Extreme free energy of stabilization of Taq DNA polymerase.

Abstract:

We have examined the chemical denaturations of the Klentaq and Klenow large-fragment domains of the Type 1 DNA polymerases from Thermus aquaticus (Klentaq) and Escherichia coli (Klenow) under identical solution conditions in order to directly compare the stabilization energetics of the two proteins. The high temperature stability of Taq DNA polymerase is common knowledge, and is the basis of its use in the polymerase chain reaction. This study, however, is aimed at understanding the thermodynamic basis for this high-temperature stability. Chemical denaturations with guanidine hydrochloride report a folding free energy (DeltaG) for Klentaq that is over 20 kcal/mol more favorable than that for Klenow under the conditions examined. This difference between the stabilization free energies of a homologous mesophilic-thermophilic protein pair is significantly larger than generally observed. This is due in part to the fact that the stabilization free energy for Klentaq polymerase, at 27.5 kcal/mol, is one of the largest ever determined for a monomeric protein. Large differences in the chemical midpoints of the unfolding (Cm) and the dependences of the unfolding free energy on denaturant concentration in the transition region (m-value) between the two proteins are also observed. Measurements of the sedimentation coefficients of the two proteins in the native and denatured states report that both proteins approximately double in hydrodynamic size upon denaturation, but that Klentaq expands somewhat more than Klenow.

Polymerases:

Topics:

Status:

new topics/pols set partial results complete validated

Results:

No results available for this paper.

Entry validated by:

Log in to edit reference All References

Using Polbase tables:

Sorting:

Tables may be sorted by clicking on any of the column titles. A second click reverses the sort order. <Ctrl> + click on the column titles to sort by more than one column (e.g. family then name).

Filtering:

It is also possible to filter the table by typing into the search box above the table. This will instantly hide lines from the table that do not contain your search text.