scholarly journals Diagnostic chemical shift markers for loop conformation and substrate and cofactor binding in dihydrofolate reductase complexes

2009 ◽  
Vol 12 (10) ◽  
pp. 2230-2238 ◽  
Author(s):  
Michael J. Osborne ◽  
Rani P. Venkitakrishnan ◽  
H. Jane Dyson ◽  
Peter E. Wright
Keyword(s):  
Chemical Shift ◽  
Dihydrofolate Reductase ◽  
Cofactor Binding ◽  
Loop Conformation

Aliphatic 1H, 13C and 15N chemical shift assignments of dihydrofolate reductase from the psychropiezophile Moritella profunda in complex with NADP+ and folate

2012 ◽  
Vol 7 (1) ◽  
pp. 61-64 ◽  
Author(s):  
E. Joel Loveridge ◽  
Stella M. Matthews ◽  
Christopher Williams ◽  
Sara B.-M. Whittaker ◽  
Ulrich L. Günther ◽  
...  
Keyword(s):  
Chemical Shift ◽  
Dihydrofolate Reductase ◽  
Chemical Shift Assignments ◽  
Moritella Profunda

1 H nuclear magnetic resonance studies of the tyrosine residues of selectively deuterated Lactobacillus casei dihydrofolate reductase

1977 ◽  
Vol 196 (1124) ◽  
pp. 267-290 ◽  
Keyword(s):  
Chemical Shift ◽  
Ligand Binding ◽  
Dihydrofolate Reductase ◽  
Conformational Changes ◽  
Lactobacillus Casei ◽  
Inhibitor Binding ◽  
Tyrosine Residues ◽  
Coenzyme Binding ◽  
Binary Complexes ◽  
Significant Difference

A selectively deuterated dihydrofolate reductase, in which all the aro­matic protons except the 2, 6-protons of the tyrosine residues have been replaced by deuterium, has been prepared from Lactobacillus casei grown on a mixture of normal and deuterated amino acids. The aromatic region of the 1 H n. m. r. spectrum of this enzyme contains only resonances from the five tyrosine residues. For each tyrosine, the 2- and 6-protons have the same chemical shift, indicating rapid interconversion of the two conformers related by 180° rotation about the C β -C γ bond. The effects of sub­strate, inhibitor and coenzyme binding on the tyrosine residues have been investigated; four of the five residues are affected by ligand binding. Using the weakly binding ligands 2, 4-diaminopyrimidine and p -nitrobenzoyl-l-glutamate to connect the spectra of the free enzyme with those of the complexes, it is possible to give a detailed description of the effects of ligand binding on individual residues. In the binary complexes, methotrexate affects three tyrosine residues, only one of which is affected by folate, indicating a significant difference in the mode of binding of substrates and inhibitors. The co-enzymes NADP + and NADPH lead to broadly similar changes in the spectrum, except for one resonance which is shifted in opposite directions by the two co-enzymes. The oxidized and reduced co­enzymes also differ in their effects on the changes produced by inhibitor binding; the spectrum of the enzyme-NADPH-methotrexate complex is similar to that of the enzyme-methotrexate complex, while that of the enzyme-NADP + -methotrexate complex is not. In contrast to the be­haviour seen in the binary complexes, the spectrum of the enzyme-NADP + -folate complex is very similar to that of enzyme-NADP + -methotrexate. Evidence is presented that some, at least, of the changes in chemical shift of the tyrosine residues are due to ligand-induced conformational changes. The binding of p -nitrobenzoyl-l-glutamate to the enzyme-2, 4-diamino-pyrimidine complex is found to be tighter than that to the enzyme alone.

Thermodynamics and Solvent Effects on Substrate and Cofactor Binding inEscherichia coliChromosomal Dihydrofolate Reductase

Biochemistry ◽  
2011 ◽  
Vol 50 (18) ◽  
pp. 3673-3685 ◽  
Author(s):  
Jordan Grubbs ◽  
Sharghi Rahmanian ◽  
Alexa DeLuca ◽  
Chetan Padmashali ◽  
Michael Jackson ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Solvent Effects ◽  
Cofactor Binding

High-resolution gold labeling

1993 ◽  
Vol 51 ◽  
pp. 330-331
Author(s):  
James F. Hainfeld ◽  
Frederic R. Furuya ◽  
Kyra Carbone ◽  
Martha Simon ◽  
Beth Lin ◽  
...  
Keyword(s):  
Dihydrofolate Reductase ◽  
Polypeptide Chain ◽  
External Surface ◽  
Gold Cluster ◽  
Macromolecular Complex ◽  
Gold Compound ◽  
Central Cavity ◽  
Chain Folding ◽  
E Coli ◽  
Chaperonin Groel

A recently developed 1.4 nm gold cluster has been found to be useful in labeling macromolecular sites to 1-3 nm resolution. The gold compound is organically derivatized to contain a monofunctional arm for covalent linking to biomolecules. This may be used to mark a specific site on a structure, or to first label a component and then reassemble a multicomponent macromolecular complex. Two examples are given here: the chaperonin groEL and ribosomes.Chaperonins are essential oligomeric complexes that mediate nascent polypeptide chain folding to produce active proteins. The E. coli chaperonin, groEL, has two stacked rings with a central hole ∽6 nm in diameter. The protein dihydrofolate reductase (DHFR) is a small protein that has been used in chain folding experiments, and serves as a model substrate for groEL. By labeling the DHFR with gold, its position with respect to the groEL complex can be followed. In particular, it was sought to determine if DHFR refolds on the external surface of the groEL complex, or whether it interacts in the central cavity.

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