Effect of pH on stability, conformation, and chaperone activity of erythroid & non-erythroid spectrin

- Both erythroid and non-erythroid spectrin form oligomeric assembly at pH ~ 4.0
- Biophysical studies showed differential oligomeric stability and structure
- Spectroscopic signatures indicate increased exposure of hydrophobic patches and burying of tryptophan residues in oligomeric core of these assemblies
- Functional implications of such structural changes are reflected in their chaperone-like activities

Spectrin, a major component of the eukaryotic membrane skeleton, has been shown to have chaperone like activity. Here we investigate the pH induced changes in the structure and stability of erythroid and brain spectrin by spectroscopic methods. We also correlate these changes with modulations of chaperone potential at different pH. We have followed the pH induced structural changes by circular dichroism spectroscopy and intrinsic tryptophan fluorescence. It is seen that lowering the pH from 9 has little effect on structure of the proteins till about pH 6. At pH 4, there is significant change of the secondary structure of the proteins, along with a 5 nm hypsochromic shift of the emission maxima. Below pH 4 the proteins undergo acid denaturation. Probing exposed hydrophobic patches on the proteins using protein-bound 8-anilinonaphthalene-1-sulfonate fluorescence demonstrates that there is higher solvent accessibility of hydrophobic surfaces in both forms of spectrin at around pH 4. Dynamic light scattering and 90° light scattering studies show that the both forms of spectrin forms oligomers at pH ~ 4. Chemical unfolding data shows that these oligomers are less stable than the tetrameric form. Aggregation studies with BSA show that at pH 4, both spectrins exhibit better chaperone activity. This enhancement of chaperone like activity appears to result from an increase in regions of solvent-exposed hydrophobicity and oligomeric state of the spectrins which in turn are induced by moderately acid pH. This may have in-vivo implications in cells facing stress conditions where cytoplasmic pH is lowered.

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