A hydrophobic cluster forms early in the folding of dihydrofolate reductase.
Abstract
The rapid kinetic phase that leads from unfolded species to transient folding intermediates in dihydrofolate reductase from Escherichia coli was examined by site-directed mutagenesis and by physicochemical means. The absence of this fluorescence-detected phase in the refolding of the Trp-74Phe mutant protein strongly implies that this early phase in refolding can be assigned to just one of the five Trp residues in the protein, Trp-74. In addition, water-soluble fluorescence quenching agents, iodide and cesium, have a much less significant effect on this early step in refolding than on the slower phases that lead to native and native-like conformers. These and other data imply that an important early event in the folding of dihydrofolate reductase is the formation of a hydrophobic cluster which protects Trp-74 from solvent. Study holds ProTherm entries: 10792, 10793 Extra Details: additive : 2-mercaptethanol(1 mM), protein folding; folding intermediates; hydrophobic effect;,tryptophan fluorescence; site-directed mutagenesis
Submission Details
ID: 6gpxRwNz
Submitter: Connie Wang
Submission Date: April 24, 2018, 8:40 p.m.
Version: 1
Publication Details
Garvey EP;Swank J;Matthews CR,Proteins (1989) A hydrophobic cluster forms early in the folding of dihydrofolate reductase. PMID:2622906Additional Information
Study Summary
| Number of data points | 3 |
| Proteins | Dihydrofolate reductase ; Dihydrofolate reductase |
| Unique complexes | 2 |
| Assays/Quantities/Protocols | Experimental Assay: ddG_H2O ; Experimental Assay: Cm |
| Libraries | Mutations for sequence MISLIAALAVDRVIGMENAMPWNLPADLAWFKRNTLDKPVIMGRHTWESIGRPLPGRKNIILSSQPGTDDRVTWVKSVDEAIAACGDVPEIMVIGGGRVYEQFLPKAQKLYLTHIDAEVEGDTHFPDYEPDDWESVFSEFHDADAQNSHSYCFEILERR |
Structure view and single mutant data analysis
Study data
| Colors: | D | E | R | H | K | S | T | N | Q | A | V | I | L | M | F | Y | W | C | G | P |
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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 |
| 6MT8 | 2019-05-15 | 1.35 | E. coli DHFR complex modeled with two ligand states |
| 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 |
| 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 |
| 6MR9 | 2019-05-15 | 1.35 | E. coli DHFR complex with a reaction intermediate |
| 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) |
| 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 |
| 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 |
| 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) |
| 5Z6K | 2018-09-19 | 1.8 | High-pressure Crystal Structure Analysis of M20 loop closed DHFR at 400 MPa |
| 1RF7 | 1997-03-12 | 1.8 | STRUCTURE OF DIHYDROFOLATE REDUCTASE COMPLEXED WITH DIHYDROFOLATE |
| 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) |
| 5Z6F | 2018-09-19 | 1.8 | High-pressure Crystal Structure Analysis of DHFR(0.1 MPa) |
| 1RA8 | 1997-03-12 | 1.8 | DIHYDROFOLATE REDUCTASE COMPLEXED WITH FOLATE AND 2-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE |
| 1DHJ | 1994-01-31 | 1.8 | LONG-RANGE STRUCTURAL EFFECTS IN A SECOND-SITE REVERTANT OF A MUTANT DIHYDROFOLATE REDUCTASE |
| 5E8Q | 2016-09-21 | 1.8 | Crystal structure of DHFR in 20% Isopropanol |
| 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 |
| 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 |
| 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 |
| 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 |
| 1DRA | 1994-01-31 | 1.9 | CRYSTAL STRUCTURE OF UNLIGANDED ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE. LIGAND-INDUCED CONFORMATIONAL CHANGES AND COOPERATIVITY IN BINDING |
| 2DRC | 1994-01-31 | 1.9 | INVESTIGATION OF THE FUNCTIONAL ROLE OF TRYPTOPHAN-22 IN ESCHERICHIA COLI DIHYDROFOLATE REDUCTASE BY SITE-DIRECTED MUTAGENESIS |
| 1JOM | 1996-11-08 | 1.9 | THE CRYSTAL STRUCTURE OF THE BINARY COMPLEX BETWEEN FOLINIC ACID (LEUCOVORIN) AND E. COLI DIHYDROFOLATE REDUCTASE |
| 1RX9 | 1997-04-01 | 1.9 | DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM) |
| 1RC4 | 1997-03-12 | 1.9 | DIHYDROFOLATE REDUCTASE COMPLEXED WITH 5,10-DIDEAZATETRAHYDROFOLATE AND NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (OXIDIZED FORM) |
| 1DHI | 1994-01-31 | 1.9 | LONG-RANGE STRUCTURAL EFFECTS IN A SECOND-SITE REVERTANT OF A MUTANT DIHYDROFOLATE REDUCTASE |
| 5UIP | 2017-11-22 | 1.9 | structure of DHFR with bound DAP, p-ABG and NADP |
| 1RA1 | 1996-12-23 | 1.9 | DIHYDROFOLATE REDUCTASE COMPLEXED WITH NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (REDUCED FORM) |
| 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 |
| 2D0K | 2006-02-28 | 1.9 | Methionine-free mutant of Escherichia coli dihydrofolate reductase |
| 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 |
| 5Z6L | 2018-09-19 | 1.9 | High-pressure Crystal Structure Analysis of M20 loop closed DHFR at 650 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 |
| 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 |
| 1RX1 | 1997-01-11 | 2.0 | DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH NICOTINAMIDE ADENINE DINUCLEOTIDE PHOSPHATE (REDUCED FORM) |
| 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) |
| 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. |
| 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 |
| 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 |
| 6RUL | 2020-02-12 | 2.2 | Crystal structure of GFP-LAMA-F98 - a GFP enhancer nanobody with cpDHFR insertion and TMP and NADPH |
| 1RX4 | 1997-01-11 | 2.2 | DIHYDROFOLATE REDUCTASE (E.C.1.5.1.3) COMPLEXED WITH 5,10-DIDEAZATETRAHYDROFOLATE AND 2'-MONOPHOSPHOADENOSINE 5'-DIPHOSPHORIBOSE |
| 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 |
| 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 |
| 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 |
| 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 |
| 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 |
| 4EIG | 2013-04-24 | 2.5 | CA1698 camel antibody fragment in complex with DHFR |
| 1TDR | 1995-07-10 | 2.5 | EXPRESSION, CHARACTERIZATION, AND CRYSTALLOGRAPHIC ANALYSIS OF TELLUROMETHIONYL DIHYDROFOLATE REDUCTASE |
| 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 |
| 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) |