Structural Analysis of Heptameric Alpha-Hemolysin under Extreme Conditions that Facilitate Nucleic Acid Translocation

Abstract

Protein nanopores are under intense investigation as sensors of various analytes, particularly for the rapid analysis of genomic material. In several important applications, notably ultrarapid sequencing, nucleic acids must be analyzed in unfolded single-stranded form. Therefore, conditions were examined that cause the denaturation of double stranded DNA (dsDNA), and single stranded DNA (ssDNA) and RNA with secondary structure. The behavior of the heptameric α-hemolysin (αHL) pore was investigated under alkaline pH conditions (8.0-13.0) and in varying concentrations of urea (0-8 M). The structural stability of the pore was examined by SDS-PAGE, intrinsic tryptophan emission fluorescence (ITFE) spectroscopy and circular dichroism (CD) spectroscopy. The pH studies revealed that the heptamer is resistant to structural change at up to pH 12.0. Electrical recordings revealed that DNA is capable of translocating through the pore at pH 11.7, a value at which its secondary structure is lost [1]. Studies conducted at various urea concentrations revealed that a substantial fraction of the heptamer remains stable in 8 M urea. At urea concentrations above 4.0 M, the secondary structure of single stranded DNA/RNA is denatured [2], permitting translocation.[1] Zimmer C. Alkaline denaturation of DNA's from various sources. Biochim Biophys Acta.161 (2), 584-586 (1968).[2] Levine, L., J. A. Gordon, and W. P. Jencks. The relationship of structure to the effectiveness of denaturing agents for deoxyribonucleic acid. Biochemistry 2, 168-175 (1963).