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

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 GlcNAcβ1,4GlcNAc (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.     

Expanding the Scope of 2′‐SCF3 Modified RNA

The 2′‐trifluoromethylthio (2′‐SCF₃) modification endows ribonucleic acids with exceptional properties and has attracted considerable interest as a reporter group for NMR spectroscopic applications. However, only modified pyrimidine nucleosides have been generated so far. Here, the syntheses of 2′‐SCF₃ adenosine and guanosine phosphoramidites of which the latter was obtained in highly efficient manner by an unconventional Boc‐protecting group strategy, are reported. RNA solid‐phase synthesis provided site‐specifically 2′‐SCF₃‐modified oligoribonucleotides that were investigated intensively. Their excellent behavior in ¹⁹F NMR spectroscopic probing of RNA ligand binding was exemplified for a noncovalent small molecule–RNA interaction. Moreover, comparably to the 2′‐SCF₃ pyrimidine nucleosides, the purine counterparts were also found to cause a significant thermodynamic destabilization when located in double helical regions. This property was considered beneficial for siRNA design under the aspect to minimize off‐target effects and their performance in silencing of the BASP1 gene was demonstrated.     

Robust Synthesis and Crystal‐Structure Analysis of 7‐Cyano‐7‐deazaguanine (PreQ0 Base) and 7‐(Aminomethyl)‐7‐deazaguanine (PreQ1 Base)

We describe robust and efficient synthetic methods for the synthesis of the preQ₀ and preQ₁ bases, which are the biosynthetic precursors of the hypermodified RNA nucleoside queuosine. The X‐ray crystal‐structure analysis of preQ₁ is also described.     

Guest–host chemistry with dendrimers: Stable polymer assemblies by rational design

A new type of guest has been designed and synthesized for the exo‐type supramolecular functionalization of adamantyl‐urea‐terminated poly(propylene imine) dendrimers. This new type of guest motif features a uriedo methane sulfonic acid moiety that binds very selectively to the surfaces of dendrimers via a combination of noncovalent interactions forming well‐defined complexes. The guest–host properties have been examined for a fifth‐generation adamantyl‐urea‐functionalized poly(propylene imine) dendrimer capable of binding 32 guest molecules and for a model host molecule that can bind only one guest molecule. The guest–host chemistry has been studied with ¹H NMR spectroscopy, nuclear Overhauser enhancement spectroscopy NMR spectroscopy, T₁‐relaxation NMR experiments, and IR spectroscopy. The 1:32 ratio with the dendrimer has been confirmed unambiguously from ¹H NMR spectra of the complex after size exclusion chromatography. Competition experiments with guests bearing a carboxylic acid instead of a sulfonic acid in the binding motif have demonstrated that the sulfonic acid has superior binding strength. Also, the importance of a combination of noncovalent interactions has been shown via competition experiments with a guest lacking the uriedo moiety. © 2004 Wiley Periodicals, Inc. J Polym Sci Part A: Polym Chem 42: 3792–3799, 2004     

The “Speedy” Synthesis of Atom‐Specific 15N Imino/Amido‐Labeled RNA

Although numerous reports on the synthesis of atom‐specific ¹⁵N‐labeled nucleosides exist, fast and facile access to the corresponding phosphoramidites for RNA solid‐phase synthesis is still lacking. This situation represents a severe bottleneck for NMR spectroscopic investigations on functional RNAs. Here, we present optimized procedures to speed up the synthesis of ¹⁵N(1) adenosine and ¹⁵N(1) guanosine amidites, which are the much needed counterparts of the more straightforward‐to‐achieve ¹⁵N(3) uridine and ¹⁵N(3) cytidine amidites in order to tap full potential of ¹H/¹⁵N/¹⁵N‐COSY experiments for directly monitoring individual Watson–Crick base pairs in RNA. Demonstrated for two preQ₁ riboswitch systems, we exemplify a versatile concept for individual base‐pair labeling in the analysis of conformationally flexible RNAs when competing structures and conformational dynamics are encountered.     

Formation of a Ligand‐Assisted Complex of Two RNA Hairpin Loops

The hairpin structure is one of the most common secondary structures in RNA and holds a central position in the stream of RNA folding from a non‐structured RNA to structurally complex and functional ribonucleoproteins. Since the RNA secondary structure is strongly correlated to the function and can be modulated by the binding of small molecules, we have investigated the modulation of RNA folding by a ligand‐assisted formation of loop–loop complexes of two RNA hairpin loops. With a ligand (NCT6), designed based on the ligand binding to the G–G mismatches in double‐stranded DNA, we successfully demonstrated the formation of both inter‐ and intra‐molecular NCT6‐assisted complex of two RNA hairpin loops. NCT6 selectively bound to the two hairpin loops containing (CGG)₃ in the loop region. Native polyacrylamide gel electrophoresis analysis of two doubly‐labeled RNA hairpin loops clearly showed the formation of intermolecular NCT6‐assisted loop–loop complex. Förster resonance energy‐transfer studies of RNA constructs containing two hairpin loops, in which each hairpin was labeled with Alexa488 and Cy3 fluorophores, showed the conformational change of the RNA constructs upon binding of NCT6. These experimental data showed that NCT6 simultaneously bound to two hairpin RNAs at the loop region, and can induce the conformational change of the RNA molecule. These data strongly support that NCT6 functions as molecular glue for two hairpin RNAs.     

Ligand‐Induced Conformational Changes in HSP90 Monitored Time Resolved and Label Free—Towards a Conformational Activity Screening for Drug Discovery

Investigation of protein–ligand interactions is crucial during early drug‐discovery processes. ATR‐FTIR spectroscopy can detect label‐free protein–ligand interactions with high spatiotemporal resolution. Here we immobilized, as an example, the heat shock protein HSP90 on an ATR crystal. This protein is an important molecular target for drugs against several diseases including cancer. With our novel approach we investigated a ligand‐induced secondary structural change. Two specific binding modes of 19 drug‐like compounds were analyzed. Different binding modes can lead to different efficacy and specificity of different drugs. In addition, the k_obs values of ligand dissociation were obtained. The results were validated by X‐ray crystallography for the structural change and by SPR experiments for the dissociation kinetics, but our method yields all data in a single and simple experiment.     

Adamantanes as spherical nanosondes in adducts with a chiral dirhodium complex-discriminating enantiomers and probing spatial proximities

Three different kinds of substituted chiral adamantane molecules—adamantanones, dioxolanoadamantanes and dithiolano—adamantanes—were studied in the dirhodium experiment (NMR measurement with 1:1 molar mixtures with Rh^(II)₂[(R)‐(+)‐MTPA]₄ in CDCl₃). Their different behavior in adduct formation is described, and the possibility of determining enantiomeric purities and absolute configurations is explored. Detailed inspection of one‐ and two‐dimensional NMR experiments allowed for an interpretation of steric and electronic intra‐adduct interaction showing that the phenyl groups of Rh* tend to enwrap the bound adamantane ligand so that through‐space effects over a range of 6–7 Å away from the binding rhodium atom can be observed. Even slight differences in the relative orientation of phenyl groups can be monitored when comparing diastereomeric adducts via NMR signal dispersion. Copyright © 2011 John Wiley & Sons, Ltd.     

Fluorinated Carbohydrates as Lectin Ligands: Dissecting Glycan–Cyanovirin Interactions by Using 19F NMR Spectroscopy

NMR spectroscopy and isothermal titration calorimetry (ITC) are powerful methods to investigate ligand–protein interactions. Here, we present a versatile and sensitive fluorine NMR spectroscopic approach that exploits the ¹⁹F nucleus of ¹⁹F‐labeled carbohydrates as a sensor to study glycan binding to lectins. Our approach is illustrated with the 11 kDa Cyanovirin‐N, a mannose binding anti‐HIV lectin. Two fluoro‐deoxy sugar derivatives, methyl 2‐deoxy‐2‐fluoro‐α‐D ‐mannopyranosyl‐(1→2)‐α‐D ‐mannopyranoside and methyl 2‐deoxy‐2‐fluoro‐α‐D ‐mannopyranosyl‐(1→2)‐α‐D ‐mannopyranosyl‐(1→2)‐α‐D ‐mannopyranoside were utilized. Binding was studied by ¹⁹F NMR spectroscopy of the ligand and ¹H–¹⁵N HSQC NMR spectroscopy of the protein. The NMR data agree well with those obtained from the equivalent reciprocal and direct ITC titrations. Our study shows that the strategic design of fluorinated ligands and fluorine NMR spectroscopy for ligand screening holds great promise for easy and fast identification of glycan binding, as well as for their use in reporting structural and/or electronic perturbations that ensue upon interaction with a cognate lectin.     

Complex Formation of the Tetracycline‐Binding Aptamer Investigated by Specific Cross‐Relaxation under DNP

While dynamic nuclear polarization (DNP) under magic‐angle spinning (MAS) is generally a powerful method capable of greatly enhancing the sensitivity of solid‐state NMR spectroscopy, hyperpolarization also gives rise to peculiar spin dynamics. Here, we elucidate how specific cross‐relaxation enhancement by active motions under DNP (SCREAM‐DNP) can be utilized to selectively obtain MAS‐NMR spectra of an RNA aptamer in a tightly bound complex with a methyl‐bearing ligand (tetracycline) due to the effective CH₃‐reorientation at an optimized sample temperature of approximately 160 K. SCREAM‐DNP can spectrally isolate the complex from non‐bound species in an RNA mixture. This selectivity allows for a competition assay between the aptamer and a mutant with compromised binding affinity. Variations in molecular structure and methyl dynamics, as observed by SCREAM‐DNP, between free tetracycline and RNA‐bound tetracycline are discussed.     

High-resolution protein hydration NMR experiments: probing how protein surfaces interact with water and other non-covalent ligands

High-resolution solution NMR experiments are extremely useful to characterize the location and the dynamics of hydrating water molecules at atomic resolution. However, these methods are severely limited by undesired incoherent transfer pathways such as those arising from exchange-relayed intra-molecular cross-relaxation. Here, we review several complementary exchange network editing methods that can be used in conjunction with other types of NMR hydration experiments such as magnetic relaxation dispersion and ¹J_NC′ measurements to circumvent these limitations. We also review several recent contributions illustrating how the original solution hydration NMR pulse sequence architecture has inspired new approaches to map other types of non-covalent interactions going well beyond the initial scope of hydration. Specifically, we will show how hydration NMR methods have evolved and have been adapted to binding site mapping, ligand screening, protein-peptide and peptide-lipid interaction profiling.     

Probing Transient Conformational States of Proteins by Solid‐State R1ρ Relaxation‐Dispersion NMR Spectroscopy

The function of proteins depends on their ability to sample a variety of states differing in structure and free energy. Deciphering how the various thermally accessible conformations are connected, and understanding their structures and relative energies is crucial in rationalizing protein function. Many biomolecular reactions take place within microseconds to milliseconds, and this timescale is therefore of central functional importance. Here we show that R_1ρ relaxation dispersion experiments in magic‐angle‐spinning solid‐state NMR spectroscopy make it possible to investigate the thermodynamics and kinetics of such exchange process, and gain insight into structural features of short‐lived states.     

Differential Epitope Mapping by STD NMR Spectroscopy To Reveal the Nature of Protein–Ligand Contacts

Saturation transfer difference (STD) NMR spectroscopy is extensively used to obtain epitope maps of ligands binding to protein receptors, thereby revealing structural details of the interaction, which is key to direct lead optimization efforts in drug discovery. However, it does not give information about the nature of the amino acids surrounding the ligand in the binding pocket. Herein, we report the development of the novel method differential epitope mapping by STD NMR (DEEP‐STD NMR) for identifying the type of protein residues contacting the ligand. The method produces differential epitope maps through 1) differential frequency STD NMR and/or 2) differential solvent (D₂O/H₂O) STD NMR experiments. The two approaches provide different complementary information on the binding pocket. We demonstrate that DEEP‐STD NMR can be used to readily obtain pharmacophore information on the protein. Furthermore, if the 3D structure of the protein is known, this information also helps in orienting the ligand in the binding pocket.     

Dynamics of ligand binding from 13C NMR relaxation dispersion at natural abundance

We show that Carr-Purcell-Meiboom-Gill (CPMG) 13Calpha NMR relaxation dispersion measurements are a viable means for profiling mus-ms ligand dynamics involved in receptor binding. Critically, the dispersion is at natural 13C abundance; this matches typical pharmaceutical research settings in which ligand isotope-labeling is often impractical. The dispersion reveals ligand 13Calpha nuclei that experience mus-ms modulation of their chemical shifts due to binding. 13Calpha shifts are dominated by local torsion angles , psi, chi1; hence, these experiments identify flexible torsion angles that may assist complex formation. Since the experiments detect the ligand, they are viable even in the absence of a receptor structure. The mus-ms dynamic information gained helps establish flexibility-activity relationships. We apply these experiments to study the binding of a phospho-peptide substrate ligand to the peptidyl-prolyl isomerase Pin1.     

Fluorescent Cross‐Linked Supramolecular Polymer Constructed by Orthogonal Self‐Assembly of Metal–Ligand Coordination and Host–Guest Interaction

A new host molecule consists of four terpyridine groups as the binding sites with zinc(II) ion and a copillar[5]arene incorporated in the center as a spacer to interact with guest molecule was designed and synthesized. Due to the 120 ° angle of the rigid aromatic segment, a cross‐linked dimeric hexagonal supramolecular polymer was therefore generated as the result of the orthogonal self‐assembly of metal–ligand coordination and host–guest interaction. UV/Vis spectroscopy, ¹H NMR spectroscopy, viscosity and dynamic light‐scattering techniques were employed to characterize and understand the cross‐linking process with the introduction of zinc(II) ion and guest molecule. More importantly, well‐defined morphology of the self‐assembled supramolecular structure can be tuned by altering the adding sequence of the two components, that is, the zinc(II) ion and the guest molecule. In addition, introduction of a competitive ligand suggested the dynamic nature of the supramolecular structure.     

Unwinding of the C‐Terminal Residues of Neuropeptide Y is critical for Y2 Receptor Binding and Activation

Despite recent breakthroughs in the structural characterization of G‐protein‐coupled receptors (GPCRs), there is only sparse data on how GPCRs recognize larger peptide ligands. NMR spectroscopy, molecular modeling, and double‐cycle mutagenesis studies were integrated to obtain a structural model of the peptide hormone neuropeptide Y (NPY) bound to its human G‐protein‐coupled Y₂ receptor (Y₂R). Solid‐state NMR measurements of specific isotope‐labeled NPY in complex with in vitro folded Y₂R reconstituted into phospholipid bicelles provided the bioactive structure of the peptide. Guided by solution NMR experiments, it could be shown that the ligand is tethered to the second extracellular loop by hydrophobic contacts. The C‐terminal α‐helix of NPY, which is formed in a membrane environment in the absence of the receptor, is unwound starting at T³² to provide optimal contacts in a deep binding pocket within the transmembrane bundle of the Y₂R.     

Star PolyMOCs with Diverse Structures,Dynamics, and Functions by Three‐Component Assembly

We report star polymer metal–organic cage (polyMOC) materials whose structures, mechanical properties, functionalities, and dynamics can all be precisely tailored through a simple three‐component assembly strategy. The star polyMOC network is composed of tetra‐arm star polymers functionalized with ligands on the chain ends, small molecule ligands, and palladium ions; polyMOCs are formed via metal–ligand coordination and thermal annealing. The ratio of small molecule ligands to polymer‐bound ligands determines the connectivity of the MOC junctions and the network structure. The use of large M₁₂L₂₄ MOCs enables great flexibility in tuning this ratio, which provides access to a rich spectrum of material properties including tunable moduli and relaxation dynamics.     

Star PolyMOCs with Diverse Structures,Dynamics, and Functions by Three‐Component Assembly

We report star polymer metal–organic cage (polyMOC) materials whose structures, mechanical properties, functionalities, and dynamics can all be precisely tailored through a simple three‐component assembly strategy. The star polyMOC network is composed of tetra‐arm star polymers functionalized with ligands on the chain ends, small molecule ligands, and palladium ions; polyMOCs are formed via metal–ligand coordination and thermal annealing. The ratio of small molecule ligands to polymer‐bound ligands determines the connectivity of the MOC junctions and the network structure. The use of large M₁₂L₂₄ MOCs enables great flexibility in tuning this ratio, which provides access to a rich spectrum of material properties including tunable moduli and relaxation dynamics.