Abstract
Protein aggregation plays an important role in biotechnology and also causes numerous diseases. Human carbonic anhydrase II is a suitable model protein for studying the mechanism of aggregation. We found that a molten globule state of the enzyme formed aggregates. The intermolecular interactions involved in aggregate formation were localized in a direct way by measuring excimer formation between each of 20 site-specific pyrene-labeled cysteine mutants. The contact area of the aggregated protein was very specific, and all sites included in the intermolecular interactions were located in the large beta-sheet of the protein, within a limited region between the central beta-strands 4 and 7. This substructure is very hydrophobic, which underlines the importance of hydrophobic interactions between specific beta-sheet containing regions in aggregate formation.
Highlights
Protein aggregation is highly important in biotechnology and biomedicine, as well as in studies in vitro focused on the mechanism of protein folding
Our folding studies have shown that Human carbonic anhydrase II (HCA II) forms aggregates during the refolding process and during incubation at elevated temperatures [13,14,15]; similar aggregation behavior has been reported for bovine carbonic anhydrase II [16, 17]
We found that the aggregation of HCA II is highly specific and involves a molten globule-like intermediate
Summary
Materials—N-(1-Pyrenemethyl)iodoacetamide was obtained from Molecular Probes. 1-(Pyrene)-maleimide was purchased from Sigma. Reagent grade GuHCl was obtained from Pierce and was treated as described previously [12], and the concentration was determined by refractive index [30].
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