Differentiation of the binding of two ligands to a tetrameric protein with a single symmetric active site by 19F NMR

		BioNMR > Educational resources > Journal club
Differentiation of the binding of two ligands to a tetrameric protein with a single symmetric active site by 19F NMR

Webservers

NMR processing:

MDD

NMR assignment:

Backbone:

Side-chains:

NOEs:

Structure from NMR restraints:

Ab initio:

Fragment-based:

Rosetta-NMR (Robetta)

Template-based:

GeNMR

I-TASSER

Refinement:

Amber

Structure from chemical shifts:

Fragment-based:

Homology-based:

Torsion angles from chemical shifts:

Preditor

TALOS

Promega- Proline

Secondary structure from chemical shifts:

CSI (via RCI server)

TALOS

MICS caps, β-turns

d2D

PECAN

Flexibility from chemical shifts:

RCI

Interactions from chemical shifts:

HADDOCK

Chemical shifts re-referencing:

NMR model quality:

NOEs, other restraints:

Chemical shifts:

RDCs:

Pseudocontact shifts:

Protein geomtery:

SAVES2 or SAVES4

Quality Control Check

NMR spectrum prediction:

FANDAS

MestReS

V-NMR

Flexibility from structure:

Backbone S2

Methyl S2

B-factor

Molecular dynamics:

Gromacs

Amber

Antechamber

Chemical shifts prediction:

From structure:

Shiftx2

Sparta+

Camshift

CH3shift- Methyl

ArShift- Aromatic

ShiftS

Proshift

PPM

CheShift-2- Cα

From sequence:

Shifty

Camcoil

Poulsen_rc_CS

Disordered proteins:

MAXOCC

Format conversion & validation:

CCPN

From NMR-STAR 3.1

Validate NMR-STAR 3.1

NMR sample preparation:

Protein disorder:

DisMeta

Protein solubility:

Isotope labeling:

Solid-state NMR:

Thread Tools

Search this Thread

Rate Thread

Display Modes

12-12-2020, 06:54 PM

nmrlearner

Senior Member

Join Date: Jan 2005

Posts: 23,777

Points: 193,617, Level: 100

Level up: 0%, 0 Points needed

Activity: 50.7%

Last Achievements

Award-Showcase

NMR Credits: 0

NMR Points: 193,617

Downloads: 0

Uploads: 0

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 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.

More...

Did you find this post helpful?

Similar Threads
Thread	Thread Starter	Forum	Replies	Last Post
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...	nmrlearner	Journal club	0	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...	nmrlearner	Journal club	0	12-04-2020 03:46 PM
[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. Related Articles Nucleotide Binding and Active Site Gate Dynamics for the Hsp90 Chaperone ATPase Domain from Benchtop and High Field 19F NMR Spectroscopy. 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...	nmrlearner	Journal club	0	03-29-2020 12:14 PM
[NMR paper] Substrate binding drives active site closing of human blood group B galactosyltransferase as revealed by hot-spot labeling and NMR experiments. Substrate binding drives active site closing of human blood group B galactosyltransferase as revealed by hot-spot labeling and NMR experiments. Related Articles Substrate binding drives active site closing of human blood group B galactosyltransferase as revealed by hot-spot labeling and NMR experiments. Chembiochem. 2018 Feb 19;: Authors: Peters T, Weissbach S, Flügge F Abstract	nmrlearner	Journal club	0	02-21-2018 12:45 AM
[NMR paper] NMR reveals double occupancy of quinone-type ligands in the catalytic quinone binding site of the Na+-translocating NADH:Quinone oxidoreductase from Vibrio cholerae. NMR reveals double occupancy of quinone-type ligands in the catalytic quinone binding site of the Na+-translocating NADH:Quinone oxidoreductase from Vibrio cholerae. http://www.bionmr.com//www.ncbi.nlm.nih.gov/corehtml/query/egifs/http:--highwire.stanford.edu-icons-externalservices-pubmed-standard-jbc_final.gif Related Articles NMR reveals double occupancy of quinone-type ligands in the catalytic quinone binding site of the Na+-translocating NADH:Quinone oxidoreductase from Vibrio cholerae. J Biol Chem. 2013 Oct 18;288(42):30597-606 Authors: Nedielkov R,...	nmrlearner	Journal club	0	01-01-2014 03:05 PM
[NMR paper] Epitope mapping and competitive binding of HSA drug site II ligands by NMR diffusion Epitope mapping and competitive binding of HSA drug site II ligands by NMR diffusion measurements. Related Articles Epitope mapping and competitive binding of HSA drug site II ligands by NMR diffusion measurements. 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)...	nmrlearner	Journal club	0	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...	nmrlearner	Journal club	0	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...	nmrlearner	Journal club	0	08-22-2010 03:33 AM

« Previous Thread | Next Thread »

Posting Rules
You may not post new threads You may not post replies You may not post attachments You may not edit your posts BB code is On Smilies are On [IMG] code is On HTML code is On Trackbacks are Off Pingbacks are Off Refbacks are Off