R67 dihydrofolate reductase (R67 DHFR) is a plasmid-encoded enzyme that confers resistance to the antibacterial drug trimethoprim. R67 DHFR is a tetramer with a single active site that is unusual as both cofactor and substrate are recognized by symmetry-related residues. Such promiscuity has limited our previous efforts to differentiate ligand binding by NMR. To address this problem, we incorporated fluorine at positions 4, 5, 6, or 7 of the indole rings of tryptophans 38 and 45 and characterized the spectra to determine which probe was optimal for studying ligand binding. Two resonances were observed for all apo proteins. Unexpectedly, the W45 resonance appeared broad, and truncation of the disordered N-termini resulted in the appearance of one sharp W45 resonance. These results are consistent with interaction of the N-terminus with W45. Binding of the cofactor broadened W38 for all fluorine probes, whereas substrate, dihydrofolate, binding resulted in the appearance of three new resonances for 4- and 5-fluoroindole labeled protein and severe line broadening for 6- and 7-fluoroindole R67 DHFR. W45 became slightly broader upon ligand binding. With only two peaks in the 19F NMR spectra, our data were able to differentiate cofactor and substrate binding to the single, symmetric active site of R67 DHFR and yield binding affinities.
Differentiation of the binding of two ligands to a tetrameric protein with a single symmetric active site by 19F NMR
Differentiation of the binding of two ligands to a tetrameric protein with a single symmetric active site by 19F NMR
Abstract
R67 dihydrofolate reductase (R67 DHFR) is a plasmid-encoded enzyme that confers resistance to the antibacterial drug trimethoprim. R67 DHFR is a tetramer with a single active site that is unusual as both cofactor and substrate are recognized by symmetry-related residues. Such promiscuity has limited our previous efforts to differentiate ligand binding by NMR. To address this problem, we incorporated fluorine at positions 4, 5, 6, or 7 of the indole rings of...
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Differentiation of the binding of two ligands to a tetrameric protein with a single symmetric active site by 19F NMR
Differentiation of the binding of two ligands to a tetrameric protein with a single symmetric active site by 19F NMR
Abstract
R67 dihydrofolate reductase (R67 DHFR) is a plasmid-encoded enzyme that confers resistance to the antibacterial drug trimethoprim. R67 DHFR is a tetramer with a single active site that is unusual as both cofactor and substrate are recognized by symmetry-related residues. Such promiscuity has limited our previous efforts to differentiate ligand binding by NMR. To address this problem, we incorporated fluorine at positions 4, 5, 6, or 7 of the indole rings of...
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12-04-2020 03:46 PM
[NMR paper] Differentiation of the binding of two ligands to a tetrameric protein with a single symmetric active site by 19 F NMR.
Differentiation of the binding of two ligands to a tetrameric protein with a single symmetric active site by 19 F NMR.
Related Articles Differentiation of the binding of two ligands to a tetrameric protein with a single symmetric active site by 19 F NMR.
Protein Sci. 2020 Dec 02;:
Authors: Fuente-Gómez GJ, Kellum CL, Miranda AC, Duff MR, Howell EE
Abstract
R67 dihydrofolate reductase (R67 DHFR) is a plasmid-encoded enzyme that confers resistance to the antibacterial drug trimethoprim. R67 DHFR is a tetramer with a single active...
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[NMR paper] Nucleotide Binding and Active Site Gate Dynamics for the Hsp90 Chaperone ATPase Domain from Benchtop and High Field 19F NMR Spectroscopy.
Nucleotide Binding and Active Site Gate Dynamics for the Hsp90 Chaperone ATPase Domain from Benchtop and High Field 19F NMR Spectroscopy.
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J Phys Chem B. 2020 Mar 25;:
Authors: Rashid S, Lee BL, Wajda B, Spyracopoulos L
Abstract
Protein turnover in cells is regulated by the ATP dependent activity of the Hsp90 chaperone. In concert with accessory proteins, ATP hydrolysis drives...
[NMR paper] Epitope mapping and competitive binding of HSA drug site II ligands by NMR diffusion
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J Am Chem Soc. 2004 Nov 3;126(43):14258-66
Authors: Lucas LH, Price KE, Larive CK
It is important to characterize drug-albumin binding during drug discovery and lead optimization as strong binding may reduce bioavailability and/or increase the drug's in vivo half-life. Despite knowing about the location of human serum albumin (HSA)...
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11-24-2010 10:03 PM
[NMR paper] NMR observation of substrate in the binding site of an active sugar-H+ symport protei
NMR observation of substrate in the binding site of an active sugar-H+ symport protein in native membranes.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--www.pubmedcentral.nih.gov-corehtml-pmc-pmcgifs-pubmed-pmc.gif Related Articles NMR observation of substrate in the binding site of an active sugar-H+ symport protein in native membranes.
Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3877-81
Authors: Spooner PJ, Rutherford NG, Watts A, Henderson PJ
NMR methods have been adopted to observe directly the characteristics of substrate...
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08-22-2010 03:33 AM
[NMR paper] NMR observation of substrate in the binding site of an active sugar-H+ symport protei
NMR observation of substrate in the binding site of an active sugar-H+ symport protein in native membranes.
http://www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--www.pubmedcentral.nih.gov-corehtml-pmc-pmcgifs-pubmed-pmc.gif Related Articles NMR observation of substrate in the binding site of an active sugar-H+ symport protein in native membranes.
Proc Natl Acad Sci U S A. 1994 Apr 26;91(9):3877-81
Authors: Spooner PJ, Rutherford NG, Watts A, Henderson PJ
NMR methods have been adopted to observe directly the characteristics of substrate...