Journal
BIOPOLYMERS
Volume 97, Issue 12, Pages 933-949Publisher
WILEY
DOI: 10.1002/bip.22110
Keywords
RNAse A; a-LA; refolding; folding intermediates; hydrophobic exposure; aggregates
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In this study, we extensively report the effect of glycine betaine during the refolding of partially folded bovine a-lactalbumin (a-LA) in presence of hexadecyl trimethyl ammonium bromide (HTAB), and Ribonuclease A (RNAse A) in presence of sodium dodecyl sulfate (SDS) by different complementary biophysical, light scattering, and microscopic techniques. Though a substantial refolding/compaction was observed in both the studied proteins, the fluorescence studies contradicted the finding obtained from circular dichroism spectroscopy (CD) in case of a-LA. CD stopped flow showed extensive presence of intermediates during the refolding of proteins which could potentially lead to aggregation. The aggregates as observed in dynamic light scattering (DLS), in a-LA were massive as compared to RNAse A and was directly proportional to betaine concentration. The zeta potential confirmed that the aggregates are a direct manifestation of strong aggregating and/or immense preferential excluding tendency of GB and not because of charge neutralization; however a possible role of conformational change as observed in FTIR spectroscopy cannot be completely ruled out. In contrary though RNAse A showed a substantial refolding, the final state of the folded protein was significantly different from the native state. These findings for a-LA and RNAse A were further supported by electron microscopic and thermodynamic studies. We thus propose that betaine has a strong macromolecular excluding tendency, primarily directed to shield the hydrophobic exposure either by refolding or aggregation, and depending on the hydrophobicity of the proteins, the functional restoration of the protein is manifested. (c) 2012 Wiley Periodicals, Inc. Biopolymers 97:933949, 2012.
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