Comparative thermal denaturation of Thermus aquaticus and Escherichia coli type 1 DNA polymerases

Abstract

Thermal denaturations of the type 1 DNA polymerases from Thermus aquaticus (Taq polymerase) and Escherichia coli (Pol 1) have been examined using differential scanning calorimetry and CD spectroscopy. The full-length proteins are single-polypeptide chains comprising a polymerase domain, a proofreading domain (inactive in Taq) and a 5' nuclease domain. Removal of the 5' nuclease domains produces the 'large fragment' domains of Pol 1 and Taq, termed Klenow and Klentaq respectively. Although the high temperature stability of Taq polymerase is well known, its thermal denaturation has never been directly examined previously. Thermal denaturations of both species of polymerase are irreversible, precluding rigorous thermodynamic analysis. However, the comparative melting behaviour of the polymerases yields information regarding domain structure, domain interactions and also the similarities and differences in the stabilizing forces for the two species of polymerase. In differential scanning calorimetry, Klenow and Klentaq denature as single peaks, with a melting temperature T(m) of 37 and 100 degrees C respectively at pH 9.5. Both full-length polymerases are found to be comprised of two thermodynamic unfolding domains with the 5' nuclease domains of each melting separately. The 5' nuclease domain of Taq denatures as a separate peak, 10 degrees C before the Klentaq domain. Melting of the 5' nuclease domain of Pol 1 overlaps with the Klenow fragment. Presence of the 5' nuclease domain stabilizes the large fragment in Pol 1, but destabilizes it in Taq. Both Klentaq and Klenow denaturations have a very similar dependence on pH and methanol, indicating similarities in the hydrophobic forces and protonation effects stabilizing the proteins. Melting monitored by CD yields slightly lower T(m) values, but almost identical van't Hoff enthalpy Delta H values, consistent with two-state unfolding followed by an irreversible kinetic step. Analysis of the denaturation scan rate dependences with Arrhenius formalism estimates a kinetic barrier to irreversible denaturation for Klentaq that is significantly higher than that for Klenow.

Kinetics of the DNA polymerase pyrococcus kodakaraensis

“…When using Taq DNA pol, the PCR yield roughly triples for every 10 °C increase until it reaches an optimum temperature of 72-75 °C (Innis et al, 1988). In retrospect, this is a curious observation since it has recently been shown that this enzyme unfolds only when the temperature reaches 95 °C (Karantzeni et al, 2003). This enzyme's long half-life at the PCR melting temperature is the consequence of its very high barrier to unfolding.…”

Minimizing loss of sequence information in SAGE ditags by modulating the temperature dependent 3′ → 5′ exonuclease activity of DNA polymerases on 3′‐terminal isoheptyl amino groups

“…In principal, fraying of the duplex ends should be more prevalent at this reaction temperature, which should increase the propensity for partitioning of the (À) strand into the exonuclease domain of KF. However, given that a population of KF would exist in a partially unfolded state at 508C (T m ¼ 458C at pH 7.5) (Karantzeni et al, 2003), selective heat inactivation of the exonuclease domain responsible for KF-mediated removal of the 3 0 -IHA may be occurring while the polymerase domain remains intact (Bailey et al, 2004).…”

Structure of a Nudix hydrolase (MutT) in the Mg2+-bound state fromBartonella henselae, the bacterium responsible for cat scratch fever

“…5(b), the melting profile was typical. A gradual increase in ellipticity at 220 nm was observed for Bh-MutT up to $323 K, followed by a more rapid increase in ellipticity that leveled off at $335 K. Visual inspection of the sample after heating to 353 K showed evidence of precipitation, indicating that the unfolding was irreversible, and consequently the CD data could not be analyzed thermodynamically (Karantzeni et al, 2003). A quantitative estimation of the T m for this transition was still possible by assuming a two-state model and taking a first derivative of the curve in Fig.…”

“…Usually, the ellipticity at a specific wavelength increases with increasing temperature as the protein unfolds and a melting temperature, T m , may be estimated for the transition between a structured and an unstructured state (Karantzeni et al, 2003). However, it has recently been observed that the thermal melt for the Nudix hydrolase DR_0079 from the bacterium Deinococcus radiodurans displays the opposite behavior, suggesting an increase in ordered structural communications structure with an increase in temperature (Buchko, 2010).…”