Project description:RNA, at the forefront of biochemical research due to its central role in biology, is recognized by proteins through various mechanisms. Analysis of the RNA-protein interface provides insight into the recognition determinants and function. As such, there is a demand for developing new methods to characterize RNA-protein interactions. Saturation transfer difference (STD) NMR can identify binding ligands for proteins in a rather short period of time, with data acquisitions of just a few hours. Two RNA-protein systems involved in RNA modification were studied using STD NMR. The N (6)-threonylcarbamoyltransferase, YrdC, with nucleoside-specific recognition, was shown to bind the anticodon stem-loop of tRNA(Lys)UUU. The points of contact on the RNA were assigned and a binding interface was identified. STD NMR was also applied to the interaction of the archaeal ribosomal protein, L7Ae, with the box C/D K-turn RNA. The distinctiveness of the two RNA-protein interfaces was evident. Both RNAs exhibited strong STD signals indicative of direct contact with the respective protein, but reflected the nature of recognition. Characterization of nucleic acid recognition determinants traditionally involves cost and time prohibitive methods. This approach offers significant insight into interaction interfaces fairly rapidly, and complements existing structural methods.
Project description:A simple method is presented that optimizes the STD NMR Gaussian pulse to deliver significant increases in STD amplification factors with minimal perturbation of the ligand. This approach is practically demonstrated using the wheat-germ agglutinin/N-acetyl-D-glucosamine protein-ligand system.
Project description:We have combined saturation transfer difference NMR (STD NMR) with chemical shift imaging (CSI) and controlled concentration gradients of small molecule ligands to develop imaging STD NMR, a new tool for the assessment of protein-ligand interactions. Our methodology allows the determination of protein-ligand dissociation constants (KD) and assessment of the binding specificity in a single NMR tube, avoiding time-consuming titrations. We demonstrate the formation of suitable and reproducible concentration gradients of ligand along the vertical axis of the tube, against homogeneous protein concentration, and present a CSI pulse sequence for the acquisition of STD NMR experiments at different positions along the sample tube. Compared to the conventional methodology in which the [ligand]/[protein] ratio is increased manually, we can perform STD NMR experiments at a greater number of ratios and construct binding epitopes in a fraction (∼20%) of the experimental time. Second, imaging STD NMR also allows us to screen for non-specific binders, by monitoring any variation of the binding epitope map at increasing [ligand]/[protein] ratios. Hence, the proposed method does carry the potential to speed up and smooth out the drug discovery process.
Project description:As nanotechnology becomes increasingly used in biomedicine, it is important to have techniques by which to examine the structure and dynamics of biologically-relevant molecules on the surface of engineered nanoparticles. Previous work has shown that Saturation-Transfer Difference (STD)-NMR can be used to explore the interaction between small molecules, including amino acids, and the surface of polystyrene nanoparticles. Here we use STD-NMR to further explore the different driving forces that are responsible for these interactions. Electrostatic effects are probed by using zwitterionic polystyrene beads and performing STD-NMR experiments at high, low, and neutral pH, as well as by varying the salt concentration and observing the effect on the STD buildup curve. The influence of dispersion interactions on ligand-nanoparticle binding is also explored, by establishing a structure-activity relationship for binding using a series of unnatural amino acids with different lengths of hydrophobic side chains. These results will be useful for predicting which residues in a peptide are responsible for binding and for understanding the driving forces for binding between peptides and nanoparticles in future studies.
Project description:Methyl ferulate (MF) is an alkyl ferulate ester that widely exists in edible plants and has application value in the food and medicine industries. Thus, its effect on biological macromolecules should be considered. In this study, we exploit saturation transfer difference NMR (STD-NMR) to characterize the interaction of all protons of MF with human serum albumin (HSA) at the molecular level. STD-NMR and K a (1.298 × 103 M-1) revealed that protons H1-6 and H8 bound to HSA with a medium affinity. Binding epitope mapping further showed that the aromatic ring played a key role in the HSA-MF interaction. STD-NMR site-marker-displacement experiments and circular dichroism spectroscopy revealed that MF prefered to bind to site II of HSA without changing the basic skeleton of HSA. Computer simulations confirmed these experimental results. Overall, this work elucidates the molecular level interaction of MF with HSA and provides new insights into the possibility of the potential applications of MF in the food and medicine industries.
Project description:The stereoselective introduction of the glycosidic bond remains one of the main challenges in carbohydrate synthesis. Characterizing the reactive intermediates of this reaction is key to develop stereoselective glycosylation reactions. Herein we report the characterization of low-populated, rapidly equilibrating mannosyl dioxanium ions that arise from participation of a C-3 acyl group using chemical exchange saturation transfer (CEST) NMR spectroscopy. Dioxanium ion structure and equilibration kinetics were measured under relevant glycosylation conditions and highly α-selective couplings were observed suggesting glycosylation took place via this elusive intermediate.
Project description:Saturation-transfer difference (STD) NMR spectroscopy is a fast and versatile method which can be applied for drug-screening purposes, allowing the determination of essential ligand binding affinities (KD). Although widely employed to study soluble proteins, its use remains negligible for membrane proteins. Here the use of STD NMR for KD determination is demonstrated for two competing substrates with very different binding affinities (low nanomolar to millimolar) for an integral membrane transport protein in both detergent-solubilised micelles and reconstituted proteoliposomes. GltPh, a homotrimeric aspartate transporter from Pyrococcus horikoshii, is an archaeal homolog of mammalian membrane transport proteins-known as excitatory amino acid transporters (EAATs). They are found within the central nervous system and are responsible for fast uptake of the neurotransmitter glutamate, essential for neuronal function. Differences in both KD's and cooperativity are observed between detergent micelles and proteoliposomes, the physiological implications of which are discussed.
Project description:Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is the etiological cause of the coronavirus disease 2019, for which no effective antiviral therapeutics are available. The SARS-CoV-2 main protease (Mpro) is essential for viral replication and constitutes a promising therapeutic target. Many efforts aimed at deriving effective Mpro inhibitors are currently underway, including an international open-science discovery project, codenamed COVID Moonshot. As part of COVID Moonshot, we used saturation transfer difference nuclear magnetic resonance (STD-NMR) spectroscopy to assess the binding of putative Mpro ligands to the viral protease, including molecules identified by crystallographic fragment screening and novel compounds designed as Mpro inhibitors. In this manner, we aimed to complement enzymatic activity assays of Mpro performed by other groups with information on ligand affinity. We have made the Mpro STD-NMR data publicly available. Here, we provide detailed information on the NMR protocols used and challenges faced, thereby placing these data into context. Our goal is to assist the interpretation of Mpro STD-NMR data, thereby accelerating ongoing drug design efforts.
Project description:Human Galectin-3 (hGal-3) is a protein that selectively binds to β-galactosides and holds diverse roles in both normal and pathological circumstances. Therefore, targeting hGal-3 has become a vibrant area of research in the pharmaceutical chemistry. As a step towards the development of novel hGal-3 inhibitors, we synthesized and investigated derivatives of thiodigalactoside (TDG) modified with different aromatic substituents. Specifically, we describe a high-yielding synthetic route of thiodigalactoside (TDG); an optimized procedure for the synthesis of the novel 3,3'-di-O-(quinoline-2-yl)methyl)-TDG and three other known, symmetric 3,3'-di-O-TDG derivatives ((naphthalene-2yl)methyl, benzyl, (7-methoxy-2H-1-benzopyran-2-on-4-yl)methyl). In the present study, using competition Saturation Transfer Difference (STD) NMR spectroscopy, we determined the dissociation constant (Kd) of the former three TDG derivatives produced to characterize the strength of the interaction with the target protein (hGal-3). Based on the Kd values determined, the (naphthalen-2-yl)methyl, the (quinolin-2-yl)methyl and the benzyl derivatives bind to hGal-3 94, 30 and 24 times more strongly than TDG. Then, we studied the binding modes of the derivatives in silico by molecular docking calculations. Docking poses similar to the canonical binding modes of well-known hGal-3 inhibitors have been found. However, additional binding forces, cation-π interactions between the arginine residues in the binding pocket of the protein and the aromatic groups of the ligands, have been established as significant features. Our results offer a molecular-level understanding of the varying affinities observed among the synthesized thiodigalactoside derivatives, which can be a key aspect in the future development of more effective ligands of hGal-3.
Project description:Thioredoxins are small ubiquitous proteins that participate in a diverse variety of redox reactions via the reversible oxidation of two cysteine thiol groups in a structurally conserved active site. Here, the NMR solution structures of a reduced and oxidized thioredoxin from Ehrlichia chaffeensis (Ec-Trx, ECH_0218), the etiological agent responsible for human monocytic ehrlichiosis, are described. The overall topology of the calculated structures is similar in both redox states and is similar to those of other thioredoxins: a five-stranded, mixed β-sheet (β1-β3-β2-β4-β5) surrounded by four α-helices. Unlike other thioredoxins studied by NMR in both redox states, the 1H-15N HSQC spectrum of reduced Ec-Trx was missing eight additional amide cross peaks relative to the spectrum of oxidized Ec-Trx. These missing amides correspond to residues Cys35-Glu39 in the active-site-containing helix (α2) and Ser72-Ile75 in a loop near the active site, and suggest a change in backbone dynamics on the millisecond-to-microsecond timescale associated with the breakage of an intramolecular Cys32-Cys35 disulfide bond in a thioredoxin. A consequence of the missing amide resonances is the absence of observable or unambiguous NOEs to provide the distance restraints necessary to define the N-terminal end of the α-helix containing the CPGC active site in the reduced state. This region adopts a well defined α-helical structure in other reported reduced thioredoxin structures, is mostly helical in oxidized Ec-Trx and CD studies of Ec-Trx in both redox states suggests there is no significant difference in the secondary structure of the protein. The NMR solution structure of reduced Ec-Trx illustrates that the absence of canonical structure in a region of a protein may be owing to unfavorable dynamics prohibiting NOE observations or unambiguous NOE assignments.