NMR-Untersuchungen zur Ligandenbindung an Dihydrofolat-Reduktase

Feeney, J.

doi:10.1002/(sici)1521-3757(20000117)112:2<298::aid-ange298>3.0.co;2-w

Angewandte Chemie

2000

DOI: 10.1002/(sici)1521-3757(20000117)112:2<298::aid-ange298>3.0.co;2-w

|View full text |Cite

NMR-Untersuchungen zur Ligandenbindung an Dihydrofolat-Reduktase

J. Feeney¹

Abstract: Einzigartige Möglichkeiten bietet die NMR‐Spektroskopie zur Untersuchung spezifischer Wechselwirkungen von Enzymen mit Substraten und Substrat‐analoga, zur Charakterisierung multipler Konformationen der Komplexe und zur Messung der Geschwindigkeiten einer Vielzahl von dynamischen Prozessen. Die umfangreichsten Studien dieser Art befassten sich mit Komplexen aus Dihydrofolat‐Reduktase und Folsäure‐Antagonisten und werden im vorliegenden Aufsatz beschrieben.

Help me understand this report

Search citation statements

Order By: Relevance

Paper Sections

Select...

Citation Types

Supporting

Mentioning

Contrasting

Year Published

2005

2010

Publication Types

Select...

Article3

Relationship

Self Cite0

Independent3

Authors

Journals

Cited by 3 publications

References 171 publications

(184 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

Searching Sequence Space: Two Different Approaches to Dihydrofolate Reductase Catalysis

Howell

2005

ChemBioChem

117

View full text Add to dashboard Cite

There are numerous examples of proteins that catalyze the same reaction while possessing different structures. This review focuses on two dihydrofolate reductases (DHFRs) that have disparate structures and discusses how the catalytic strategies of these two DHFRs are driven by their respective scaffolds. The two proteins are E. coli chromosomal DHFR (Ec DHFR) and a type II R-plasmid-encoded DHFR, typified by R67 DHFR. The former has been described as a very well evolved enzyme with an efficiency of 0.15, while the latter has been suggested to be a model for a "primitive" enzyme that has not yet been optimized by evolution. This comparison underlines what is important to catalysis in these two enzymes and concurrently highlights fundamental issues in enzyme catalysis.

show abstract

Searching Sequence Space: Two Different Approaches to Dihydrofolate Reductase Catalysis

Howell

2005

ChemBioChem

117

View full text Add to dashboard Cite

show abstract

Binding of Topotecan to a Nicked DNA Oligomer in Solution

Bocian

Kawęcki

Bednarek

et al. 2008

Chemistry A European J

View full text Add to dashboard Cite

Topotecan (TPT) is in clinical use as an antitumor agent. It acts by binding to the covalent complex formed between nicked DNA and topoisomerase I, and inserts itself into the single-strand nick, thereby inhibiting the religation of the nick and acting as a poison. A crystal structure analysis of the ternary complex has shown how the drug binds (B. L. Staker, K. Hjerrild, M. D. Feese, C. A. Behnke, A. B. Burgin, L. Stewart, Proc. Natl. Acad. Sci. U.S.A., 2002, 99, 15 387-15 392), but has left a number of unanswered questions. Herein, we use NMR spectroscopy and molecular modeling to show that the solution structure of a complex of TPT with nicked natural DNA is similar, but not identical to the crystal conformation, and that other geometries are of very low population. We also show that the lactone form of TPT binds approximately 40 times more strongly than the ring-opened carboxylate.

show abstract

Ligand–Receptor Binding Affinities from Saturation Transfer Difference (STD) NMR Spectroscopy: The Binding Isotherm of STD Initial Growth Rates

Angulo¹,

Enríquez-Navas²,

Nieto³

2010

Chemistry A European J

181

213

View full text Add to dashboard Cite

The direct evaluation of dissociation constants (K(D)) from the variation of saturation transfer difference (STD) NMR spectroscopy values with the receptor-ligand ratio is not feasible due to the complex dependence of STD intensities on the spectral properties of the observed signals. Indirect evaluation, by competition experiments, allows the determination of K(D), as long as a ligand of known affinity is available for the protein under study. Herein, we present a novel protocol based on STD NMR spectroscopy for the direct measurements of receptor-ligand dissociation constants (K(D)) from single-ligand titration experiments. The influence of several experimental factors on STD values has been studied in detail, confirming the marked impact on standard determinations of protein-ligand affinities by STD NMR spectroscopy. These factors, namely, STD saturation time, ligand residence time in the complex, and the intensity of the signal, affect the accumulation of saturation in the free ligand by processes closely related to fast protein-ligand rebinding and longitudinal relaxation of the ligand signals. The proposed method avoids the dependence of the magnitudes of ligand STD signals at a given saturation time on spurious factors by constructing the binding isotherms using the initial growth rates of the STD amplification factors, in a similar way to the use of NOE growing rates to estimate cross relaxation rates for distance evaluations. Herein, it is demonstrated that the effects of these factors are cancelled out by analyzing the protein-ligand association curve using STD values at the limit of zero saturation time, when virtually no ligand rebinding or relaxation takes place. The approach is validated for two well-studied protein-ligand systems: the binding of the saccharides GlcNAc and GlcNAcbeta1,4GlcNAc (chitobiose) to the wheat germ agglutinin (WGA) lectin, and the interaction of the amino acid L-tryptophan to bovine serum albumin (BSA). In all cases, the experimental K(D) measured under different experimental conditions converged to the thermodynamic values. The proposed protocol allows accurate determinations of protein-ligand dissociation constants, extending the applicability of the STD NMR spectroscopy for affinity measurements, which is of particular relevance for those proteins for which a ligand of known affinity is not available.

show abstract

scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.

Contact Info

customersupport@researchsolutions.com

10624 S. Eastern Ave., Ste. A-614

Henderson, NV 89052, USA

This site is protected by reCAPTCHA and the Google Privacy Policy and Terms of Service apply.

Blog Terms and Conditions API Terms Privacy Policy Contact Cookie Preferences Do Not Sell or Share My Personal Information

Made with 💙 for researchers

Part of the Research Solutions Family.

NMR-Untersuchungen zur Ligandenbindung an Dihydrofolat-Reduktase

Cited by 3 publications

References 171 publications

Searching Sequence Space: Two Different Approaches to Dihydrofolate Reductase Catalysis

Searching Sequence Space: Two Different Approaches to Dihydrofolate Reductase Catalysis

Binding of Topotecan to a Nicked DNA Oligomer in Solution

Ligand–Receptor Binding Affinities from Saturation Transfer Difference (STD) NMR Spectroscopy: The Binding Isotherm of STD Initial Growth Rates

Contact Info

Product

Resources

About