The role of surface amino acid residues in protein folding and stability

Abstract

Colicin A is a plasmid encoded bacteriocin produced by Escherichia coli (E. coli) regarded as a potential antibiotic. Colicin A is unusual in that it requires acidic lipids for its activity and its pore-forming domain (ColA-P) forms an acidic molten globule state. Previously it was shown that surface aspartyl groups are essential for the stability of colicin A in solution. Learning the role of these surface amino acids in folding and stability of Colicin A pore-forming domain requires knowledge of the folded and unfolded states. To do this the aspartates were replaced by alanine, asparagine, glutamate and glutamine respectively. The folded and unfolded states of the protein were probed experimentally by near and far UV CD spectra and NMR 1H-15N HSQC-maps. The urea unfolding processes were studied and analysed by fluorescence emission spectra. This showed the unexpected result that this single domain protein unfolds in two distinct transitions, the first one being destabilized, and the second one being stabilized, by low pH. The thermal denaturation transitions of the protein were measured by differential scanning calorimetry (DSC) and CD spectroscopy at 209 nm and 295 nm. Unexpectedly the DSC data showed that the measured ΔHtr was always half the ΔHvt’H (van’t Hoff) value and possible reasons for this are discussed. The data show that the aspartate to alanine replacements destabilize the protein significantly and the asparagine mutations do not. ColA-P D216, D274 and D372 are N-cap residues in the full length protein and this may explain why protonated Asp destabilizes the protein at low pH. The glutamate and glutamine mutants stabilize the protein more than expected for true N-capping residues. The biochemical function of the surface aspartates were studied comparing the NMR 1H-15N HSQC-maps of the proteins. The NMR experiments show that the aspartate to alanine mutation destabilize the protein in the same way as the acidic environment.