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. Study holds ProTherm entries: 17071, 17072, 17073, 17074 Extra Details: chemical denaturation; thermophilic; Taq polymerase; protein unfolding; Klenow polymerase; sedimentation coefficient

Submission Details

ID: ZmafVnaM4

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:49 p.m.

Version: 1

Publication Details
Schoeffler AJ;Joubert AM;Peng F;Khan F;Liu CC;LiCata VJ,Proteins (2004) Extreme free energy of stabilization of Taq DNA polymerase. PMID:14997557
Additional Information

Study Summary

Number of data points 12
Proteins DNA polymerase I ; DNA polymerase I, thermostable ; DNA polymerase I ; DNA polymerase I, thermostable
Unique complexes 2
Assays/Quantities/Protocols Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O ; Experimental Assay: Cm ; Experimental Assay: m ; Experimental Assay: dG_H2O
Libraries Mutations for sequence SPKALEEAPWPPPEGAFVGFVLSRKEPMWADLLALAAARGGRVHRAPEPYKALRDLKEARGLLAKDLSVLALREGLGLPPGDDPMLLAYLLDPSNTTPEGVARRYGGEWTEEAGERAALSERLFANLWGRLEGEERLLWLYREVERPLSAVLAHMEATGVRLDVAYLRALSLEVAEEIARLEAEVFRLAGHPFNLNSRDQLERVLFDELGLPAIGKTEKTGKRSTSAAVLEALREAHPIVEKILQYRELTKLKSTYIDPLPDLIHPRTGRLHTRFNQTATATGRLSSSDPNLQNIPVRTPLGQRIRRAFIAEEGWLLVALDYSQIELRVLAHLSGDENLIRVFQEGRDIHTETASWMFGVPREAVDPLMRRAAKTINFGVLYGMSAHRLSQELAIPYEEAQAFIERYFQSFPKVRAWIEKTLEEGRRRGYVETLFGRRRYVPDLEARVKSVREAAERMAFNMPVQGTAADLMKLAMVKLFPRLEEMGARMLLQVHDELVLEAPKERAEAVARLAKEVMEGVYPLAVPLEVEVGIGEDWLSAKE ; Mutations for sequence VISYDNYVTILDEETLKAWIAKLEKAPVFAFDTETDSLDNISANLVGLSFAIEPGVAAYIPVAHDYLDAPDQISRERALELLKPLLEDEKALKVGQNLKYDRGILANYGIELRGIAFDTMLESYILNSVAGRHDMDSLAERWLKHKTITFEEIAGKGKNQLTFNQIALEEAGRYAAEDADVTLQLHLKMWPDLQKHKGPLNVFENIEMPLVPVLSRIERNGVKIDPKVLHNHSEELTLRLAELEKKAHEIAGEEFNLSSTKQLQTILFEKQGIKPLKKTPGGAPSTSEEVLEELALDYPLPKVILEYRGLAKLKSTYTDKLPLMINPKTGRVHTSYHQAVTATGRLSSTDPNLQNIPVRNEEGRRIRQAFIAPEDYVIVSADYSQIELRIMAHLSRDKGLLTAFAEGKDIHRATAAEVFGLPLETVTSEQRRSAKAINFGLIYGMSAFGLARQLNIPRKEAQKYMDLYFERYPGVLEYMERTRAQAKEQGYVETLDGRRLYLPDIKSSNGARRAAAERAAINAPMQGTAADIIKRAMIAVDAWLQAEQPRVRMIMQVHDELVFEVHKDDVDAVAKQIHQLMENCTRLDVPLLVEVGSGENWDQAH

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1D8Y 1999-10-26T00:00:00+0000 2.08 CRYSTAL STRUCTURE OF THE COMPLEX OF DNA POLYMERASE I KLENOW FRAGMENT WITH DNA
1D9D 1999-10-27T00:00:00+0000 2.18 CRYSTALL STRUCTURE OF THE COMPLEX OF DNA POLYMERASE I KLENOW FRAGMENT WITH SHORT DNA FRAGMENT CARRYING 2'-0-AMINOPROPYL-RNA MODIFICATIONS 5'-D(TCG)-AP(AUC)-3'
1D9F 1999-10-27T00:00:00+0000 3.0 CRYSTAL STRUCTURE OF THE COMPLEX OF DNA POLYMERASE I KLENOW FRAGMENT WITH DNA TETRAMER CARRYING 2'-O-(3-AMINOPROPYL)-RNA MODIFICATION 5'-D(TT)-AP(U)-D(T)-3'
1DPI 1987-08-11T00:00:00+0000 2.8 STRUCTURE OF LARGE FRAGMENT OF ESCHERICHIA COLI DNA POLYMERASE I COMPLEXED WITH D/TMP
1KFD 1993-09-23T00:00:00+0000 3.9 CRYSTAL STRUCTURES OF THE KLENOW FRAGMENT OF DNA POLYMERASE I COMPLEXED WITH DEOXYNUCLEOSIDE TRIPHOSPHATE AND PYROPHOSPHATE
1KFS 1997-08-18T00:00:00+0000 2.1 DNA POLYMERASE I KLENOW FRAGMENT (E.C.2.7.7.7) MUTANT/DNA COMPLEX
1KLN 1994-05-24T00:00:00+0000 3.2 DNA POLYMERASE I KLENOW FRAGMENT (E.C.2.7.7.7) MUTANT/DNA COMPLEX
1KRP 1997-08-19T00:00:00+0000 2.2 DNA polymerase I Klenow fragment (E.C.2.7.7.7) mutant/DNA complex
1KSP 1997-08-19T00:00:00+0000 2.3 DNA polymerase I Klenow fragment (E.C.2.7.7.7) mutant/DNA complex
1QSL 1999-06-22T00:00:00+0000 2.2 KLENOW FRAGMENT COMPLEXED WITH SINGLE-STRANDED SUBSTRATE AND EUROPIUM (III) ION

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
93.9 DNA polymerase I Q9F173 DPO1_SALTY
100.0 DNA polymerase I P00582 DPO1_ECOLI
100.0 DNA polymerase I, thermostable P19821 DPO1_THEAQ