Transient intermediates in the folding of dihydrofolate reductase as detected by far-ultraviolet circular dichroism spectroscopy.


Abstract

The kinetics of the reversible folding and unfolding of Escherichia coli dihydrofolate reductase have been studied by stopped-flow circular dichroism in the peptide region at pH 7.8 and 15 degrees C. The reactions were induced by concentration jumps of a denaturant, urea. The method can detect various intermediates transiently populated in the reactions although the equilibrium unfolding of the protein is apparently approximated by a two-state reaction. The results can be summarized as follows. (1) From transient circular dichroism spectra measured as soon as the refolding is started, a substantial amount of secondary structure is formed in the burst phase, i.e., within the dead time of stopped-flow mixing (18 ms). (2) The kinetics from this burst-phase intermediate to the native state are multiphasic, consisting of five phases designated as tau 1, tau 2, tau 3, tau 4, and tau 5 in increasing order of the reaction rate. Measurements of the kinetics at various wavelengths have provided kinetic difference circular dichroism spectra for the individual phases. (3) The tau 5 phase shows a kinetic difference spectrum consistent with an exciton contribution of two aromatic residues in the peptide CD region. The absence of the tau 5 phase in a mutant protein, in which Trp 74 is replaced by leucine, suggests that Trp 74 is involved in the exciton pair and that the tau 5 phase reflects the formation of a hydrophobic cluster around Trp 74. From the similarity of the kinetic difference spectrum to the difference between the native spectra of the mutant and wild-type proteins, it appears that Trp 47 is the partner in the exciton pair and that the structure formed in the tau 5 phase persists during the later stages of folding. (4) The later stages of folding show kinetic difference spectra that can be interpreted by rearrangement of secondary structure, particularly the central beta sheet of the protein. The pairwise similarities in the spectrum between the tau 3 and tau 4 phases, and between the tau 1 and tau 2 phases, also suggest the presence of two parallel folding channels for refolding. (5) The unfolding kinetics show three to four phases and are interpreted in terms of the presence of multiple native species. The total ellipticity change in kinetic unfolding reaction, however, agrees with the ellipticity difference between the native and unfolding states, indicating the absence of the burst phase in unfolding.(ABSTRACT TRUNCATED AT 400 WORDS) Study holds ProTherm entries: 4002 Extra Details: two-state reaction; burst phase; aromatic residues; exciton pair;,secondary structure; hydrophobic cluster; folding channels

Submission Details

ID: p2DYnE29

Submitter: Connie Wang

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

Version: 1

Publication Details
Kuwajima K;Garvey EP;Finn BE;Matthews CR;Sugai S,Biochemistry (1991) Transient intermediates in the folding of dihydrofolate reductase as detected by far-ultraviolet circular dichroism spectroscopy. PMID:1868049
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
5UII 2018-01-31 1.35 structure of DHFR with bound buformin and NADP
4NX6 2014-01-15 1.35 single room temperature model of DHFR
4KJK 2013-08-21 1.35 Room Temperature WT DHFR
6MTH 2019-05-15 1.35 E. coli DHFR complex modeled with three ligand states
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
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)
6RUM 2020-02-12 1.6 Crystal structure of GFP-LAMA-G97 - a GFP enhancer nanobody with cpDHFR insertion and TMP and NADPH
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
4X5F 2016-01-13 1.7 ecDHFR complexed with folate and NADP+ at 0.1 MPa
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
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
3QL3 2011-04-27 1.8 Re-refined coordinates for PDB entry 1RX2
5Z6J 2018-09-19 1.8 High-pressure Crystal Structure Analysis of M20 loop closed DHFR at 220 MPa
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
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
5Z6F 2018-09-19 1.8 High-pressure Crystal Structure Analysis of DHFR(0.1 MPa)
1DHJ 1994-01-31 1.8 LONG-RANGE STRUCTURAL EFFECTS IN A SECOND-SITE REVERTANT OF A MUTANT DIHYDROFOLATE REDUCTASE
4X5I 2016-01-13 1.8 ecDHFR complexed with folate and NADP+ at 660 MPa
1RA3 1996-12-23 1.8 DIHYDROFOLATE REDUCTASE COMPLEXED WITH METHOTREXATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
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)
1RA8 1997-03-12 1.8 DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE AND 2-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE
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
5UIP 2017-11-22 1.9 structure of DHFR with bound DAP, p-ABG and NADP
1RX9 1997-04-01 1.9 DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH 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
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)
2DRC 1994-01-31 1.9 INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS
2D0K 2006-02-28 1.9 Methionine-free mutant of Escherichia coli dihydrofolate reductase
1JOM 1996-11-08 1.9 THE CRYSTAL STRUCTURE OF THE BINARY COMPLEX BETWEEN FOLINIC ACID (LEUCOVORIN) AND E. COLI DIHYDROFOLATE REDUCTASE
1DHI 1994-01-31 1.9 LONG-RANGE STRUCTURAL EFFECTS IN A SECOND-SITE REVERTANT OF A MUTANT DIHYDROFOLATE REDUCTASE
1RA1 1996-12-23 1.9 DIHYDROFOLATE REDUCTASE COMPLEXED WITH NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (REDUCED FORM)
3DRC 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
5Z6L 2018-09-19 1.9 High-pressure Crystal Structure Analysis of M20 loop closed DHFR at 650 MPa
4X5H 2016-01-13 1.9 ecDHFR complexed with folate and NADP+ at 500 MPa
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
1DRB 1994-01-31 1.96 CRYSTAL STRUCTURE OF UNLIGANDED ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE. LIGAND-INDUCED CONFORMATIONAL CHANGES AND COOPERATIVITY IN BINDING
1JOL 1996-11-08 1.96 THE CRYSTAL STRUCTURE OF THE BINARY COMPLEX BETWEEN FOLINIC ACID (LEUCOVORIN) AND E. COLI DIHYDROFOLATE REDUCTASE
1RX1 1997-01-11 2.0 DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (REDUCED FORM)
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
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
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)
1RX4 1997-01-11 2.2 DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH 5,10-DIDEAZATETRAHYDROFOLATE AND 2'-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE
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
6RUL 2020-02-12 2.2 Crystal structure of GFP-LAMA-F98 - a GFP enhancer nanobody with cpDHFR insertion and TMP and NADPH
1DDS 1995-10-15 2.2 MOLECULE: DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH METHOTREXATE
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
1RB3 1997-03-12 2.3 DIHYDROFOLATE REDUCTASE COMPLEXED WITH METHOTREXATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM)
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
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
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
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
4FHB 2013-04-24 2.8 Enhancing DHFR catalysis by binding of an allosteric regulator nanobody (Nb179)
1RX8 1997-03-12 2.8 DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE AND 2'-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE

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