained value [75].


Probing conformational stability and dynamics of erythroid and nonerythroid spectrin: effects of urea and guanidine hydrochloride.

Patra M, Mukhopadhyay C, Chakrabarti A - PLoS ONE (2015)

Compactness of erythroid and nonerythroid spectrin under different conditions.Histograms show size distribution of spectrin species obtained by DLS measurements of erythroid spectrin in (A) native; (B) unfolded in 8M urea and (C) unfolded in 6M GuHCl and of non-erythroid spectrin in (D) native; (E) unfolded in 8M urea and (F) unfolded in 6M GuHCl.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4305312&req=5

pone.0116991.g005: Compactness of erythroid and nonerythroid spectrin under different conditions.Histograms show size distribution of spectrin species obtained by DLS measurements of erythroid spectrin in (A) native; (B) unfolded in 8M urea and (C) unfolded in 6M GuHCl and of non-erythroid spectrin in (D) native; (E) unfolded in 8M urea and (F) unfolded in 6M GuHCl.
Mentions: DLS measurements were carried out to monitor the changes in size of the erythroid and nonerythroid spectrin in presence and absence of urea or GuHCl. The changes in the apparent hydrodynamic radii (Rh) of spectrin against increasing concentrations of urea and GuHCl, are shown in Fig. 4. Surprisingly, the apparent hydrodynamic radii of dimeric erythroid and tetrameric nonerythroid spectrin were estimated to be 60±10 nm and 40±5 nm respectively, which are in good agreement with Budzynski and co-workers [55, 56]. The hydrodynamic radius of both spectrin remained almost same up to 1M urea and beyond that the particle size significantly increased with denaturant concentration up to 4M. At a concentration of 8M urea, the hydrodynamic radii of completely unfolded erythroid and nonerythroid spectrin were 95±10 nm and 85±7 nm respectively, shown in Fig. 5. The hydrodynamic radii increased by the factor f = Rh unfold / Rh nat of 1.6 and 2.0 respectively for erythroid and nonerythroid spectrin in 6M GuHCl. Previous reports showed that the hydrodynamic stokes radius of lysozyme and RNase A increased by a factor 1.45 and 1.37 respectively in GuHCl induced denatured state [72, 73, 74]. The hydrodynamic radius (Rh ) measured for BSA in urea and GuHCl are shown in S6 Fig., used as control. The DLS profile of native BSA shows two peaks with hydrodynamic radii of 3.8±0.2 nm and 80 nm respectively, corresponding to the monomeric BSA and that of oligomeric BSA. On the other hand, the completely unfolded BSA showed a hydrodynamic radius of 8.5±0.5 nm in higher than 7M urea and 9.5±0.5 nm in higher than 3M GuHCl which is comparable to the previously obtained value [75].

Bottom Line: The folded erythroid spectrin and non-erythroid spectrin were directly converted to unfolded monomer without formation of any intermediate.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 (ΔGuH20) for the dimeric spectrin has been 20 kcal/mol lesser than the tetrameric from.

View Article: PubMed Central - PubMed

Affiliation: Chemistry Department, University of Calcutta, Kolkata, West Bengal, India.

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 (ΔGuH20) for the dimeric spectrin has been 20 kcal/mol lesser than the tetrameric from.

Show MeSH
Related in: MedlinePlus