Dynamic NMR Research Articles

Enhanced EphB2-Specific Peptide Inhibitors through Stabilization of Polyproline II Helical Structure.

Ephrin (Eph) receptors are the largest family of receptor tyrosine kinases. Interactions between Eph receptors and their membrane-bound ephrin protein ligands are associated with many developmental processes as well as various cancers and neurodegenerative diseases. With significant crosstalk between different Eph receptors and ephrin ligands, there is an urgent need for high-affinity ligands that bind specifically to individual Eph receptors to interrogate and modulate their functions. Here, we describe the rational development of potent EphB2 receptor inhibitors derived from the EphB2 receptor-specific SNEW peptide. To improve inhibitory potency, we evaluated 20+ cross-linkers with the goal of spanning and stabilizing a single polyproline II helical turn observed when SNEW binds to the EphB2 receptor. Of the cross-linkers evaluated, an 11-atom cross-linker, composed of a rigid 2,7-dimethylnaphthyl moiety between two cysteine residues, was found to yield the most potent inhibitor. Analysis of the cyclized region of this peptide by NMR and molecular dynamics simulations suggests that cross-linking stabilizes the receptor-bound polyproline II helix structure observed in the receptor-peptide complex. Cross-linked SNEW variants retained binding specificity for EphB2 and showed cross-linker-dependent resistance to trypsin proteolysis. Beyond the discovery of more potent EphB2 receptor inhibitors, these studies illustrate a novel cyclization approach with potential to stabilize polyproline II helical structure in various peptides for specific targeting of the myriad protein-protein interactions (PPIs) mediated by polyproline II helices.

Stability and dynamics of extradenticle modulates its function

Extradenticle (EXD) is a partner protein of the HOX transcription factors and plays an important role in the development of Drosophila. It confers increased affinity and specificity of DNA-binding to the HOX proteins. However, the DNA-binding homeodomain of EXD has a significantly weaker affinity to DNA compared to the HOX homeodomains. Here, we show that a glycine residue (G290) in the middle of the EXD DNA-binding helix primarily results in this weaker binding. Glycine destabilizes helices. To probe its role in the stability and function of the protein, G290 was mutated to alanine. The intrinsic stability of the DNA-binding helix increased in the G290A mutant as observed by NMR studies and molecular dynamics (MD) simulation. Also, NMR dynamics and MD simulation show that dynamic motions present in the wild-type protein are quenched in the mutant. This in turn resulted in increased stability of the entire homeodomain (ΔΔGG→A of -2.6 kcal/mol). Increased protein stability resulted in three-fold better DNA-binding affinity of the mutant as compared to the wild-type protein. Molecular mechanics with generalized Born and surface area solvation (MMGBSA) analysis of our MD simulation on DNA-bound models of both wild-type and mutant proteins shows that the contribution to binding is enhanced for most of the interface residues in the mutant compared to the wild-type. Interestingly, the flexible N-terminal arm makes more stable contact with the DNA minor groove in the mutant. We found that the two interaction sites i.e. the DNA-binding helix and the unstructured N-terminal arm influence each other via the bound DNA. These results provide an interesting conundrum: alanine at position 290 enhances both the stability and the DNA-binding affinity of the protein, however, evolution prefers glycine at this position. We have provided several plausible explanations for this apparent conundrum. The function of the EXD as a HOX co-factor requires its ability to discriminate similar DNA sequences, which is most likely comprom

Probing Homogeneous Catalysts and Precatalysts in Solution by Exchange-Mediated Overhauser Dynamic Nuclear Polarization NMR.

Triphenylphosphine (PPh3) is a ubiquitous ligand in organometallic chemistry that has been shown to give enhanced 31P NMR signals at high magnetic field via a scalar-dominated Overhauser effect dynamic nuclear polarization (OE DNP). However, PPh3 can only be polarized via DNP in the free form, while the coordinated form is DNP-inactive. Here, we demonstrate the possibility of enhancing the 31P NMR signals of coordinated PPh3 in metal complexes in solution at room temperature by combining Overhauser effect DNP and chemical exchange between the free and coordinated PPh3 forms. With this method, we successfully obtain 31P DNP enhancements of up to 2 orders of magnitude for the PPh3 ligands in Rh(I), Ru(II), Pd(II), and Pt(II) complexes, and we show that the DNP enhancements can be used to determine the activation energy of the ligand exchange reaction.

Formation of Stable Radicals by Mechanochemistry and Their Application for Magic Angle Spinning Dynamic Nuclear Polarization Solid-State NMR Spectroscopy.

High-field magic angle spinning (MAS) dynamic nuclear polarization (DNP) is becoming a common technique for improving the sensitivity of solid-state nuclear magnetic resonance (SSNMR) by the hyperpolarization of nuclear spins. Recently, we have shown that gamma irradiation is capable of creating long-lived free radicals that are amenable to MAS DNP in quartz and a variety of organic solids. Here, we demonstrate that ball milling is able to generate millimolar concentrations of stable radical species in diverse materials such as polystyrene, cellulose, borosilicate glass, and fused quartz. High-field electron paramagnetic resonance (EPR) was used to obtain further insight into the nature of the radicals formed in ball milled quartz and borosilicate glass. We further show that radicals generated in quartz by ball milling can be used for solid-effect DNP. We obtained 29Si DNP enhancements of approximately 114 and 33 at 110 K and room temperature, respectively, from a sample of ball milled quartz.

Beyond N-Alkylation: Synthesis, Structure, and Function of N-Amino Peptides.

ConspectusThe growing list of physiologically important protein-protein interactions (PPIs) has amplified the need for compounds to target topologically complex biomolecular surfaces. In contrast to small molecules, peptide and protein mimics can exhibit three-dimensional shape complementarity across a large area and thus have the potential to significantly expand the "druggable" proteome. Strategies to stabilize canonical protein secondary structures without sacrificing side-chain content are particularly useful in the design of peptide-based chemical probes and therapeutics.Substitution of the backbone amide in peptides represents a subtle chemical modification with profound effects on conformation and stability. Studies focused on N-alkylation have already led to broad-ranging applications in peptidomimetic design. Inspired by nonribosomal peptide natural products harboring amide N-oxidations, we envisioned that main-chain hydrazide and hydroxamate bonds would impose distinct conformational preferences and offer unique opportunities for backbone diversification. This Account describes our exploration of peptide N-amination as a strategy for stabilizing canonical protein folds and for the structure-based design of soluble amyloid mimics.We developed a general synthetic protocol to access N-amino peptides (NAPs) on solid support. In an effort to stabilize β-strand conformation, we designed stitched peptidomimetics featuring covalent tethering of the backbone N-amino substituent to the preceding residue side chain. Using a combination of NMR, X-ray crystallography, and molecular dynamics simulations, we discovered that backbone N-amination alone could significantly stabilize β-hairpin conformation in multiple models of folding. Our studies revealed that the amide NH2 substituent in NAPs participates in cooperative noncovalent interactions that promote β-sheet secondary structure. In contrast to Cα-substituted α-hydrazino acids, we found that N-aminoglycine and its N'-alkylated derivatives instead stabilize polyproline II (PPII) conformation. The reactivity of hydrazides also allows for late-stage peptide macrocyclization, affording novel covalent surrogates of side-chain-backbone H-bonds.The pronounced β-sheet propensity of Cα-substituted α-hydrazino acids prompted us to target amyloidogenic proteins using NAP-based β-strand mimics. Backbone N-amination was found to render aggregation-prone lead sequences soluble and resistant to proteolysis. Inhibitors of Aβ and tau identified through N-amino scanning blocked protein aggregation and the formation of mature fibrils in vitro. We further identified NAP-based single-strand and cross-β tau mimics capable of inhibiting the prion-like cellular seeding activity of recombinant and patient-derived tau fibrils.Our studies establish backbone N-amination as a valuable addition to the peptido- and proteomimetic tool kit. α-Hydrazino acids show particular promise as minimalist β-strand mimics that retain side-chain information. Late-stage derivatization of hydrazides also provides facile entry into libraries of backbone-edited peptides. We anticipate that NAPs will thus find applications in the development of optimally constrained folds and modulators of PPIs.

Composition and Conformation of Hetero- versus Homo-Fluorinated Triazolamers Influence their Activity on Islet Amyloid Polypeptide Aggregation.

Novel fluorinated foldamers based on aminomethyl-1,4-triazolyl-difluoroacetic acid (1,4-Tz-CF2) units were synthesized and their conformational behaviour was studied by NMR and molecular dynamics. Their activity on the aggregation of the human islet amyloid polypeptide (hIAPP) amyloid protein was evaluated by fluorescence spectroscopy and mass spectrometry. The fluorine labelling of these foldamers allowed the analysis of their interaction with the target protein. We demonstrated that the preferred extended conformation of homotriazolamers of 1,4-Tz-CF2 unit increases the aggregation of hIAPP, while the hairpin-like conformation of more flexible heterotriazolamers containing two 1,4-Tz-CF2 units mixed with natural amino acids from the hIAPP sequence reduces it, and more efficiently than the parent natural peptide. The longer heterotriazolamers having three 1,4-Tz-CF2 units adopting more folded hairpin-like and ladder-like structures similar to short multi-stranded β-sheets have no effect. This work demonstrates that a good balance between the structuring and flexibility of these foldamers is necessary to allow efficient interaction with the target protein.

Supramolecular citrate poly allylamine hydrochloride nanoparticles for citrate delivery and calcium oxalate nanocrystal dissolution

HypothesisRenal calculi (kidney stones) are mainly made by calcium oxalate and can cause different complications including malfunction of the kidney. The most important urinary stone inhibitors are citrate molecules. Unfortunately, the amount of citrate reaching the kidney after oral ingestion is low. We hypothesized that nanoparticles of polyallylamine hydrochloride (CIT-PAH) carrying citrate ions could simultaneously deliver citrates while PAH would complex oxalate triggering dissolution and removal of CaOx nanocrystals. ExperimentsWe successfully prepared nanoparticles of citrate ions with polyallylamine hydrochloride (CIT-PAH), PAH with oxalate (OX-PAH) and characterize them by Small Angle X ray Scattering (SAXS), Transmission Electron Microscopy (TEM), Dynamic Light Scattering (DLS) and NMR. Dissolution of CaOx nanocrystals in presence of CIT-PAH have been followed with Wide Angle Xray Scattering (WAXS), DLS and Confocal Raman Microscopy. Raman spectroscopy was used to study the dissolution of crystals in synthetic urine samples. The release of citrate from CIT-PAH was followed by diffusion NMR. Molecular dynamics (MD) simulations were carried out to study the interaction of CIT and OX ions with PAH. FindingsCIT-PAH nanoparticles dissolves CaOx nanocrysPAH.as shown by NMR, DLS, TEM and WAXS in water and by Raman spectroscopy in artificial human urine. WAXS and Raman show that the crystal structure of CaOx disappears in the presence of CIT-PAH. DLS shows that the time required for CaOX dissolution will depend on the concentration of CIT-PAH NPs. NMR proves that citrate ions are released from the CIT PAH NPs during CaOX dissolution, MD simulations showed that oxalates exhibit a stronger interaction for PAH than citrate, explaining the removal of oxalate ions and replacement of the citrate in the polymer nanoparticles.

Preparation of indapamide-HP-β-CD and indapamide-PVP nanoparticles by supercritical antisolvent technology: Experimental and DPD simulations

The poor aqueous solubility of indapamide (INP) limits its applicability in various biomedical applications. To overcome this issue, nanosized INP particles (INP-HP-β-CD NPs and INP-PVP-K30 NPs) were fabricated using the supercritical antisolvent (SAS) approach to improve the bioavailability of INP. The process, formula, and supercritical antisolvent process were optimized using single-factor and orthogonal methods. The tiny INP-HP-β-CD NPs with a particle size of 142.5nm and INP-PVP-K30 NPs with a particle size of 150.4nm were obtained under optimum conditions. Further analysis and characterization, such as SEM, FTIR, XRD, DSC, 1H NMR, and dissipative particle dynamics (DPD) simulations, were carried out to verify the formation mechanism of the INP complex nanoparticles. The altered physical state of INP from crystalline to amorphous loaded onto the polymer resulted in a significant improvement in the solubility and dissolution rate and high biocompatibility with cells compared to raw INP. Therefore, we propose that INP nanoparticles processed by a supercritical antisolvent process can be used as an advanced drug delivery system, especially for insoluble drugs.

Deciphering the Multi-state Conformational Equilibrium of HDM2 in the Regulation of p53 Binding: Perspectives from Molecular Dynamics Simulation and NMR Analysis.

HDM2 negatively regulates the activity of the tumor suppressor p53. Previous NMR studies have shown that apo-HDM2 interconverts between an "open" state in which the N-terminal "lid" is disordered and a "closed" state in which the lid covers the p53-binding site in the core region. Molecular dynamics (MD) simulation studies have been performed to elucidate the conformational dynamics of HDM2, but the direct relevance of the experimental and computational analyses is unclear. In addition, how the phosphorylation of S17 in the lid contributes to the inhibition of p53 binding remains controversial. Here, we used both NMR and MD simulations to investigate the conformational dynamics of apo-HDM2. The NMR analysis revealed that apo-HDM2 exists in a fast-exchanging equilibrium within two closed states, closed 1 and closed 2, in addition to a previously demonstrated slow-exchanging "open-closed" equilibrium. MD simulations visualized two characteristic closed states, where the spatial orientation of the key residues corresponds well to the chemical shift changes of the NMR spectra. Furthermore, the phosphorylation of S17 induced an equilibrium shift toward closed 1, thereby suppressing the binding of p53 to HDM2. This study reveals a multi-state equilibrium of apo-HDM2 and provides new insights into the regulation mechanism of HDM2-p53 interactions.

Study of the Reversible Hawthorne Rearrangement between Isomeric Forms of the Octadecahydroeicosaborate Anion Using Dynamic 11B NMR Spectroscopy

Study of the Reversible Hawthorne Rearrangement between Isomeric Forms of the Octadecahydroeicosaborate Anion Using Dynamic 11B NMR Spectroscopy

Cellular Applications of DNP Solid-State NMR - State of the Art and a Look to the Future.

Sensitivity enhanced dynamic nuclear polarization solid-state NMR is emerging as a powerful technique for probing the structural properties of conformationally homogenous and heterogenous biomolecular species irrespective of size at atomic resolution within their native environments. Herein we detail advancements that have made acquiring such data, specifically within the confines of intact bacterial and eukaryotic cell a reality and further discuss the type of structural information that can presently be garnered by the technique's exploitation. Subsequently, we discuss bottlenecks that have thus far curbed cellular DNP-ssNMR's broader adoption namely due a lack of sensitivity and spectral resolution. We also explore possible solutions ranging from utilization of new pulse sequences, design of better performing polarizing agents, and application of additional biochemical/ cell biological methodologies.

A Synthetic Strategy toward S-, N-, and O-Heterocyclooctynes Facilitates Bioconjugation Using Multifunctional Handles.

Over the past two decades, the introduction of bioorthogonal reactions has transformed the ways in which chemoselective labeling, isolation, imaging, and drug delivery are carried out in a complex biological milieu. A key feature of a good bioorthogonal probe is the ease with which it can be attached to a target compound through bioconjugation. This paper describes the expansion of the utility of a class of unique S-, N-, and O-containing heterocyclooctynes (SNO-OCTs), which show chemoselective reactivity with type I and type II dipoles and divergent reactivities in response to electronic tuning of the alkyne. Currently, bioconjugation of SNO-OCTs to a desired target is achieved through an inconvenient aryl or amide linker at the sulfamate nitrogen. Herein, a new synthetic approach toward general SNO-OCT scaffolds is demonstrated that enables the installation of functional handles at both propargylic carbons of the heterocycloalkyne. This capability increases the utility of SNO-OCTs as labeling reagents through the design of bifunctional bioorthogonal probes with expanded capabilities. NMR kinetics also revealed up to sixfold improvement in cycloaddition rates of new analogues compared to first-generation SNO-OCTs.

Insights into Molecular Diversity within the FUS/EWS/TAF15 Protein Family: Unraveling Phase Separation of the N-Terminal Low-Complexity Domain from RNA-Binding Protein EWS.

The FET protein family, comprising FUS, EWS, and TAF15, plays crucial roles in mRNA maturation, transcriptional regulation, and DNA damage response. Clinically, they are linked to Ewing family tumors and neurodegenerative diseases such as amyotrophic lateral sclerosis. The fusion protein EWS::FLI1, the causative mutation of Ewing sarcoma, arises from a genomic translocation that fuses a portion of the low-complexity domain (LCD) of EWS (EWSLCD) with the DNA binding domain of the ETS transcription factor FLI1. This fusion protein modifies transcriptional programs and disrupts native EWS functions, such as splicing. The exact role of the intrinsically disordered EWSLCD remains a topic of active investigation, but its ability to phase separate and form biomolecular condensates is believed to be central to EWS::FLI1's oncogenic properties. Here, we used paramagnetic relaxation enhancement NMR, microscopy, and all-atom molecular dynamics (MD) simulations to better understand the self-association and phase separation tendencies of the EWSLCD. Our NMR data and mutational analysis suggest that a higher density and proximity of tyrosine residues amplify the likelihood of condensate formation. MD simulations revealed that the tyrosine-rich termini exhibit compact conformations with unique contact networks and provided critical input on the relationship between contacts formed within a single molecule (intramolecular) and inside the condensed phase (intermolecular). These findings enhance our understanding of FET proteins' condensate-forming capabilities and underline differences between EWS, FUS, and TAF15.

Comparative Conformational Analysis of Acyclic Sugar Alcohols Ribitol, Xylitol and d-Arabitol by Solution NMR and Molecular Dynamics Simulations.

Ribitol (C5H12O5) is an acyclic sugar alcohol that was recently identified in O-mannose glycan on mammalian α-dystroglycan. The conformation and dynamics of acyclic sugar alcohols such as ribitol are dependent on the stereochemistry of the hydroxyl groups; however, the dynamics are not fully understood. To gain insights into the conformation and dynamics of sugar alcohols, we carried out comparative analyses of ribitol, d-arabitol and xylitol by a crystal structure database search, solution NMR analysis and molecular dynamics (MD) simulations. The crystal structures of the sugar alcohols showed a limited number of conformations, suggesting that only certain stable conformations are prevalent among all possible conformations. The three-bond scholar coupling constants and exchange rates of hydroxyl protons were measured to obtain information on the backbone torsion angle and possible hydrogen bonding of each hydroxyl group. The 100 ns MD simulations indicate that the ribitol backbone has frequent conformational transitions with torsion angles between 180∘ and ±60∘, while d-arabitol and xylitol showed fewer conformational transitions. Taking our experimental and computational data together, it can be concluded that ribitol is more flexible than d-arabitol or xylitol, and the flexibility is at least in part defined by the configuration of the OH groups, which may form intramolecular hydrogen bonds.

Forensic implications of novel synthesis of cathinone derivatives by Neber and modified Neber rearrangements

Cathinone and its synthetic analogues are known compounds of clandestine interest. Investigation into novel pathways for synthesising cathinone derivatives has potential for forensic analysis and tracking. The known Neber rearrangement of commercially available phenylpropanones that were evaluated yielded amides described herein and was not viable for clandestine synthesis of cathinone derivatives. Whereas the known modified Neber rearrangement of phenylpropanones that were evaluated via stable aziridine salts and subsequent treatment with acid, gave cathinone derivatives described herein in poor to low yields (2–17%). Assessment of the reagents, equipment, and procedures required for the modified Neber rearrangement was considered as only viable for more advanced clandestine operations. An improved understanding of the potential by-product formation from the modified Neber rearrangement was determined by density functional theory (DFT) of hydrazone to azirine to aziridine intermediates and attempted dynamic NMR spectroscopy of a hydrazone described herein. The substantially lower energy of the azirine step compared to the starting hydrazonium salt step of the reaction mechanism implied that the azirine structure was a short-lived intermediate, and unable to be experimentally determined. New mass-spectral fragmentation data of compounds described herein was reported, where differentiation was observed for some individual compounds at the GC-EIMS fragmentation pattern level. From this study, individual mass-spectrometry fragmentation of key compounds evaluated from the modified Neber rearrangement of commercially available phenylpropanones indicates potential for forensic profiling analysis applications.

Insight into Formation, Synchronized Release and Stability of Co-Amorphous Curcumin-Piperine by Integrating Experimental-Modeling Techniques

Intermolecular interactions between drug and co-former are crucial in the formation, release and physical stability of co-amorphous system. However, the interactions remain difficult to investigate with only experimental tools. In this study, intermolecular interactions of co-amorphous curcumin-piperine (i.e., CUR-PIP CM) during formation, dissolution and storage were explored by integrating experimental and modeling techniques. The formed CUR-PIP CM exhibited the strong hydrogen bond interaction between the phenolic OH group of CUR and the CO group of PIP as confirmed by FTIR, ss 13C NMR and molecular dynamics (MD) simulation. In comparison to crystalline CUR, crystalline PIP and their physical mixture, CUR-PIP CM performed significantly increased dissolution accompanied by the synchronized release of CUR and PIP, which arose from the greater interaction energy of H2O-CUR molecules and H2O-PIP molecules than CUR-PIP molecules, breaking the hydrogen bond between CUR and PIP molecules, and then causing a pair-wise solvation of CUR-PIP CM at the molecular level. Furthermore, the stronger intermolecular interaction between CUR and PIP was revealed by higher binding energy of CUR-PIP molecules, which contributed to the excellent physical stability of CUR-PIP CM over amorphous CUR or PIP. The study provides a unique insight into the formation, release and stability of co-amorphous system from MD perspective. Meanwhile, this integrated technique can be used as a practical methodology for the future design of co-amorphous formulations.

Bis(N-xylyl-imino)phenyl “NCN” iridium pincer complexes. Thermodynamics of ligand binding and C[sbnd]C bond cleavage

Iridium dibromide complexes of the phenyldiimine ligand 2,6-bis(1-((2,6-dimethylphenyl)imino)ethyl)phenyl, trans-(XyPhDI)IrBr2L, have been synthesized, and relative Ir-L BDFEs have been experimentally determined for a wide range of corresponding adducts of ligands L. An estimate of the absolute enthalpy of Ir-L binding has been obtained from dynamic NMR measurements. The results of DFT calculations are in very good agreement with the relative and absolute experimental values. Computational studies were extended to the formation of adducts of (XyPhDI)IrH2 and (XyPhDI)IrI, as well as other (pincer)IrI fragments, (Phebox)IrI and (PCP)IrI, to enable a comparison of electronic and steric effects with these archetypal pincer ligands. Attempts to reduce (XyPhDI)IrBr2(MeCN) to a hydride or an IrI complex yielded a dinuclear CN-bridged complex with a methyl ligand on the cyanide-C-bound Ir center (characterized by scXRD), indicating that C-CN bond cleavage took place at that Ir center. DFT calculations indicate that the C-CN bond cleavage occurs at one Ir center with strong assistance by coordination of the CN nitrogen to the other Ir center.

Kinetic behavior of the network structure changes in funoran gelation studied by rheological and NMR measurements

Funoran is a promising food polysaccharide with unique gelling properties but understanding its gelation behaviors remains inadequate. This study aimed to investigate the influence of cooling and heating rates on funoran gelation properties and study its gelation mechanism by dynamic rheology and NMR studies. With constant heating rates, the melting point was unaffected by changing cooling rates, whereas higher cooling and heating rates enhance the gel strength and thermal hysteresis. The gelation kinetics mechanism of the funoran solution was elucidated at different quenching temperatures. The rheological measurement results indicated that the funoran solution quenched to lower temperatures resulted in higher G` in the gel state, and faster gelation rate compared with the solution quenched at higher temperatures. 1H NMR results revealed a slow decay in funoran signal intensity at higher temperatures (20 °C) and a fast signal decay at 2 °C. A theoretical model involving second and first-order reactions was proposed to describe the gelation kinetic process of funoran at different quenching temperatures. It was shown longer helix lengths were required for stable intermolecular association and gel formation at higher temperatures than at lower temperatures. These results offer valuable insights for optimizing the funoran gelation properties for various food applications.

Evaluating binding and interaction of selected pesticides with serum albumin proteins by Raman, 1H NMR, mass spectrometry and molecular dynamics simulation

Addressing the acute pesticide poisoning and toxicity to humans, is a global challenge of top priority. Serum albumin is the most abundant plasma protein, capable of binding with herbicide and pesticide residues. This study reports multifaceted approaches for in-depth and robust investigation of the molecular interactions of selected pesticides, including propanil (PPL), bromoxynil (BXL), metolachlor (MLR) and glyphosate (GPE) with bovine serum albumin (BSA) proteins using experimental (Raman and FTIR spectroscopy, native mass spectrometry and high field 1H NMR), molecular dynamics (MD) simulation and principal component analysis (PCA). The binding of pesticides with BSA resulted in BSA amide I and amide II Raman spectral shifts. PCA of Raman spectra of serum-pesticide complexes showed the grouping of pesticides on the score plot based on the similarities and differences in pesticides’ chemical structures. Native mass spectrometry results revealed strong adduct formation of the pesticides with the protein. The observed changes in chemical shifts, peak broadening or peak disappearance of characteristic proton signals of the pesticides, indicated altered chemical environments due to binding BSA-pesticides interactions. The results of MD simulation conducted for over 500 ns revealed strong pesticides interaction with LEU197, LEU218, LEU237, TRP213, SER286 and ILE289 residues to the site I of BSA. Free energy landscapes provided insights into the conformational changes in BSA on the binding of pesticides. Overall, the experimental and computational results are in consonant and indicate the binding of pesticides into the site I and site II (sub-domain IIA) of the BSA via hydrogen bonding, non-covalent and hydrophobic interactions. Communicated by Ramaswamy H. Sarma

One touch is all it takes: the supramolecular interaction between ubiquitin and lanthanide complexes revisited by paramagnetic NMR and molecular dynamics.

The supramolecular interaction between lanthanide complexes and proteins is at the heart of numerous chemical and biological studies. Some of these complexes have demonstrated remarkable interaction properties with proteins or peptides in solution and in the crystalline state. Here we have used the paramagnetism of lanthanide ions to characterize the affinity of two lanthanide complexes for ubiquitin. As the interaction process is dynamic, the acquired NMR data only reflect the time average of the different steps. We have used molecular dynamics (MD) simulations to get a deeper insight into the detailed interaction scenario at the microsecond scale. This NMR/MD approach enabled us to establish that the tris-dipicolinate complex interacts specifically with arginines and lysines, while the crystallophore explores the protein surface through weak interactions with carboxylates. These observations shed new light on the dynamic interaction properties of these complexes, which will ultimately enable us to propose a crystallization mechanism.