A protocol based on saturation transfer difference (STD) NMR spectra was developed to characterize the binding interactions at an atom level, termed group epitope mapping (GEM). As an example we chose the well-studied system of galactose binding to the 120-kDa lectin Ricinus communis agglutinin I (RCA120). As ligands we used methyl β-d-galactoside and a biantennary decasaccharide. Analysis of the saturation transfer effects of methyl β-d-galactoside showed that the H2, H3, and H4 protons are saturated to the highest degree, giving evidence of their close proximity to protons of the RCA120 lectin. The direct interaction of the lectin with this region of the galactose is in excellent agreement with results obtained from the analysis of the binding specificities of many chemically modified galactose derivatives (Bhattacharyya, L.; Brewer, C. F. Eur. J. Biochem. 1988, 176, 207−212). This new NMR technique can identify the binding epitope of even complex ligands very quickly, which is a great improvement over ...

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Group Epitope Mapping by Saturation Transfer Difference NMR To Identify Segments of a Ligand in Direct Contact with a Protein Receptor

Carbohydrate structural determination by NMR spectroscopy: modern methods and limitations

WaterLOGSY represents a powerful method for primary NMR screening in the identification of compounds interacting with macromolecules, including proteins and DNA or RNA fragments. Several relay pathways are used constructively in the experiment for transferring bulk water magnetization to the ligand. The method is particularly useful for the identification of novel scaffolds of micromolar affinity that can be then optimized using directed screening, combinatorial chemistry, medicinal chemistry and structure-based drug design. The practical aspects and range of applicability of the WaterLOGSY experiment are analyzed in detail here. Competition binding and titration WaterLOGSY permit, after proper correction, the evaluation of the dissociation binding constant. The high sensitivity of the technique in combination with the easy deconvolution of the mixtures for the identification of the active components, significantly reduces the amount of material and time needed for the NMR screening process.

WaterLOGSY as a method for primary NMR screening: practical aspects and range of applicability.

The identification of extracellular phospholipid vesicles as conveyors of cellular information has created excitement in the field of drug delivery. Biological therapeutics, including short interfering RNA and recombinant proteins, are prone to degradation, have limited ability to cross biological membranes, and may elicit immune responses. Therefore, delivery systems for such drugs are under intensive investigation. Exploiting extracellular vesicles as carriers for biological therapeutics is a promising strategy to overcome these issues and to achieve efficient delivery to the cytosol of target cells. Exosomes are a well studied class of extracellular vesicles known to carry proteins and nucleic acids, making them especially suitable for such strategies. However, the considerable complexity and the related high chance of off-target effects of these carriers are major barriers for translation to the clinic. Given that it is well possible that not all components of exosomes are required for their proper functioning, an alternative strategy would be to mimic these vesicles synthetically. By assembly of liposomes harboring only crucial components of natural exosomes, functional exosome mimetics may be created. The low complexity and use of well characterized components strongly increase the pharmaceutical acceptability of such systems. However, exosomal components that would be required for the assembly of functional exosome mimetics remain to be identified. This review provides insights into the composition and functional properties of exosomes, and focuses on components which could be used to enhance the drug delivery properties of exosome mimetics.

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Exosome mimetics: a novel class of drug delivery systems

A powerful screening by NMR methodology (WaterLOGSY), based on transfer of magnetization from bulk water, for the identification of compounds that interact with target biomolecules (proteins, RNA and DNA fragments) is described The method exploits efficiently the large reservoir of H2O magnetization The high sensitivity of the technique reduces the amount of biomolecule and ligands needed for the screening, which constitutes an important requirement for high throughput screening by NMR of large libraries of compounds Application of the method to a compound mixture against the cyclin-dependent kinase 2 (cdk2) protein is presented

Identification of compounds with binding affinity to proteins via magnetization transfer from bulk water.

Detecting binding affinity to immobilized receptor proteins in compound libraries by hr-mas std nmr

About 30% of the proteins in mammalian systems are membrane bound or integrated (e.g., GPCRs). It is inherently difficult to investigate receptor−ligand interactions on a molecular level in their natural membrane environment. Here, we present a new method based on saturation transfer difference (STD) NMR to characterize at an atomic level binding interactions of cell surface proteins in living cells. Implemented as a double difference technique, STD NMR allows the direct observation of binding events and the definition of the binding epitopes of ligands. The binding of the pentapeptide cyclo(RGDfV) to the surface glycoprotein integrin αIIbβ3 of intact human blood platelets can be detected by saturation transfer double difference (STDD) NMR in less than an hour. A 5-fold higher STD response reflects a significantly higher affinity of integrin αIIbβ3 in native platelets than in liposomes, which demonstrates the importance of studying membrane proteins in their natural environment. Also, the binding mode of ...

Direct Observation of Ligand Binding to Membrane Proteins in Living Cells by a Saturation Transfer Double Difference (STDD) NMR Spectroscopy Method Shows a Significantly Higher Affinity of Integrin αIIbβ3 in Native Platelets than in Liposomes

Saturation transfer difference (STD) NMR is a fast and versatile method to screen compound mixtures in the presence of a receptor for binding affinity and to characterize the ligand's binding epitope. Here we demonstrate that ligand interactions with integral membrane proteins can be investigated by STD NMR if the receptor is embedded into the lipid bilayer of a liposome. The integrin alphaIIbbeta3, also termed GPIIb-IIIa, is a platelet surface glycoprotein that plays a pivotal role in platelet aggregation and that interacts with proteins and peptides presenting the peptide recognition motif RGD. Purified human integrin alphaIIbbeta3 was incorporated into liposomes, and the binding of RGD peptides was analyzed by STD NMR techniques. Cyclo(RGDfV) gave STD NMR effects in the presence of liposomes containing the integrin. The magnitude of the STD effect as a function of the ligand's concentration gave a value for the dissociation constant of 30-60 microM. Adding the weakly binding RGD to the solution of cyclo(RGDfV) resulted in STD effects of the stronger ligand cyclo(RGDfV) only. This demonstrates in agreement with literature that the peptide RGD is a much weaker ligand to the integrin than the peptide cyclo(RGDfV) that largely replaces the RGD peptides from the binding site. The binding epitope of the ligand cyclo(RGDfV) was characterized by STD NMR to contain sections of the D-Phe, the Val methyl groups, Arg alpha, beta, and gamma protons, one Hbeta of Asp, and one Halpha of Gly.

Determination of the Binding Specificity of an Integral Membrane Protein by Saturation Transfer Difference NMR: RGD Peptide Ligands Binding to Integrin αIIbβ3†

We have studied the potential of saturation transfer nuclear magnetic resonance (NMR) experiments in different mode to screen compound mixtures for binding activity and to characterize binding epitopes on the ligand. We have developed a protocol based on the transfer of saturation from the protein to bound ligands which by dissociation is moved into solution where it is detected (Fig. 1; Mayer and Meyer 1999; Peters and Meyer). By subtracting a spectrum, where the protein is saturated from one without protein saturation, a spectrum is produced where only signals of the ligand(s) remain in the difference spectrum. The irradiation frequency is set to a value where only protein resonances and no resonances of free ligands are located. Usually, irradiation frequencies around 1.5 ppm are practical because no ligand resonances are found in this spectral region, whereas the significant line width of protein signals still allows selective saturation. If the ligands show no resonances in the aromatic spectral region, the saturation frequency may also be placed there.

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Robert Meinecke

Papers

Detecting binding affinity to immobilized receptor proteins in compound libraries by hr-mas std nmr

Direct Observation of Ligand Binding to Membrane Proteins in Living Cells by a Saturation Transfer Double Difference (STDD) NMR Spectroscopy Method Shows a Significantly Higher Affinity of Integrin αIIbβ3 in Native Platelets than in Liposomes

Determination of the Binding Specificity of an Integral Membrane Protein by Saturation Transfer Difference NMR: RGD Peptide Ligands Binding to Integrin αIIbβ3†

Saturation Transfer Difference NMR Spectroscopy for Identifying Ligand Epitopes and Binding Specificities