The effect of relaxation on the epitope mapping by saturation transfer difference NMR

doi:10.1016/S1090-7807(03)00106-X

Journal of Magnetic Resonance

Volume 163, Issue 2, August 2003, Pages 270-276

https://doi.org/10.1016/S1090-7807(03)00106-X Get rights and content

Abstract

The effect of longitudinal relaxation of ligand protons on saturation transfer difference (STD) was investigated by using a known binding system, dihydrofolate reductase and trimethoprim. The results indicate that T1 relaxation of ligand protons has a severe interference on the epitope map derived from a STD measurement. When the T1s of individual ligand protons are distinctly different, STD experiments may not give an accurate epitope map for the ligand–target interactions. Measuring the relaxation times prior to mapping is strongly advised. A saturation time shorter than T1s is suggested for improving the potential epitope map. Reduction in temperature was seen to enhance the saturation efficiency in small to medium size targets.

Introduction

In biochemical and pharmaceutical research, identification of the binding activity and characterization of the binding epitope of ligands is important to lead validation and optimization in drug discovery. Many NMR-based methods have been developed for detecting the binding interactions between small ligands and biomolecular targets. Target-based methods rely on the perturbations of ¹H/¹⁵N/¹³C chemical shifts due to ligand binding and can be utilized to map the binding site on the protein target [1], [2], [3]. By directly detecting the ligand signals, several different methodologies have been developed to determine binding affinities of potential candidates. These ligand-based techniques include transferred NOE [4], [5], [6], [7], [8], inter-ligand NOE [9], [10], [11], diffusion [12], [13], [14], relaxation [15], [16], [17], NOE-pumping [18], [19], ligand release [20], [21], waterLOGSY [22], [23], and saturation transfer difference [24], [25], [26]. Although NMR-based techniques are not as sensitive as other commonly used methods in high-throughput drug screening, such as mass spectrometry, NMR has been considered comparatively robust in drug discovery since it is less prone to give artifacts [21]. In addition, NMR is able to supply more detailed structural information, such as the binding epitope for both target and ligand using target-based and ligand-based methods, respectively.

Saturation transfer difference (STD) is a fast and versatile method for screening binding components from a mixture [25], [26]. It is based on magnetization transfer by protein signal saturation and its relayed effect to ligand. Macromolecules have a large network of protons that are tightly coupled by dipole–dipole interactions. Saturation of a single protein resonance can result in a rapid spread of the saturation over the entire protein if spin diffusion within the protein is efficient. During the saturation period, progressive saturation transfers from the protein to the ligand protons if the ligand binds to the target. The ligand protons nearest to the protein should be saturated to the highest degree and therefore, have the strongest signal in the STD spectrum. The ligand protons, which are further from the target surface will be saturated to lower degree and their STD intensities will be weaker. Therefore, the degree of saturation of individual ligand protons reflect their proximities to the protein surface and can be used as an epitope method to describe the target–ligand interactions [7], [27], [28], [29]. Here we present a study of STD epitope mapping using a well-known system, dihydrofolate reductase (DHFR) and trimethoprim (TMP) [30], [31], [32], [33], [34], [35], [36]. It is found that the STD epitope values are greatly affected by longitudinal relaxation. When the longitudinal relaxation time of individual protons are significantly different, the resulting STD does not produce an accurate epitope map for the ligand–target interaction.

Section snippets

Methods

One times phosphate-buffered saline (PBS) of D₂O containing 10 mM sodium phosphate, 0.16 M NaCl, and 3 μM NaN₃ at pH 7.3 was used to prepare all the NMR samples. Dihydrofolate reductase (EC 1.5.1.3) from bovine liver and trimethoprim were purchased from Sigma. Prior to use, DHFR was dialyzed extensively and TMP was dissolved into PBS buffer. 4.2 mM of TMP, 4.2 mM of TMP with 120 μM DHFR, and 100 μM DHFR solutions were prepared to test the ligand–target binding.

All NMR experiments were performed on

Results

In principle, the saturation degree of an individual ligand proton reflects the proximity of this proton to the target surface. When creating an epitope map, the STD intensity relative to the reference is used to create the binding epitope maps, this is usually described by the STD factor A_STD, as shown by Eq. (1): $A_{std} = I_{0} −I_{sat} I_{0} = I_{STD} I_{0} .$

If different systems are compared, ligand excess should be used to normalize the differences of protein concentration [27]. Fig. 1 presents the STD spectrum and

Conclusion

During the presaturation period, saturation diffuses over the entire protein and transfers progressively from the target to the ligand. However, all magnetizations, including the transferred saturation, decay because of relaxation. The relaxation mechanism during the saturation period is related not only to the observed ligand but also to the saturated protein. Several other mechanisms, such as spin diffusion within the target, magnetization transferring from the target to the ligand, and spin

Acknowledgements

The authors thank Dr. Cynthia K. Larive for useful discussions.

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View full text

The effect of relaxation on the epitope mapping by saturation transfer difference NMR

Abstract

Introduction

Section snippets

Methods

Results

Conclusion

Acknowledgements

Prog. Nucl. Magn. Reson.

J. Magn. Reson.

J. Mol. Biol.

J. Biol. Chem.

J. Mag. Res.

FEBS Lett.

Discovering high-affinity ligands for proteins: SAR by NMR

Science

NMR-based screening of proteins containing 13C-labeled methyl groups

J. Am. Chem. Soc.

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J. Med. Chem.

Transfer NOE experiments for the study of carbohydrate–protein interactions

Carbohydr. Chem. Biol.

Transferred cross-correlated relaxation complements transferred NOE: structure of an IL-4R-derived peptide bound to STAT-6

J. Am. Chem. Soc.

Mapping the active site of angiotensin-converting enzyme by transferred NOE spectroscopy

J. Med. Chem.

Application of 3D-TOCSY-trNOESY for the assignment of bioactive ligands from mixtures

Angew. Chem. Int. Ed. Eng.

The inter-ligand Overhauser effect: a powerful new NMR approach for mapping structural relationships of macromolecular ligands

J. Biol. NMR

Interligand overhauser effects in Type II dihydrofolate reductase

Biochemistry

Epitope mapping of ligand–receptor interactions by diffusion NMR

J. Am. Chem. Soc.

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J. Org. Chem.

Diffusion-edited NMR-affinity NMR for direct observation of molecular interactions

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J. Comb. Chem.

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