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
6FBC 2018-09-26 1.54 KlenTaq DNA polymerase processing a modified primer - bearing the modification at the 3'-terminus of the primer.
4BWJ 2013-09-18 1.55 KlenTaq mutant in complex with DNA and ddCTP
4C8M 2013-12-11 1.57 Binary complex of the large fragment of DNA polymerase I from Thermus Aquaticus with the aritificial base pair d5SICS-dNaM at the postinsertion site (sequence context 2)
6FBE 2018-09-26 1.59 KlenTaq DNA polymerase processing a modified primer - bearing the modification upstream at the third primer nucleotide.
4DFK 2012-05-16 1.65 large fragment of DNA Polymerase I from Thermus aquaticus in a closed ternary complex with 5-(N-(10-hydroxydecanoyl)-aminopentinyl)-2-dUTP
4N5S 2015-01-21 1.67 Ternary complex structure of Klenow fragment of Taq DNA polymerase I707L mutant (Cs3C KlenTaq) with DNA and ddCTP
6FBG 2018-09-26 1.7 KlenTaq DNA polymerase processing a modified primer - bearing the modification upstream at the fifth primer nucleotide.
5NKL 2017-06-28 1.7 Crystal structure of the large fragment of DNA polymerase I from Thermus Aquaticus in a closed ternary complex with the artificial base pair dDs-dPxTP
4C8L 2013-12-11 1.7 Binary complex of the large fragment of DNA polymerase I from Thermus Aquaticus with the artificial base pair dNaM-d5SICS at the postinsertion site (sequence context 1)
3PY8 2011-06-15 1.74 Crystal structure of a mutant of the large fragment of DNA polymerase I from thermus aquaticus in a closed ternary complex with DNA and ddCTP
4BWM 2013-09-18 1.75 KlenTaq mutant in complex with a RNA/DNA hybrid
4C8O 2013-12-11 1.75 Binary complex of the large fragment of DNA polymerase I from Thermus Aquaticus with the aritificial base pair dNaM-d5SICS at the postinsertion site (sequence context 2)
4ELU 2013-03-27 1.8 Snapshot of the large fragment of DNA polymerase I from Thermus Aquaticus processing modified pyrimidines
6FBH 2018-09-26 1.8 KlenTaq DNA polymerase processing a modified primer - bearing the modification upstream at the sixth primer nucleotide.
5SZT 2016-12-14 1.8 Crystal structure of the large fragment of DNA Polymerase I from Thermus aquaticus in a closed ternary complex with 7-(N-(10-hydroxydecanoyl)-aminopentenyl)-7-deaza-2'-dATP
5O7T 2017-11-29 1.8 Crystal structure of KlenTaq mutant M747K in a closed ternary complex with a dG:dCTP base pair
5E41 2016-12-07 1.8 Crystal structure of the large fragment of DNA Polymerase I from Thermus aquaticus in a closed ternary complex with 5-(N-(10-hydroxydecanoyl)-aminopentenyl)-2'-deoxyuridine-triphosphate
3RRH 2012-02-15 1.8 Ternary Structure of the large fragment of Taq DNA polymerase bound to an abasic site and a ddTTP
3PO4 2011-06-15 1.8 Structure of a mutant of the large fragment of DNA polymerase I from Thermus aquaticus in complex with a blunt-ended DNA and ddATP
4C8N 2013-12-11 1.88 Binary complex of the large fragment of DNA polymerase I from Thermus Aquaticus with the aritificial base pair dNaM-d5SICS at the postinsertion site (sequence context 3)
4DLG 2012-11-28 1.89 Ternary Structure of the large Fragment of Taq DNA polymerase
4DFM 2012-05-16 1.89 Crystal structure of the large fragment of DNA polymerase I from Thermus aquaticus in ternary complex with 5-(aminopentinyl)-2-dCTP
4DFJ 2012-05-16 1.9 Crystal structure of the large fragment of DNA Polymerase I from Thermus aquaticus in a closed ternary complex with 5-(aminopentinyl)-dTTP
3OJS 2010-12-15 1.9 Snapshots of the large fragment of DNA polymerase I from Thermus Aquaticus processing C5 modified thymidines
3T3F 2012-02-15 1.9 Ternary Structure of the large fragment of Taq DNA polymerase bound to an abasic site and dNITP
4ELV 2013-04-24 1.9 Snapshot of the large fragment of DNA polymerase I from Thermus Aquaticus processing modified pyrimidines
3RTV 2012-05-30 1.9 Crystal structure of the large fragment of DNA polymerase I from Thermus Aquaticus in a closed ternary complex with natural primer/template DNA
6FBI 2018-09-26 1.9 KlenTaq DNA polymerase in a closed, ternary complex with dGpNHpp bound in the active site
5Z3N 2018-11-21 1.91 Structure of large fragment of DNA Polymerase I from Thermus aquaticus Host-Guest complex with the unnatural base 5fC pair with dA
3RR7 2012-02-15 1.95 Binary Structure of the large fragment of Taq DNA polymerase bound to an abasic site
3SYZ 2012-06-06 1.95 Crystal structure of the large fragment of DNA polymerase I from Thermus Aquaticus in an open binary complex with dNaM as templating nucleobase
5YTF 2018-11-21 1.98 Structure of large fragment of DNA Polymerase I from Thermus aquaticus Host-Guest complex with the unnatural base M-fC pair with dA
3SV4 2012-06-06 1.99 Crystal structure of the large fragment of DNA polymerase I from Thermus Aquaticus in an open binary complex with dT as templating nucleobase
3M8R 2010-07-07 2.0 Crystal structure of the large fragment of DNA polymerase I from Thermus aquaticus in a closed ternary complex with trapped 4'-ethylated dTTP
5OXJ 2017-11-29 2.0 Crystal structure of KlenTaq mutant M747K in a closed ternary complex with a O6-MeG:BenziTP base pair
4DFP 2012-05-16 2.0 Crystal structure of the large fragment of DNA Polymerase I from Thermus aqauticus in a ternary complex with 7-(aminopentinyl)-7-deaza-dGTP
3OJU 2010-12-15 2.0 Snapshot of the large fragment of DNA polymerase I from Thermus Aquaticus processing c5 modified thymidies
6FBF 2018-09-26 2.0 KlenTaq DNA polymerase processing a modified primer - bearing the modification upstream at the fourth primer nucleotide.
5YTD 2018-11-21 2.0 large fragment of DNA Polymerase I from Thermus aquaticus in a closed ternary complex with the natural base pair 5fC:dGTP
4DF8 2012-05-16 2.0 Crystal structure of the large fragment of DNA Polymerase I from Thermus aquaticus in a closed ternary complex with aminopentinyl-7-deaza-2-dATP
6Q4V 2019-02-27 2.01 KlenTaq DNA polymerase in complex with dATP
6Q4U 2019-02-27 2.01 KlenTaq DNA pol in a closed ternary complex with 7-deaza-7-(2-(2-hydroxyethoxy)-N-(prop-2-yn-1-yl)acetamide)-2-dATP
2KFZ 1998-11-11 2.03 KLENOW FRAGMENT WITH BRIDGING-SULFUR SUBSTRATE AND ZINC ONLY
2KFN 1998-11-11 2.03 KLENOW FRAGMENT WITH BRIDGING-SULFUR SUBSTRATE AND MANGANESE
5YTG 2018-11-21 2.07 Structure of large fragment of DNA Polymerase I from Thermus aquaticus Host-Guest complex with the unnatural base I-fC pair with dA
1D8Y 1999-12-02 2.08 CRYSTAL STRUCTURE OF THE COMPLEX OF DNA POLYMERASE I KLENOW FRAGMENT WITH DNA
6FBD 2018-09-26 2.1 KlenTaq DNA polymerase processing a modified primer - bearing the modification upstream at the second primer nucleotide.
1KFS 1998-02-25 2.1 DNA POLYMERASE I KLENOW FRAGMENT (E.C.2.7.7.7) MUTANT/DNA COMPLEX
3SV3 2012-06-06 2.1 Crystal structure of the large fragment of DNA polymerase I from Thermus Aquaticus in a closed ternary complex with the artificial base pair dNaM-d5SICSTP
3SZ2 2012-06-06 2.15 Crystal structure of the large fragment of DNA polymerase I from Thermus Aquaticus in an open binary complex with dG as templating nucleobase
4C8K 2013-12-11 2.17 Crystal structure of the large fragment of DNA polymerase I from Thermus Aquaticus in a partially closed complex with the artificial base pair d5SICS-dNaMTP
1D9D 1999-12-02 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'
3LWM 2010-05-05 2.19 Structure of the large fragment of thermus aquaticus DNA polymerase I in complex with a blunt-ended DNA and ddATP
3M8S 2010-07-07 2.2 Crystal structure of the large fragment of DNA polymerase I from Thermus aquaticus in a closed ternary complex with trapped 4'-methylated dTTP
4N56 2015-01-21 2.2 Binary complex structure of Klenow fragment of Taq DNA polymerase I707L mutant (Cs3C KlenTaq) with DNA
1QSL 1999-06-30 2.2 KLENOW FRAGMENT COMPLEXED WITH SINGLE-STRANDED SUBSTRATE AND EUROPIUM (III) ION
1KRP 1998-02-25 2.2 DNA polymerase I Klenow fragment (E.C.2.7.7.7) mutant/DNA complex
4DF4 2012-05-16 2.2 Crystal structure of the large fragment of DNA Polymerase I from Thermus aquaticus in a closed ternary complex with 7-(N-(10-hydroxydecanoyl)-aminopentinyl)-7-deaza-2 -dATP
4ELT 2013-03-27 2.2 Snapshot of the large fragment of DNA polymerase I from Thermus Aquaticus processing modified pyrimidines
5YTE 2018-11-21 2.21 Large fragment of DNA Polymerase I from Thermus aquaticus in a closed ternary complex with with natural dT:dATP base pair
3LWL 2010-05-05 2.25 Structure of Klenow fragment of Taq polymerase in complex with an abasic site
2KZZ 1999-12-14 2.25 KLENOW FRAGMENT WITH NORMAL SUBSTRATE AND ZINC ONLY
5YTC 2018-11-21 2.28 Large fragment of DNA Polymerase I from Thermus aquaticus in a closed ternary complex with the unnatural base M-fC pair with dATP in the active site
3KTQ 1999-01-13 2.3 CRYSTAL STRUCTURE OF AN ACTIVE TERNARY COMPLEX OF THE LARGE FRAGMENT OF DNA POLYMERASE I FROM THERMUS AQUATICUS
1BGX 1998-10-14 2.3 TAQ POLYMERASE IN COMPLEX WITH TP7, AN INHIBITORY FAB
3RRG 2012-02-15 2.3 Ternary Structure of the large fragment of Taq DNA polymerase bound to an abasic site and a ddGTP
1QTM 1999-08-16 2.3 DDTTP-TRAPPED CLOSED TERNARY COMPLEX OF THE LARGE FRAGMENT OF DNA POLYMERASE I FROM THERMUS AQUATICUS
1QSS 1999-08-16 2.3 DDGTP-TRAPPED CLOSED TERNARY COMPLEX OF THE LARGE FRAGMENT OF DNA POLYMERASE I FROM THERMUS AQUATICUS
2KTQ 1999-01-13 2.3 OPEN TERNARY COMPLEX OF THE LARGE FRAGMENT OF DNA POLYMERASE I FROM THERMUS AQUATICUS
1KSP 1998-02-25 2.3 DNA polymerase I Klenow fragment (E.C.2.7.7.7) mutant/DNA complex
1QSY 1999-08-16 2.3 DDATP-Trapped closed ternary complex of the large fragment of DNA Polymerase I from thermus aquaticus
5W6K 2018-07-18 2.34 Structure of mutant Taq Polymerase incorporating unnatural base pairs Z:P
3PO5 2011-06-15 2.39 Structure of a mutant of the large fragment of DNA polymerase I from Thermus Auqaticus in complex with an abasic site and ddATP
3RR8 2012-02-15 2.4 Ternary Structure of the large fragment of Taq DNA polymerase bound to an abasic site and a ddGTP
1TAQ 1996-12-07 2.4 STRUCTURE OF TAQ DNA POLYMERASE
4DLE 2012-11-28 2.44 Ternary Structure of the large Fragment of Taq DNA Polymerase: 4-Fluoroproline Variant
4XIU 2015-01-21 2.5 Binary complex structure of Klenow fragment of Taq DNA polymerase I707L mutant with DNA containing TTT overhang
4KTQ 1999-01-13 2.5 BINARY COMPLEX OF THE LARGE FRAGMENT OF DNA POLYMERASE I FROM T. AQUATICUS BOUND TO A PRIMER/TEMPLATE DNA
1KTQ 1996-11-08 2.5 DNA POLYMERASE
5KTQ 1998-09-30 2.5 LARGE FRAGMENT OF TAQ DNA POLYMERASE BOUND TO DCTP
5YTH 2018-11-21 2.53 Structure of large fragment of DNA Polymerase I from Thermus aquaticus Host-Guest complex with the unnatural base M-fC pair with dG
4CCH 2013-12-11 2.55 Crystal structure of the large fragment of DNA polymerase I from Thermus Aquaticus in an open binary complex with d5SICS as templating nucleotide
2KZM 1999-02-16 2.6 KLENOW FRAGMENT WITH NORMAL SUBSTRATE AND ZINC AND MANGANESE
5W6Q 2018-07-18 2.66 Structural basis for recognition of artificial DNA by an evolved KlenTaq variant
1DPI 1987-10-16 2.8 STRUCTURE OF LARGE FRAGMENT OF ESCHERICHIA COLI DNA POLYMERASE I COMPLEXED WITH D/TMP
1TAU 1997-04-18 3.0 TAQ POLYMERASE (E.C.2.7.7.7)/DNA/B-OCTYLGLUCOSIDE COMPLEX
1D9F 1999-12-02 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'
1KLN 1994-11-30 3.2 DNA POLYMERASE I KLENOW FRAGMENT (E.C.2.7.7.7) MUTANT/DNA COMPLEX
1KFD 1994-06-22 3.9 CRYSTAL STRUCTURES OF THE KLENOW FRAGMENT OF DNA POLYMERASE I COMPLEXED WITH DEOXYNUCLEOSIDE TRIPHOSPHATE AND PYROPHOSPHATE

Relevant UniProtKB Entries

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