| Summary: | <p>Alpha-hemolysin (αHL) is a membrane protein derived from Staphylococcus aureus. The structural stability of the monomeric and heptameric protein was investigated under high pH conditions (8.0−13.0) and in various concentrations of urea (0−8.0 M). The dimensions of the αHL heptameric pore are ideal for the translocation of single-stranded polynucleotides such as DNA or RNA. Therefore, it could potentially be used as a DNA sequencing device. Exploring the pore’s ability to withstand harsh conditions would help determine whether it could be used in the translocation of double stranded DNA (dsDNA), which dissociates into single-stranded DNA (ssDNA) at high pH or ssDNA that loses its secondary structure in the presence of urea. Studying the behavior of monomeric αHL under various denaturing and renaturing conditions will aid in improving the conditions used to purify and refold mutated forms of αHL.</p><p>The pH and urea studies revealed that the heptamer is more resistant to structural change than the monomer. The heptamer remained stable up to pH 12.0, which is ideal since dsDNA dissociates into ssDNA at pH 11.7. The majority of the heptamer’s structure was lost at pH conditions above 12.5. Structural studies on the heptamer at various urea concentrations revealed that a fraction of the heptamer was still intact even at 8.0 M urea whereas the monomer seems to unfold completely at high urea concentrations. However, upon refolding via dilution, the monomer seems to regain its structure and function. Urea was determined as an ideal denaturant even after incubating the monomer in guanidinium chloride (GdnCl) or organic acids.</p><p>The structural stability of the αHL monomer and heptamer was elucidated using techniques such as circular dichroism, intrinsic tryptophan emission spectroscopy, SDS-PAGE, light scattering hemolytic assays and single-channel recording experiments.</p>
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