Determination of protein-ligand binding affinity by NMR: observations from serum albumin model systems

Fielding, Lee A.; Rutherford, Samantha H.; Fletcher, Dan

doi:10.1002/mrc.1574

Cited by 109 publications

(115 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Comparing the experimental data in Table 2 with the K D value reported in the literature for the interaction of l-tryptophan with BSA (K D = 125-230 mm) [17] , it can be inferred that 1) the closest apparent K D measured from a given ligand proton STD is again the one obtained by using the lowest saturation time and 2) related to STD intensities, the isotherms yielding the best approximation to the reported values of K D are those constructed by using the least intense STD signal. Once more, unfortunately, our results show that the best conditions for better signal-to-noise ratio, and hence, for more accurate determination of K D , that is, the most intense STD signals, give, in fact, the largest deviations in the determination of the dissociation constant.…”

Section: Wwwchemeurjorgmentioning

confidence: 94%

“…[15] The feasibility of the binding isotherm of STD initial growth rates approach for obtaining accurate experimental values of ligand-receptor dissociation constants is demonstrated for two well-studied proteinligand systems, affinities of which have been reported in the literature: the binding of N-acetylglucosamine (GlcNAc) www.chemeurj.org and N,N'-diacetylglucosamine (GlcNAcb1,4GlcNAc, chitobiose) to the plant lectin wheat germ agglutinin (WGA), [16] and the interaction of l-tryptophan to bovine serum albumin (BSA). [17] …”

Section: Introductionmentioning

confidence: 97%

See 1 more Smart Citation

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

180

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

Section: Wwwchemeurjorgmentioning

confidence: 94%

Section: Introductionmentioning

confidence: 97%

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

180

213

View full text Add to dashboard Cite

show abstract

“…Aptamer dissociation constants were determined by titrating ATP or GTP into a constant amount of aptamer. The fraction of ligand-bound aptamer was determined by NMR line width or waterLOGSY methods (51,30,31, and SI Text).…”

Section: Methodsmentioning

confidence: 99%

“…To characterize the binding affinity of the MNA ATP aptamer 74, we employed two NMR-based ligand titration methods. Both the water-ligand observed gradient spectroscopy (waterLOGSY) NMR technique (30,31) and the line-width method (31) require only 15-50 nmol of aptamer and are appropriate for detecting low micromolar to low millimolar binding interactions. In these methods, aptamer in the free and bound states can be determined by titration of ligand into a constant amount of aptamer until binding saturation is observed (SI Text).…”

Section: Synthesis Of Transcripts Containing Both Deoxy-and Ribonuclementioning

confidence: 99%

Evolution of functional nucleic acids in the presence of nonheritable backbone heterogeneity

Trevino

Zhang

Elenko

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

View full text Add to dashboard Cite

Multiple lines of evidence support the hypothesis that the early evolution of life was dominated by RNA, which can both transfer information from generation to generation through replication directed by base-pairing, and carry out biochemical activities by folding into functional structures. To understand how life emerged from prebiotic chemistry we must therefore explain the steps that led to the emergence of the RNA world, and in particular, the synthesis of RNA. The generation of pools of highly pure ribonucleotides on the early Earth seems unlikely, but the presence of alternative nucleotides would support the assembly of nucleic acid polymers containing nonheritable backbone heterogeneity. We suggest that homogeneous monomers might not have been necessary if populations of heterogeneous nucleic acid molecules could evolve reproducible function. For such evolution to be possible, function would have to be maintained despite the repeated scrambling of backbone chemistry from generation to generation. We have tested this possibility in a simplified model system, by using a T7 RNA polymerase variant capable of transcribing nucleic acids that contain an approximately 1∶1 mixture of deoxy-and ribonucleotides. We readily isolated nucleotide-binding aptamers by utilizing an in vitro selection process that shuffles the order of deoxy-and ribonucleotides in each round. We describe two such RNA/DNA mosaic nucleic acid aptamers that specifically bind ATP and GTP, respectively. We conclude that nonheritable variations in nucleic acid backbone structure may not have posed an insurmountable barrier to the emergence of functionality in early nucleic acids.abiogenesis | genetic takeover | RNA progenitor | origin of life G iven that RNA is likely to have played a key role early in the evolution of life (1), understanding the origins of the RNAbased biosphere is a critical aspect of understanding the origin of life. The difficulties associated with the prebiotic synthesis of a macromolecule as complicated and fragile as RNA have stimulated interest in the hypothesis that RNA was preceded by simpler and/or more stable progenitor nucleic acids (2-4). The systematic exploration of nucleic acids that are structurally related to RNA has revealed that the formation of stable WatsonCrick base-paired, antiparallel duplex structures is compatible with a surprising degree of variation in the sugar-phosphate backbone (5). These studies imply that many distinct nucleic acids are in principle capable of mediating the inheritance of genetic information. On the other hand, recent advances in the prebiotic chemistry leading to the pyrimidine ribonucleotides (6) have revived the prospects of the RNA-first model, with the attendant advantage of avoiding a difficult genetic takeover. Both RNAfirst and RNA-late models assume that life began with a single, well-defined genetic polymer. Perhaps the greatest problem implicit in this assumption is the challenge of explaining the origin of pure nucleotide monomers on the early Earth. Indeed, the supp...

show abstract

“…To compensate for the magnetic field inhomogeneity, the determined linewidths were normalized to the linewidth of 2,2-dimethyl-2-silapentane-5-sulfonic acid (DSS) in a particular spectrum. The estimation of K D was accomplished using non-linear least squares fitting in a home written MS Excel workbook kindly provided by Dr. Lee Fielding 19 .…”

Section: Nmr Measurementsmentioning

confidence: 99%

Inhibition of carnitine acetyltransferase by mildronate, a regulator of energy metabolism

Jaudzems

Kuka

Gutsaits

et al. 2009

Journal of Enzyme Inhibition and Medicinal Chemistry

View full text Add to dashboard Cite

Determination of protein-ligand binding affinity by NMR: observations from serum albumin model systems

Cited by 109 publications

References 36 publications

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

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

Evolution of functional nucleic acids in the presence of nonheritable backbone heterogeneity

Inhibition of carnitine acetyltransferase by mildronate, a regulator of energy metabolism

Contact Info

Product

Resources

About