Why is Taq DNA Polymerase so Stable? A Reduced Entropic Folding Penalty and the Denatured State Ensemble

Abstract

The thermal stability of Taq DNA polymerase is well known, and is the basis for its use in PCR. A comparative thermodynamic characterization of the large fragment domains of Taq (Klentaq) and E. coli (Klenow) DNA polymerases reveals that Klentaq's extreme free energy of folding originates from a significantly decreased entropic folding penalty. A parallel solution-based structural analysis reveals that the denatured state of the mesophilic polymerase (Klenow) is significantly more elongated than the denatured state of the thermophilic Taq polymerase and thus has a significantly larger radius of gyration. The combined data indicate that the folded state of the thermophilic polymerase is not more intrinsically stable: instead, its more globular denatured state has a considerably reduced entropic and structural barrier between the denatured and native states, resulting in a much more favorable ΔG of folding. The data also indicate that the stability-linked evolutionary differences between the two proteins are expressed primarily in the denatured state. This is the first direct structural demonstration of denatured state size/shape differences in a mesophilic-thermophilic protein pair. Despite their demonstrably different sizes and structures, both denatured state ensembles still fall within the range of random-coil behavior.