A T1 rho-filtered two-dimensional transferred NOE spectrum for studying antibody interactions with peptide antigens

Scherf, Tali; Anglister, Jacob

doi:10.1016/s0006-3495(93)81436-x

Cited by 92 publications

(70 citation statements)

References 22 publications

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“…Zero filling and Fourier transformation gave 4 KX2 K spectra. After the first n/2 pulse, a spin lock field of approximately 3.5 kHz was applied to remove disturbing protein signal background [13]. The mixing time was set at 400ms (Fig.…”

Section: Methodsmentioning

confidence: 99%

Screening Mixtures for Biological Activity by NMR

Meyer¹,

Weimar²,

Peters³

1997

European Journal of Biochemistry

163

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Development of new drugs often involves the screening of compound libraries for biological activity. Currently, the biologically active component can only be identified if either a pure compound is being tested or if the components of a mixture are spatially separated, for example, on beads. Here, we present an NMR technique based on the transferred nuclear Overhauser effect (transfer NOE) that allows identification and structural characterization of biologically active molecules from a mixture. As an example we demonstrate that from mixtures of oligosaccharides only a-~-Fuc-(1+6)-p-~-GlcNA~-OMe binds to Aleuria auruntia agglutinin. The sign of transferred NOEs is opposite to NOEs of small molecules that do not bind to the protein and, thus, an unequivocal identification of molecules with binding activity is possible. Normally, the selection of bound ligands is further facilitated in that the absolute intensity of transfer NOEs is much greater than that of NOEs of non-binding molecules. In addition, transfer NOEs provide information on the three-dimensional structure of the ligands in the bound state. Therefore, measuring transfer NOEs of mixtures of small molecules in the presence of large molecules, like proteins, should significantly enhance the options for screening mixtures of compounds for biological activity.Keywords: screening of mixtures ; biological activity ; transfer NOE; receptor; oligosaccharide.Lead substances for pharmaceutical research are normally low-molecular-mass molecules that interact with a protein receptor. With the advent of compound libraries, powerful screening protocols became of principal importance and a variety of such protocols based on ELISA, RIA, or immunoblotting, for example, have therefore been developed. None of these techniques allows the identification of biologically active compounds in a mixture. Therefore, the value of using libraries could be significantly enhanced if it were possible to identify an active compound directly from a mixture and to determine its three-dimensional structure.One physical parameter that distinguishes free and bound molecules is the tumbling time z, . Low or medium molecular mass molecules (< 1-2 kDa) have a short tumbling time z, and, as a consequence, such molecules exhibit positive NOEs, no NOEs, or very small negative NOEs depending on their molecular mass, shape and the field strength. When a small molecule is liganded with a large-molecular-mass protein, relaxation is governed by the slow tumbling time zc of the protein resulting in strong negative NOEs, so-called transfer NOEs. The transfer NOEs also reflect the bound conformation of the ligand. Furthermore, the discrimination between transfer NOEs and NOEs of the ligand in solution is facilitated by the fact that transfer NOEs are built up much faster. Therefore, the maximum enhancement for transfer NOEs is observed at significantly lower mixing than is the case for the isolated ligand in solution. The principles of transferred NOEs were originally observed and described more than tw...

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Section: Methodsmentioning

confidence: 99%

Screening Mixtures for Biological Activity by NMR

Meyer¹,

Weimar²,

Peters³

1997

European Journal of Biochemistry

163

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“…1a) is conceptually similar to that presented by Scherf and Anglister. 20 The T1ρ-filtered NOESY results in a spectrum with binding ligand signals in the first dimension and protein signals in the second dimension. The technique is based on T1ρ-filtering using a spin-lock pulse before the evolution period (t1), such that the broad resonances of the protein protons decay before the evolution period.…”

Section: Resultsmentioning

confidence: 99%

Segment Identification of a Ligand Binding with a Protein Receptor Using Multidimensional T1ρ-, Diffusion-Filtered and Diffusion-Ordered NOESY Experiments

et al. 2003

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Protein-ligand, protein-protein and protein-nucleic acid interactions are of primary interest in pharmacology and structural biology, and often provide useful clues for understanding the physiological function of drugs and macromolecules. Specific characterization of direct contact in a complex is currently left largely to X-ray analyses. Because Xray studies are often prevented by a difficulty to obtain crystals suitable for diffraction, NMR spectroscopy is an attractive alternative for characterizing macromolecular complexes and for determining compounds with binding affinity to macromolecules. There is a growing need for efficient methods capable of reliably identifying compounds with the desired binding activity, and several NMR-based screening methods have been developed using chemical-shift perturbation, [1][2][3][4][5][6][7][8][9] transferred NOE 10-13 and diffusion and relaxation editing. [14][15][16] In the development of NMR techniques for measuring binding affinity, the main aim is to reduce the experiment time and material usage without a need for isotope labeling. Although a high-resolution structure of the protein-ligand complex is desirable in rational drug design, a map of the interaction sites obtained by NMR techniques often provides valuable insights for identifying lead compounds, or making a judgment as to whether different compounds share a common binding mode.Recently it was shown that 1D NOE pumping 17 and 1D reverse NOE pumping 18 experiments can be effectively used to distinguish molecules that bind to macromolecules from nonbinding compounds. The 1D saturation transfer difference NMR technique using a T1ρ-filter was also developed to obtain information about the binding specificity at the atomic level. 19 Although these 1D experiments are very effective for rapidly characterizing binding processes, it is not easy to identify the binding interface in the complex. Here, we present 2D T1ρ-and diffusion-filtered NOESY experiments and 3D DOSY-NOESY experiment to identify segments of ligand binding with a protein receptor without the need for isotope labeling. The ability of these 2D techniques to identify the binding epitopes of a ligand has been demonstrated with the human serum albumin (HSA)-salicylic acid system. HSA is the most abundant protein in serum plasma, and is capable of transporting fatty acids and of binding a variety of metabolites and drugs. It consists of three repeating domains (I, II and III); domain III possesses a high affinity for small anionic aromatic compounds and the primary binding site for medium-long chain fatty acids. ExperimentalAll NMR experiments were performed on a JEOL ECA600 spectrometer equipped with a 5-mm inverse triple-resonance probe head at 25˚C. All of the spectra were recorded using 10 mM salicylic acid and 0.1 mM HSA in 99.9% 2 H2O containing PBS buffer (pH 7.4). HSA and salicylic acid were purchased from Wako (Osaka, Japan). The pulse sequences of the 2D T1ρ-and diffusion-filtered NOESY, and 3D DOSY-NOESY experiments are shown in Fig. 1. The exp...

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“…The broad signals had been cleared out by introducing an additional delay into the standard NOESY pulse sequence after the first 90 ° pulse. A non-selective 180 ° radiofrequency pulse or continious radiofrequency field have been used during this delay to prevent a distortion of the cross-peak phases [18,19]. We tested the pulse sequence MLEV-16, continious radiofrequency field as well as a train of rapidly repeated non-selective 180 ° radiofrequency pulses to attain the same goal.…”

Section: Resultsmentioning

confidence: 99%

Conformation of surface exposed N‐terminus part of bacteriorhodopsin studied by transferred NOE technique

et al. 1996

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Interaction of the monoclonal antibody A5 raised against native bacteriorhodopsin (BR) with the synthetic peptide pGlul-Ala-Gln-Ile-Thr-Gly-ArgT-NH2, corresponding to the amino acid sequence 1-7 was studied by transferred nuclear Overhauser effect (TRNOE) spectroscopy. The denaturing reagents and the specially designed pulse sequences which eliminate broad signals from the TRNOE spectra were used to favour evaluation of the TRNOE peaks. On the basis of the data obtained, the conformation of peptide bound with A5 was calculated. A model of the mutual arrangement of bacteriorhodopsin N-terminus and the first transmembrane c~-helical segment 8-32 was proposed.

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A T1 rho-filtered two-dimensional transferred NOE spectrum for studying antibody interactions with peptide antigens

Cited by 92 publications

References 22 publications

Screening Mixtures for Biological Activity by NMR

Screening Mixtures for Biological Activity by NMR

Segment Identification of a Ligand Binding with a Protein Receptor Using Multidimensional T1ρ-, Diffusion-Filtered and Diffusion-Ordered NOESY Experiments

Conformation of surface exposed N‐terminus part of bacteriorhodopsin studied by transferred NOE technique

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