Combinatorial Synthesis and Multidimensional NMR Spectroscopy: An Approach to Understanding Protein–Ligand Interactions

Combinatorial chemistry is a laboratory emulation of natural recombination and selection processes. Strategies in this developing discipline involve the generation of diverse, molecular libraries through combinatorial synthesis and the selection of compounds that possess a desired property. Such approaches can facilitate the identification of ligands that bind to biological receptors, promoting our chemical understanding of cellular processes. This article illustrates that the coupling of combinatorial synthesis, multidimensional NMR spectroscopy, and biochemical methods has enhanced our understanding of a protein receptor used commonly in signal transduction, the Src Homology 3 (SH3) domain. This novel approach to studying molecular recognition has revealed a set of rules that govern SH3–ligand interactions, allowing models of receptor–ligand complexes to be constructed with only a knowledge of the polypeptide sequences. Combining combinatorial synthesis with structural methods provides a powerful new approach to understanding how proteins bind their ligands in general.     

Characterization of Ligand Binding by Saturation Transfer Difference NMR Spectroscopy

Fast identification of binding activity directly from mixtures of potential ligands is possible with the NMR method described, which is based on saturation transfer to molecules in direct contact to a protein. In addition, the ligand's binding epitope is easily identified. High sensitivity and ease of use are the principal advantages of this method. The picture shows the normal 1D NMR spectrum of a mixture and the spectrum obtained by applying the STD method, which exclusively shows signals from molecules with binding affinity.     

Discrete Supramolecular Stacks Based on Multinuclear Tweezer-Type Rhodium Complexes

By taking advantage of self-complementary π–π stacking and CH–π interactions, a series of discrete quadruple stacks were constructed through the self-aggregation of U-shaped dirhodium metallotweezer complexes featuring various planar polyaromatic ligands. By altering the conjugate stacking strength and bridging ligands, assemblies with a range of topologies were obtained, including a binuclear D-shaped macrocycle, tetranuclear open-ended cagelike frameworks, and duplex metallotweezer stacking structures. Furthermore, a rare stacking interaction resulting in selective C−H activation was observed during the self-assembly process of these elaborate architectures.     

Quantifying the Thermodynamics of Ligand Binding to CsPbBr3 Quantum Dots

Cesium lead halide perovskites are an emerging class of quantum dots (QDs) that have shown promise in a variety of applications; however, their properties are highly dependent on their surface chemistry. To this point, the thermodynamics of ligand binding remain unstudied. Herein, ¹H NMR methods were used to quantify the thermodynamics of ligand exchange on CsPbBr₃ QDs. Both oleic acid and oleylamine native ligands dynamically interact with the CsPbBr₃ QD surface, having individual surface densities of 1.2–1.7 nm^?2. 10‐Undecenoic acid undergoes an exergonic exchange equilibrium with bound oleate (K_eq=1.97) at 25 °C while 10‐undecenylphosphonic acid undergoes irreversible ligand exchange. Undec‐10‐en‐1‐amine exergonically exchanges with oleylamine (K_eq=2.52) at 25 °C. Exchange occurs with carboxylic acids, phosphonic acids, and amines on CsPbBr₃ QDs without etching of the nanocrystal surface; increases in the steady‐state PL intensities correlate with more strongly bound conjugate base ligands.     

On the Role of Flexibility in Protein–Ligand Interactions: the Example of p53 Tetramerization Domain

The recognition of protein surfaces by designed ligands has become an attractive approach in drug discovery. However, the variable nature and irregular behavior of protein surfaces defy this new area of research. The easy to understand “lock‐and‐key” model is far from being the ideal paradigm in biomolecular interactions and, hence, any new finding on how proteins and ligands behave in recognition events paves a step of the way. Herein, we illustrate a clear example on how an increase in flexibility of both protein and ligand can result in an increase in the stability of the macromolecular complex. The biophysical study of the interaction between a designed flexible tetraguanidinium‐calix[4]arene and the tetramerization domain of protein p53 (p53TD) and its natural mutant p53TD‐R337H shows how the floppy mutant domain interacts more tightly with the ligand than the well‐packed wild‐type protein. Moreover, the flexible calixarene ligand interacts with higher affinity to both wild‐type and mutated protein domains than a conformationally rigid calixarene analog previously reported. These findings underscore the crucial role of flexibility in molecular recognition processes, for both small ligands and large biomolecular surfaces.     

Probing Helical Hydrophobic Binding Sites in Branched Starch Polysaccharides Using NMR Spectroscopy

Branched starch polysaccharides are capable of binding multiple hydrophobic guests, but their exploitation as multivalent hosts and in functional materials is limited by their structural complexity and diversity. Linear α(1–4)‐linked glucose oligosaccharides are known to bind hydrophobic guests inside left‐handed single helices in solution and the solid state. Here, we describe the development of an amphiphilic probe that binds to linear α(1–4)‐linked glucose oligosaccharides and undergoes a conformational switch upon complexation, which gives rise to dramatic changes in the ¹H NMR spectrum of the probe. We use this probe to explore hydrophobic binding sites in the branched starch polysaccharides amylopectin and β‐limit dextrin. Diffusion‐ordered (DOSY), nuclear Overhauser effect (NOESY) and chemical shift perturbation (HSQC) NMR experiments are utilised to provide evidence that, in aqueous solution, branched polysaccharides bind hydrophobic guests in well‐defined helical binding sites, similar to those reported for complexation by linear oligosaccharides. By examining the binding affinity of the probe to systematically enzymatically degraded polysaccharides, we deduce that the binding sites for hydrophobic guests can be located on internal as well as external branches and that proximal α(1–6)‐linked branch points weaken but do not prevent complexation.     

Paramagnetic DOSY: An Accurate Tool for the Analysis of the Supramolecular Interactions between Lanthanide Complexes and Proteins

Diffusion ordered NMR is implemented to determine accurately the mobility of paramagnetic tris‐dipicolinate lanthanide complexes that are versatile probes of protein structure. It is shown that diffusion coefficient ratios can be measured with an accuracy of 1 % using a standard BPPLED pulse sequence, which allows for observing significant, though weak, variations when different species are interacting with the paramagnetic compound. We demonstrate that this approach is complementary to classical chemical shift titration experiments, and that it can be applied successfully to probe the supramolecular dynamic interactions between lanthanide complexes and small molecules on the one hand, or to determine rapidly their affinity for a targeted protein.     

Selective Interaction of Dopamine with the Self‐Assembled Fibrillar Network of a Molecular Hydrogel Revealed by STD‐NMR

A molecular hydrogel formed by a derivative of L ‐valine with pendant isonicotinoyl moieties interacts selectively with protonated dopamine in the presence of related compounds such as 3‐methylcatechol, and protonated or neutral phenethylamine. A two‐point interaction with the gel fibers is postulated to explain the results. The conclusions are obtained from nuclear magnetic resonance saturation transfer experiments (STD‐NMR), illustrating how this technique is perfectly suited to monitor the interaction of substrates with the fibrillar network of a molecular gel.     

Molecular Recognition of Rosmarinic Acid from Salvia sclareoides Extracts by Acetylcholinesterase: A New Binding Site Detected by NMR Spectroscopy

Acetylcholinesterase (AChE) inhibition is one of the most currently available therapies for the management of Alzheimer’s disease (AD) symptoms. In this context, NMR spectroscopy binding studies were accomplished to explain the inhibition of AChE activity by Salvia sclareoides extracts. HPLC‐MS analyses of the acetone, butanol and water extracts eluted with methanol and acidified water showed that rosmarinic acid is present in all the studied samples and is a major constituent of butanol and water extracts. Moreover, luteolin 4′‐O‐glucoside, luteolin 3′,7‐di‐O‐glucoside and luteolin 7‐O‐(6′′‐O‐acetylglucoside) were identified by MS² and MS³ data acquired during the LC‐MSⁿ runs. Quantification of rosmarinic acid by HPLC with diode‐array detection (DAD) showed that the butanol extract is the richest one in this component (134 μg mg^?1 extract). Saturation transfer difference (STD) NMR spectroscopy binding experiments of S. sclareoides crude extracts in the presence of AChE in buffer solution determined rosmarinic acid as the only explicit binder for AChE. Furthermore, the binding epitope and the AChE‐bound conformation of rosmarinic acid were further elucidated by STD and transferred NOE effect (trNOESY) experiments. As a control, NMR spectroscopy binding experiments were also carried out with pure rosmarinic acid, thus confirming the specific interaction and inhibition of this compound against AChE. The binding site of AChE for rosmarinic acid was also investigated by STD‐based competition binding experiments using Donepezil, a drug currently used to treat AD, as a reference. These competition experiments demonstrated that rosmarinic acid does not compete with Donepezil for the same binding site. A 3D model of the molecular complex has been proposed. Therefore, the combination of the NMR spectroscopy based data with molecular modelling has permitted us to detect a new binding site in AChE, which could be used for future drug development.     

Diffusion NMR spectroscopy in supramolecular and combinatorial chemistry: an old parameter--new insights

Intermolecular interactions in solution play an important role in molecular recognition, which lies at the heart of supramolecular and combinatorial chemistry. Diffusion NMR spectroscopy gives information over such interactions and has become the method of choice for simultaneously measuring diffusion coefficients of multicomponent systems. The diffusion coefficient reflects the effective size and shape of a molecular species. Applications of this technique include the estimation of association constants and mapping the intermolecular interactions in multicomponent systems as well as investigating aggregation, ion pairing, encapsulation, and the size and structure of labile systems. Diffusion NMR spectroscopy can also be used to virtually separate mixtures and screen for specific ligands of different receptors, and may assist in finding lead compounds.     

Posttranslational Modifications of Intact Proteins Detected by NMR Spectroscopy: Application to Glycosylation

Posttranslational modifications (PTMs) are an integral part of the majority of proteins. The characterization of structure and function of PTMs can be very challenging especially for glycans. Existing methods to analyze PTMs require complicated sample preparations and suffer from missing certain modifications, the inability to identify linkage types and thus chemical structure. We present a direct, robust, and simple NMR spectroscopy method for the detection and identification of PTMs in proteins. No isotope labeling is required, nor does the molecular weight of the studied protein limit the application. The method can directly detect modifications on intact proteins without sophisticated sample preparation. This approach is well suited for diagnostics of proteins derived from native organisms and for the quality control of biotechnologically produced therapeutic proteins.     

Microsecond Timescale Dynamics of GDP‐Bound Ras Underlies the Formation of Novel Inhibitor‐Binding Pockets

The recent discovery of inhibitory compounds binding to distinct pockets on GDP‐bound Ras has renewed the view on the druggability of this crucial cancer driver. However, the origin of these pockets, which are not readily formed in the crystal structure in the absence of the compounds, is yet unclear. Herein, we explored the intrinsic flexibility of Ras?GDP on microsecond to millisecond timescales using relaxation‐based NMR experiments, and identified substantial slow dynamics with τ_ex of 34 μs at 5 °C, which maps to the regions showing a high level of correlation with those displaying conformational differences between the inhibitor‐bound and free states. These findings, which have been demonstrated in both wild‐type Ras and the oncogenic mutant (G12V), support the mechanism of extended conformational selection for Ras–inhibitor interactions where the small molecules redistribute the protein conformational ensemble favoring the final bound states.     

A Chiral Lanthanide Tag for Stable and Rigid Attachment to Single Cysteine Residues in Proteins for NMR,EPR and Time-Resolved Luminescence Studies

A lanthanide-binding tag site-specifically attached to a protein presents a tool to probe the protein by multiple spectroscopic techniques, including nuclear magnetic resonance, electron paramagnetic resonance and time-resolved luminescence spectroscopy. Here a new stable chiral Ln^III tag, referred to as C12 , is presented for spontaneous and quantitative reaction with a cysteine residue to generate a stable thioether bond. The synthetic protocol of the tag is relatively straightforward, and the tag is stable for storage and shipping. It displays greatly enhanced reactivity towards selenocysteine, opening a route towards selective tagging of selenocysteine in proteins containing cysteine residues. Loaded with Tb^III or Tm^III ions, the C12 tag readily generates pseudocontact shifts (PCS) in protein NMR spectra. It produces a relatively rigid tether between lanthanide and protein, which is beneficial for interpretation of the PCSs by single magnetic susceptibility anisotropy tensors, and it is suitable for measuring distance distributions in double electron–electron resonance experiments. Upon reaction with cysteine or other thiol compounds, the Tb^III complex exhibits a 100-fold enhancement in luminescence quantum yield, affording a highly sensitive turn-on luminescence probe for time-resolved FRET assays and enzyme reaction monitoring.     

New Insights into Glycopeptide Antibiotic Binding to Cell Wall Precursors using SPR and NMR Spectroscopy

Glycopeptide antibiotics, such as vancomycin and teicoplanin, are used to treat life‐threatening infections caused by multidrug‐resistant Gram‐positive pathogens. They inhibit bacterial cell wall biosynthesis by binding to the D ‐Ala‐D ‐Ala C‐terminus of peptidoglycan precursors. Vancomycin‐resistant bacteria replace the dipeptide with the D ‐Ala‐D ‐Lac depsipeptide, thus reducing the binding affinity of the antibiotics with their molecular targets. Herein, studies of the interaction of teicoplanin, teicoplanin‐like A40926, and of their semisynthetic derivatives (mideplanin, MDL63,246, dalbavancin) with peptide analogues of cell‐wall precursors by NMR spectroscopy and surface plasmon resonance (SPR) are reported. NMR spectroscopy revealed the existence of two different complexes in solution, when the different glycopeptides interact with Ac2Kd AlaD AlaOH. Despite the NMR experimental conditions, which are different from those employed for the SPR measurements, the NMR spectroscopy results parallel those deduced in the chip with respect to the drastic binding difference existing between the D ‐Ala and the D ‐Lac terminating analogues, confirming that all these antibiotics share the same primary molecular mechanism of action and resistance. Kinetic analysis of the interaction between the glycopeptide antibiotics and immobilized AcKd AlaD AlaOH by SPR suggest a dimerization process that was not observed by NMR spectroscopy in DMSO solution. Moreover, in SPR, all glycopeptides with a hydrophobic acyl chain present stronger binding with a hydrophobic surface than vancomycin, indicating that additional interactions through the employed surface are involved. In conclusion, SPR provides a tool to differentiate between vancomycin and other glycopeptides, and the calculated binding affinities at the surface seem to be more relevant to in vitro antimicrobial activity than the estimations from NMR spectroscopy analysis.     

Site‐Resolved Backbone and Side‐Chain Intermediate Dynamics in a Carbohydrate‐Binding Module Protein Studied by Magic‐Angle Spinning NMR Spectroscopy

Magic‐angle spinning solid‐state NMR spectroscopy has been applied to study the dynamics of CBM3b–Cbh9A from Clostridium thermocellum (ctCBM3b), a cellulose binding module protein. This 146‐residue protein has a nine‐stranded β‐sandwich fold, in which 35 % of the residues are in the β‐sheet and the remainder are composed of loops and turns. Dynamically averaged ¹H‐¹³C dipolar coupling order parameters were extracted in a site‐specific manner by using a pseudo‐three‐dimensional constant‐time recoupled separated‐local‐field experiment (dipolar‐chemical shift correlation experiment; DIPSHIFT). The backbone‐Cα and Cβ order parameters indicate that the majority of the protein, including turns, is rigid despite having a high content of loops; this suggests that restricted motions of the turns stabilize the loops and create a rigid structure. Water molecules, located in the crystalline interface between protein units, induce an increased dynamics of the interface residues thereby lubricating crystal water‐mediated contacts, whereas other crystal contacts remain rigid.