Characterization of structural changes in alpha/beta-type small, acid-soluble spore proteins (SASP) upon binding to DNA

Date of Completion

January 2000

Keywords

Biology, Molecular|Biology, Microbiology|Chemistry, Biochemistry

Degree

Ph.D.

Abstract

The α/β-type small, acid-soluble spore proteins (SASP) are a family of highly conserved, non-specific DNA binding proteins which are found at very high levels within the spores of Bacillus and Clostridium species. These proteins change the conformation and chemical reactivity of spore DNA, and are important factors in protecting spore DNA from UV radiation, heat, and peroxides. The α/β-type SASP are monomeric and essentially unstructured in the absence of double-stranded DNA, but acquire significant secondary structure upon binding to DNA. The α/β-type SASP were very susceptible to two types of covalent protein damage, asparagine deamidation and methionine oxidation, which typically occur in unstructured and solvent accessible regions of proteins, respectively. However, DNA bound (α/β-type SASP were much more resistant to these forms of protein damage in vitro and in vivo, indicating that DNA bound α/β-type SASP are structurally more compact and have reduced solvent accessibility compared to unbound α/β-type SASP. Circular dichroism (CD) spectroscopic measurements confirmed that unbound α/β-type SASP are unstructured, and indicate that α/β-type SASP acquire a significant amount of α-helical secondary structure (∼50% of amino acid residues) upon binding to DNA. CD based equilibrium binding studies under stoichiometric conditions determined a site size of 4 base pairs per α/β-type SASP monomer. Equilibrium binding studies carried out with SspCTyr under non-stoichiometric conditions [5 mM sodium phosphate (pH 7.5), 40 mM NaCl] determined apparent binding constants (Kω) ranging from 1.3 × 105 M−1 to >10 7 M−1 for several synthetic DNAs. DNA binding was mildly salt sensitive and increased salt concentrations decreased both the intrinsic binding constant (K) and the cooperativity factor (ω) of SspCTyr-DNA binding. The binding cooperativity appears to be due in part to short range protein-protein interactions that occur in the DNA bound state. These interactions are between the N-terminal portions of adjacent DNA bound α/β-type SASP, and involve a significant electrostatic component which contributes to complex stability. ^

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