Multistate equilibrium unfolding of Escherichia coli dihydrofolate reductase: thermodynamic and spectroscopic description of the native, intermediate, and unfolded ensembles.


Abstract

The thermodynamic and spectroscopic properties of a cysteine-free variant of Escherichia coli dihydrofolate reductase (AS-DHFR) were investigated using the combined effects of urea and temperature as denaturing agents. Circular dichroism (CD), absorption, and fluorescence spectra were recorded during temperature-induced unfolding at different urea concentrations and during urea-induced unfolding at different temperatures. The first three vectors obtained by singular-value decomposition of each set of unfolding spectra were incorporated into a global analysis of a unique thermodynamic model. Although individual unfolding profiles can be described as a two-state process, a simultaneous fit of 99 vectors requires a three-state model as the minimal scheme to describe the unfolding reaction along both perturbation axes. The model, which involves native (N), intermediate (I), and unfolded (U) states, predicts a maximum apparent stability, DeltaG degrees (NU), of 6 kcal mol(-)(1) at 15 degrees C, an apparent m(NU) value of 2 kcal mol(-)(1) M(-)(1), and an apparent heat capacity change, DeltaC(p)()(-NU), of 2.5 kcal mol(-)(1) K(-)(1). The intermediate species has a maximum stability of approximately 2 kcal mol(-)(1) and a compactness closer to that of the native than to that of the unfolded state. The population of the intermediate is maximal ( approximately 70%) around 50 degrees C and falls below the limits of detection of > or =2 M urea or at temperatures of <35 or >65 degrees C. The fluorescence properties of the equilibrium intermediate resemble those of a transient intermediate detected during refolding from the urea-denatured state, suggesting that a tryptophan-containing hydrophobic cluster in the adenosine-binding domain plays a key role in both the equilibrium and kinetic reactions. The CD spectroscopic properties of the native state reveal the presence of two principal isoforms that differ in ligand binding affinities and in the packing of the adenosine-binding domain. The relative populations of these species change slightly with temperature and do not depend on the urea concentration, implying that the two native isoforms are well-structured and compact. Global analysis of data from multiple spectroscopic probes and several methods of unfolding is a powerful tool for revealing structural and thermodynamic properties of partially and fully folded forms of DHFR. Study holds ProTherm entries: 8559, 8560 Extra Details: additive : K2EDTA(0.2 mM),C85A/C152S cysteine-free double mutant of,dihydrofolate reductase. N-><-I

Submission Details

ID: kibdqBnn3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:36 p.m.

Version: 1

Publication Details
Ionescu RM;Smith VF;O'Neill JC;Matthews CR,Biochemistry (2000) Multistate equilibrium unfolding of Escherichia coli dihydrofolate reductase: thermodynamic and spectroscopic description of the native, intermediate, and unfolded ensembles. PMID:10924151
Additional Information

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
4PDJ 2015-04-15 Neutron crystal Structure of E.coli Dihydrofolate Reductase complexed with folate and NADP+
6CW7 2019-01-09 1.03 E. coli DHFR product complex with (6S)-5,6,7,8-TETRAHYDROFOLATE
6CXK 2019-01-09 1.11 E. coli DHFR substrate complex with Dihydrofolate
4NX7 2014-01-15 1.15 single cryogenic temperature model of DHFR
4KJJ 2013-08-21 1.15 Cryogenic WT DHFR
5CC9 2015-08-05 1.2 L28F E.coli dihydrofolate reductase complexed with 5,10-dideazatetrahydrofolate and oxidized nicotinamide adenine dinucleotide phosphate
6CYV 2019-01-09 1.3 E. coli DHFR ternary complex with NADP and dihydrofolate
4KJK 2013-08-21 1.35 Room Temperature WT DHFR
6MR9 2019-05-15 1.35 E. coli DHFR complex with a reaction intermediate
5UII 2018-01-31 1.35 structure of DHFR with bound buformin and NADP
6MT8 2019-05-15 1.35 E. coli DHFR complex modeled with two ligand states
6MTH 2019-05-15 1.35 E. coli DHFR complex modeled with three ligand states
4NX6 2014-01-15 1.35 single room temperature model of DHFR
4KJL 2013-08-21 1.38 Room Temperature N23PPS148A DHFR
5W3Q 2017-08-09 1.4 L28F E.coli DHFR in complex with NADPH
5CCC 2015-08-05 1.5 wild-type E.coli dihydrofolate reductase complexed with 5,10-dideazatetrahydrofolate and oxidized nicotinamide adenine dinucleotide phosphate
3DAU 2009-04-14 1.5 Crystal structure of the ternary MTX NADPH complex of Escherichia coli dihydrofolate reductase
1RA9 1996-12-23 1.55 DIHYDROFOLATE REDUCTASE COMPLEXED WITH NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
1RA2 1996-12-23 1.6 DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
4I13 2013-11-27 1.6 Nanobody ca1697 binding to the DHFR.folate binary complex
5UIH 2017-11-22 1.65 structure of DHFR with bound phenformin and NADP
4DFR 1982-10-21 1.7 CRYSTAL STRUCTURES OF ESCHERICHIA COLI AND LACTOBACILLUS CASEI DIHYDROFOLATE REDUCTASE REFINED AT 1.7 ANGSTROMS RESOLUTION. I. GENERAL FEATURES AND BINDING OF METHOTREXATE
5EAJ 2016-09-21 1.7 Crystal structure of DHFR in 0% Isopropanol
4X5F 2016-01-13 1.7 ecDHFR complexed with folate and NADP+ at 0.1 MPa
4EJ1 2013-04-24 1.75 Binding of Nb113 camelid antibody fragment with the binary DHFR:folate complex
3OCH 2010-12-08 1.79 Chemically Self-assembled Antibody Nanorings (CSANs): Design and Characterization of an Anti-CD3 IgM Biomimetic
3QYL 2012-01-18 1.79 Sensitivity of receptor internal motions to ligand binding affinity and kinetic off-rate
1DHJ 1994-01-31 1.8 LONG-RANGE STRUCTURAL EFFECTS IN A SECOND-SITE REVERTANT OF A MUTANT DIHYDROFOLATE REDUCTASE
5Z6J 2018-09-19 1.8 High-pressure Crystal Structure Analysis of M20 loop closed DHFR at 220 MPa
3QL3 2011-04-27 1.8 Re-refined coordinates for PDB entry 1RX2
1RF7 1997-03-12 1.8 STRUCTURE OF DIHYDROFOLATE REDUCTASE COMPLEXED WITH DIHYDROFOLATE
1RA8 1997-03-12 1.8 DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE AND 2-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE
5E8Q 2016-09-21 1.8 Crystal structure of DHFR in 20% Isopropanol
5UJX 2017-12-27 1.8 Crystal structure of DHFR in 20% Isopropanol
1RA3 1996-12-23 1.8 DIHYDROFOLATE REDUCTASE COMPLEXED WITH METHOTREXATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
5Z6F 2018-09-19 1.8 High-pressure Crystal Structure Analysis of DHFR(0.1 MPa)
5Z6K 2018-09-19 1.8 High-pressure Crystal Structure Analysis of M20 loop closed DHFR at 400 MPa
1RX2 1997-01-11 1.8 DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH WITH FOLATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
4X5I 2016-01-13 1.8 ecDHFR complexed with folate and NADP+ at 660 MPa
4GH8 2013-06-05 1.85 Crystal structure of a 'humanized' E. coli dihydrofolate reductase
4X5J 2016-01-13 1.85 ecDHFR complexed with folate and NADP+ at 750 MPa
1DYJ 1995-06-03 1.85 ISOMORPHOUS CRYSTAL STRUCTURES OF ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE, 5-DEAZAFOLATE AND 5,10-DIDEAZATETRAHYDROFOLATE: MECHANISTIC IMPLICATIONS
4I1N 2013-11-27 1.89 R104A-ca1697 nanobody binding to the binary DHFR.folate complex
2D0K 2006-02-28 1.9 Methionine-free mutant of Escherichia coli dihydrofolate reductase
5Z6L 2018-09-19 1.9 High-pressure Crystal Structure Analysis of M20 loop closed DHFR at 650 MPa
1RA1 1996-12-23 1.9 DIHYDROFOLATE REDUCTASE COMPLEXED WITH NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (REDUCED FORM)
2DRC 1994-01-31 1.9 INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS
4X5H 2016-01-13 1.9 ecDHFR complexed with folate and NADP+ at 500 MPa
3DRC 1994-01-31 1.9 INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS
5UIP 2017-11-22 1.9 structure of DHFR with bound DAP, p-ABG and NADP
1DYI 1995-06-03 1.9 ISOMORPHOUS CRYSTAL STRUCTURES OF ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE, 5-DEAZAFOLATE AND 5,10-DIDEAZATETRAHYDROFOLATE: MECHANISTIC IMPLICATIONS
1JOM 1996-11-08 1.9 THE CRYSTAL STRUCTURE OF THE BINARY COMPLEX BETWEEN FOLINIC ACID (LEUCOVORIN) AND E. COLI DIHYDROFOLATE REDUCTASE
4X5G 2016-01-13 1.9 ecDHFR complexed with folate and NADP+ at 270 MPa
1DYH 1995-06-03 1.9 ISOMORPHOUS CRYSTAL STRUCTURES OF ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE, 5-DEAZAFOLATE AND 5,10-DIDEAZATETRAHYDROFOLATE: MECHANISTIC IMPLICATIONS
1DHI 1994-01-31 1.9 LONG-RANGE STRUCTURAL EFFECTS IN A SECOND-SITE REVERTANT OF A MUTANT DIHYDROFOLATE REDUCTASE
1RC4 1997-03-12 1.9 DIHYDROFOLATE REDUCTASE COMPLEXED WITH 5,10-DIDEAZATETRAHYDROFOLATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
1DRA 1994-01-31 1.9 CRYSTAL STRUCTURE OF UNLIGANDED ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE. LIGAND-INDUCED CONFORMATIONAL CHANGES AND COOPERATIVITY IN BINDING
1RX9 1997-04-01 1.9 DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
5UIO 2017-11-22 1.93 structure of DHFR with bound DAP, p-ABG and NADP
3K74 2010-10-20 1.95 Disruption of protein dynamics by an allosteric effector antibody
1JOL 1996-11-08 1.96 THE CRYSTAL STRUCTURE OF THE BINARY COMPLEX BETWEEN FOLINIC ACID (LEUCOVORIN) AND E. COLI DIHYDROFOLATE REDUCTASE
1DRB 1994-01-31 1.96 CRYSTAL STRUCTURE OF UNLIGANDED ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE. LIGAND-INDUCED CONFORMATIONAL CHANGES AND COOPERATIVITY IN BINDING
1RX6 1997-01-11 2.0 DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH 5,10-DIDEAZATETRAHYDROFOLATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (REDUCED FORM)
1RG7 1997-03-12 2.0 DIHYDROFOLATE REDUCTASE COMPLEXED WITH METHOTREXATE
1RX1 1997-01-11 2.0 DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (REDUCED FORM)
3KFY 2010-12-08 2.08 Dynamic switching and partial occupancies of a small molecule inhibitor complex of DHFR
3QYO 2012-01-18 2.09 Sensitivity of receptor internal motions to ligand binding affinity and kinetic off-rate
3R33 2012-01-25 2.09 Evidence for dynamic motion in proteins as a mechanism for ligand dissociation
1RB2 1997-03-12 2.1 DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
2ANQ 2006-07-25 2.13 Crystal Structure of E.coli DHFR in complex with NADPH and the inhibitor compound 10a.
5Z6M 2018-09-19 2.2 High-pressure Crystal Structure Analysis of M20 loop closed DHFR at 800 MPa
1DDS 1995-10-15 2.2 MOLECULE: DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH METHOTREXATE
6CQA 2019-01-09 2.2 E. coli DHFR complex with inhibitor AMPQD
1RX3 1997-01-11 2.2 DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH METHOTREXATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (REDUCED FORM)
2INQ 2007-02-06 2.2 Neutron Crystal Structure of Escherichia coli Dihydrofolate Reductase Bound to the Anti-cancer drug, Methotrexate
4EIZ 2013-04-24 2.2 Structure of Nb113 bound to apoDHFR
1RX4 1997-01-11 2.2 DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH 5,10-DIDEAZATETRAHYDROFOLATE AND 2'-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE
1RX5 1997-01-11 2.3 DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH 5,10-DIDEAZATETRAHYDROFOLATE
1RB3 1997-03-12 2.3 DIHYDROFOLATE REDUCTASE COMPLEXED WITH METHOTREXATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
1RX7 1997-03-12 2.3 STRUCTURE OF DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE
1DRH 1995-02-07 2.3 ISOMORPHOUS CRYSTAL STRUCTURES OF ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE, 5-DEAZAFOLATE AND 5,10-DIDEAZATETRAHYDROFOLATE: MECHANISTIC IMPLICATIONS
5DFR 1990-07-15 2.3 CRYSTAL STRUCTURE OF UNLIGANDED ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE. LIGAND-INDUCED CONFORMATIONAL CHANGES AND COOPERATIVITY IN BINDING
1RH3 1997-03-12 2.4 DIHYDROFOLATE REDUCTASE COMPLEXED WITH METHOTREXATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (REDUCED FORM)
6DFR 1990-07-15 2.4 CRYSTAL STRUCTURES OF ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE. THE NADP+ HOLOENZYME AND THE FOLATE(DOT)NADP+ TERNARY COMPLEX. SUBSTRATE BINDING AND A MODEL FOR THE TRANSITION STATE
1DDR 1995-10-15 2.45 MOLECULE: DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH METHOTREXATE AND UREA
7DFR 1990-07-15 2.5 CRYSTAL STRUCTURES OF ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE. THE NADP+ HOLOENZYME AND THE FOLATE(DOT)NADP+ TERNARY COMPLEX. SUBSTRATE BINDING AND A MODEL FOR THE TRANSITION STATE
1TDR 1995-07-10 2.5 EXPRESSION, CHARACTERIZATION, AND CRYSTALLOGRAPHIC ANALYSIS OF TELLUROMETHIONYL DIHYDROFOLATE REDUCTASE
4EIG 2013-04-24 2.5 CA1698 camel antibody fragment in complex with DHFR
1RD7 1996-12-23 2.6 DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE
1DRE 1997-03-12 2.6 DIHYDROFOLATE REDUCTASE COMPLEXED WITH METHOTREXATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
1RE7 1996-12-23 2.6 DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE
2ANO 2006-07-25 2.68 Crystal structure of E.coli dihydrofolate reductase in complex with NADPH and the inhibitor MS-SH08-17
1RX8 1997-03-12 2.8 DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE AND 2'-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE
4FHB 2013-04-24 2.8 Enhancing DHFR catalysis by binding of an allosteric regulator nanobody (Nb179)

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
91.8 Dihydrofolate reductase P31074 DYR_KLEAE
96.2 Dihydrofolate reductase P31073 DYR_CITFR
100.0 Dihydrofolate reductase P0ABQ6 DYR_SHIFL
100.0 Dihydrofolate reductase P0ABQ4 DYR_ECOLI
100.0 Dihydrofolate reductase P0ABQ5 DYR_ECOL6