Effects of the difference in the unfolded-state ensemble on the folding of Escherichia coli dihydrofolate reductase.


Abstract

The unfolded state of a protein is an ensemble of a large number of conformations ranging from fully extended to compact structures. To investigate the effects of the difference in the unfolded-state ensemble on protein folding, we have studied the structure, stability, and folding of "circular" dihydrofolate reductase (DHFR) from Escherichia coli in which the N and C-terminal regions are cross-linked by a disulfide bond, and compared the results with those of disulfide-reduced "linear" DHFR. Equilibrium studies by circular dichroism, difference absorption spectra, solution X-ray scattering, and size-exclusion chromatography show that whereas the native structures of both proteins are essentially the same, the unfolded state of circular DHFR adopts more compact conformations than the unfolded state of the linear form, even with the absence of secondary structure. Circular DHFR is more stable than linear DHFR, which may be due to the decrease in the conformational entropy of the unfolded state as a result of circularization. Kinetic refolding measurements by stopped-flow circular dichroism and fluorescence show that under the native conditions both proteins accumulate a burst-phase intermediate having the same structures and both fold by the same complex folding mechanism with the same folding rates. Thus, the effects of the difference in the unfolded state of circular and linear DHFRs on the refolding reaction are not observed after the formation of the intermediate. This suggests that for the proteins with close termini in the native structure, early compaction of a protein molecule to form a specific folding intermediate with the N and C-terminal regions in close proximity is a crucial event in folding. If there is an enhancement in the folding reflecting the reduction in the breadth of the unfolded-state ensemble for circular DHFR, this acceleration must occur in the sub-millisecond time-range. Study holds ProTherm entries: 16275, 16276 Extra Details: (i) 0.2mM K2EDTA was added in the experiment,(ii) Circular DHFR, which is mutated amino acids, C85A/C152S and has extended amino acid residues at N and C termini with C-C bond between them. protein folding; dihydrofolate reductase; circular protein; folding intermediate;,unfolded state

Submission Details

ID: za8bCwon3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:47 p.m.

Version: 1

Publication Details
Arai M;Kataoka M;Kuwajima K;Matthews CR;Iwakura M,J. Mol. Biol. (2003) Effects of the difference in the unfolded-state ensemble on the folding of Escherichia coli dihydrofolate reductase. PMID:12787677
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
6MT8 2019-05-15 1.35 E. coli DHFR complex modeled with two ligand states
5UII 2018-01-31 1.35 structure of DHFR with bound buformin and NADP
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
3DAU 2009-04-14 1.5 Crystal structure of the ternary MTX NADPH complex of Escherichia coli dihydrofolate reductase
5CCC 2015-08-05 1.5 wild-type E.coli dihydrofolate reductase complexed with 5,10-dideazatetrahydrofolate and oxidized nicotinamide adenine dinucleotide phosphate
1RA9 1996-12-23 1.55 DIHYDROFOLATE REDUCTASE COMPLEXED WITH NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
4I13 2013-11-27 1.6 Nanobody ca1697 binding to the DHFR.folate binary complex
1RA2 1996-12-23 1.6 DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
5UIH 2017-11-22 1.65 structure of DHFR with bound phenformin and NADP
5EAJ 2016-09-21 1.7 Crystal structure of DHFR in 0% Isopropanol
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
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
1RA8 1997-03-12 1.8 DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE AND 2-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE
5Z6J 2018-09-19 1.8 High-pressure Crystal Structure Analysis of M20 loop closed DHFR at 220 MPa
5E8Q 2016-09-21 1.8 Crystal structure of DHFR in 20% Isopropanol
1RF7 1997-03-12 1.8 STRUCTURE OF DIHYDROFOLATE REDUCTASE COMPLEXED WITH DIHYDROFOLATE
5Z6K 2018-09-19 1.8 High-pressure Crystal Structure Analysis of M20 loop closed DHFR at 400 MPa
1DHJ 1994-01-31 1.8 LONG-RANGE STRUCTURAL EFFECTS IN A SECOND-SITE REVERTANT OF A MUTANT DIHYDROFOLATE REDUCTASE
5Z6F 2018-09-19 1.8 High-pressure Crystal Structure Analysis of DHFR(0.1 MPa)
3QL3 2011-04-27 1.8 Re-refined coordinates for PDB entry 1RX2
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)
1RA3 1996-12-23 1.8 DIHYDROFOLATE REDUCTASE COMPLEXED WITH METHOTREXATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
5UJX 2017-12-27 1.8 Crystal structure of DHFR in 20% Isopropanol
4X5I 2016-01-13 1.8 ecDHFR complexed with folate and NADP+ at 660 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
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
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
4X5H 2016-01-13 1.9 ecDHFR complexed with folate and NADP+ at 500 MPa
2DRC 1994-01-31 1.9 INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS
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
4X5G 2016-01-13 1.9 ecDHFR complexed with folate and NADP+ at 270 MPa
1JOM 1996-11-08 1.9 THE CRYSTAL STRUCTURE OF THE BINARY COMPLEX BETWEEN FOLINIC ACID (LEUCOVORIN) AND E. COLI DIHYDROFOLATE REDUCTASE
1RA1 1996-12-23 1.9 DIHYDROFOLATE REDUCTASE COMPLEXED WITH NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (REDUCED FORM)
1RX9 1997-04-01 1.9 DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
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
1RC4 1997-03-12 1.9 DIHYDROFOLATE REDUCTASE COMPLEXED WITH 5,10-DIDEAZATETRAHYDROFOLATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
5Z6L 2018-09-19 1.9 High-pressure Crystal Structure Analysis of M20 loop closed DHFR at 650 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
1DHI 1994-01-31 1.9 LONG-RANGE STRUCTURAL EFFECTS IN A SECOND-SITE REVERTANT OF A MUTANT DIHYDROFOLATE REDUCTASE
1DRA 1994-01-31 1.9 CRYSTAL STRUCTURE OF UNLIGANDED ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE. LIGAND-INDUCED CONFORMATIONAL CHANGES AND COOPERATIVITY IN BINDING
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
1RG7 1997-03-12 2.0 DIHYDROFOLATE REDUCTASE COMPLEXED WITH METHOTREXATE
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)
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.
1RX4 1997-01-11 2.2 DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH 5,10-DIDEAZATETRAHYDROFOLATE AND 2'-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE
1DDS 1995-10-15 2.2 MOLECULE: DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH METHOTREXATE
5Z6M 2018-09-19 2.2 High-pressure Crystal Structure Analysis of M20 loop closed DHFR at 800 MPa
4EIZ 2013-04-24 2.2 Structure of Nb113 bound to apoDHFR
2INQ 2007-02-06 2.2 Neutron Crystal Structure of Escherichia coli Dihydrofolate Reductase Bound to the Anti-cancer drug, 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)
1RB3 1997-03-12 2.3 DIHYDROFOLATE REDUCTASE COMPLEXED WITH METHOTREXATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
5DFR 1990-07-15 2.3 CRYSTAL STRUCTURE OF UNLIGANDED ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE. LIGAND-INDUCED CONFORMATIONAL CHANGES AND COOPERATIVITY IN BINDING
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
1RX5 1997-01-11 2.3 DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH 5,10-DIDEAZATETRAHYDROFOLATE
1RX7 1997-03-12 2.3 STRUCTURE OF DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE
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
1RH3 1997-03-12 2.4 DIHYDROFOLATE REDUCTASE COMPLEXED WITH METHOTREXATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (REDUCED FORM)
1DDR 1995-10-15 2.45 MOLECULE: DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH METHOTREXATE AND UREA
4EIG 2013-04-24 2.5 CA1698 camel antibody fragment in complex with DHFR
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
1DRE 1997-03-12 2.6 DIHYDROFOLATE REDUCTASE COMPLEXED WITH METHOTREXATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
1RD7 1996-12-23 2.6 DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE
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