Transferred Nuclear Overhauser Effect Research Articles

Identifying and Overcoming Artifacts in 1 H-Based Saturation Transfer NOE NMR Experiments.

Magnetization transfer experiments are versatile nuclear magnetic resonance (NMR) tools providing site-specific information. We have recently discussed how saturation magnetization transfer (SMT) experiments could leverage repeated repolarizations arising from exchanges between labile and water protons to enhance connectivities revealed via the nuclear Overhauser effect (NOE). Repeated experience with SMT has shown that a number of artifacts may arise in these experiments, which may confound the information being sought - particularly when seeking small NOEs among closely spaced resonances. One of these pertains to what we refer to as "spill-over" effects, originating from the use of long saturation pulses leading to changes in the signals of proximate peaks. A second, related but in fact different effect, derives from what we describe as NOE "oversaturation", a phenomenon whereby the use of overtly intense RF fields overwhelms the cross-relaxation signature. The origin and ways to avoid these two effects are described. A final source of potential artifact arises in applications where the labile 1Hs of interest are bound to 15N-labeled heteronuclei. SMT's long 1H saturation times will then be usually implemented while under 15N decoupling based on cyclic schemes leading to decoupling sidebands. Although these sidebands usually remain invisible in NMR, they may lead to a very efficient saturation of the main resonance when touched by SMT frequencies. All of these phenomena are herein experimentally demonstrated, and solutions to overcome them are proposed.

E6AP AZUL interaction with UBQLN1/2 in cells, condensates, and an AlphaFold-NMR integrated structure

The E3 ligase E6AP/UBE3A has a dedicated binding site in the 26S proteasome provided by the RAZUL domain of substrate receptor hRpn10/S5a/PSMD4. Guided by RAZUL sequence similarity, we test and demonstrate here that the E6AP AZUL binds transiently to the UBA of proteasomal shuttle factor UBQLN1/2. Despite a weak binding affinity, E6AP AZUL is recruited to UBQLN2 biomolecular condensates invitro and E6AP interacts with UBQLN1/2 in cellulo. Steady-state and transfer nuclear Overhauser effect (NOE) experiments indicate direct interaction of AZUL with UBQLN1UBA. Intermolecular contacts identified by NOE spectroscopy (NOESY) data were combined with AlphaFold2-Multimer predictions to yield an AZUL:UBA model structure. We additionally identify an oligomerization domain directly adjacent to UBQLN1/2UBA (UBA adjacent [UBAA]) that is α-helical and allosterically reconfigured by AZUL binding to UBA. These data lead to a model of E6AP recruitment to UBQLN1/2 by AZUL:UBA interaction and provide fundamental information on binding requirements for interactions in condensates and cells.

Structural Basis of Artemisinin Binding Sites in Serum Albumin with the Combined Use of NMR and Docking Calculations.

Artemisinin is known to bind to the main plasma protein carrier serum albumin (SA); however, there are no atomic level structural data regarding its binding mode with serum albumin. Herein, we employed a combined strategy of saturation transfer difference (STD), transfer nuclear Overhauser effect spectroscopy (TR-NOESY), STD–total correlation spectroscopy (STD-TOCSY), and Interligand Noes for PHArmacophore Mapping (INPHARMA) NMR methods and molecular docking calculations to investigate the structural basis of the interaction of artemisinin with human and bovine serum albumin (HSA/BSA). A significant number of inter-ligand NOEs between artemisinin and the drugs warfarin and ibuprofen as well as docking calculations were interpreted in terms of competitive binding modes of artemisinin in the warfarin (FA7) and ibuprofen (FA4) binding sites. STD NMR experiments demonstrate that artemisinin is the main analyte for the interaction of the A. annua extract with BSA. The combined strategy of NMR and docking calculations of the present work could be of general interest in the identification of the molecular basis of the interactions of natural products with their receptors even within a complex crude extract.

Defining specific residue‐to‐residue interactions between the gp120 bridging sheet and the N‐terminal segment of CCR5: applications of transferred NOE NMR

Infection by HIV-1 requires protein-protein interactions involving gp120, CD4 and CCR5. We have previously demonstrated that the transferred nuclear Overhauser effect (TRNOE), in combination with asymmetric deuteration of a protein and a peptide ligand can be used to detect intermolecular interactions in large protein complexes with molecular weights up to ~ 100 kDa. Here, using this approach, we reveal interactions between tyrosine residues of a 27-residue peptide corresponding to the N-terminal segment of the CCR5 chemokine receptor, and a dimeric extended core YU 2 gp120 envelope protein of HIV-1 complexed with a CD4-mimic miniprotein. The TRNOE crosspeaks in the ternary complex were assigned to the specific Tyr protons in the CCR5 peptide and to methyl protons of isoleucine, leucine and/or valine residues of gp120. Site directed mutagenesis combined with selective deuteration and TRNOE resulted in the first discernment by a biophysical method of specific pairwise interactions between gp120 residues in the bridging sheet of gp120 and the N-terminus of CCR5.

Detection of intermolecular transferred NOEs in large protein complexes using asymmetric deuteration: HIV-1 gp120 in complex with a CCR5 peptide.

Weak protein-protein and protein-ligand interactions play important roles in biological recognition. In many cases, simplification of structural studies of large protein complexes is achieved by investigation of the interaction between the protein and a weakly binding segment of its protein ligand. Detection of pairwise interactions in such complexes is a major challenge for both X-ray crystallography and nuclear magnetic resonance. We demonstrate that transferred nuclear Overhauser effect (TRNOE), in combination with asymmetric deuteration of a protein and a peptide ligand can be used to detect intermolecular interactions in large protein complexes with molecular weights up to ~ 100 kDa. Using this approach, we revealed interactions between tyrosine residues of a 27-residue peptide (deuterated at Ile and Val residues) corresponding to the N-terminal segment of the human C-C chemokine receptor 5 (CCR5) chemokine receptor, and a 43 kDa construct of gp120 envelope protein of human immunodeficiency virus type 1 (deuterated on all aromatics) complexed with a cluster of differentiation 4-mimic miniprotein. The complex was present mostly as a dimer as determined by T2 relaxation measurements. The TRNOE crosspeaks in the ternary complex were assigned to the specific Tyr protons in the CCR5 peptide and to methyl protons, predominantly of isoleucine residues, and also of leucine and/or valine residues of gp120. The TRNOE/asymmetric deuteration method benefits from the sensitivity of the homonuclear NOESY experiment and does not suffer the sensitivity losses associated with isotope-edited/isotope-filtered approaches that rely on magnetization transfer between protons and heteronuclei that are bonded to them. The technique can be widely applied for studying large protein complexes that exhibit fast off-rates.

Lipopolysaccharide-bound structure of the antimicrobial peptide cecropin P1 determined by nuclear magnetic resonance spectroscopy.

Antimicrobial peptides (AMPs) are components of the innate immune system and may be potential alternatives to conventional antibiotics because they exhibit broad-spectrum antimicrobial activity. The AMP cecropin P1 (CP1), isolated from nematodes found in the stomachs of pigs, is known to exhibit antimicrobial activity against Gram-negative bacteria. In this study, we investigated the interaction between CP1 and lipopolysaccharide (LPS), which is the main component of the outer membrane of Gram-negative bacteria, using circular dichroism (CD) and nuclear magnetic resonance (NMR). CD results showed that CP1 formed an α-helical structure in a solution containing LPS. For NMR experiments, we expressed (15) N-labeled and (13) C-labeled CP1 in bacterial cells and successfully assigned almost all backbone and side-chain proton resonance peaks of CP1 in water for transferred nuclear Overhauser effect (Tr-NOE) experiments in LPS. We performed (15) N-edited and (13) C-edited Tr-NOE spectroscopy for CP1 bound to LPS. Tr-NOE peaks were observed at the only C-terminal region of CP1 in LPS. The results of structure calculation indicated that the C-terminal region (Lys15-Gly29) formed the well-defined α-helical structure in LPS. Finally, the docking study revealed that Lys15/Lys16 interacted with phosphate at glucosamine I via an electrostatic interaction and that Ile22/Ile26 was in close proximity with the acyl chain of lipid A.

Thymosin α1 Interacts with Exposed Phosphatidylserine in Membrane Models and in Cells and Uses Serum Albumin as a Carrier.

Thymosin α1 is a peptidic hormone with pleiotropic activity and is used in the therapy of several diseases. It is unstructured in water solution and interacts with negative regions of vesicles by assuming two tracts of helical conformation with a structural break between them. This study reports on Thymosin α1's interaction with mixed phospholipids phosphatidylcholine and phosphatidylserine, the negative component of the membranes, by ¹H and natural abundance ¹⁵N nuclear magnetic resonance (NMR). The results indicate that interaction occurs when the membrane is negatively charged by exposing phosphatidylserine. Moreover, the direct interaction of Thymosin α1 with K562 cells with an overexposure of phosphatidylserine as a consequence of resveratrol-induced apoptosis was conducted. Thymosin α1's interaction with human serum albumin was also investigated by NMR spectroscopy. Steady-state saturation transfer, transfer nuclear Overhauser effect spectroscopy, and diffusion-ordered spectroscopy methodologies all reveal that the C-terminal region of Thymosin α1 is involved in the interaction with serum albumin. These results may shed more light on Thymosin α1's mechanism of action by its insertion in negative regions of membranes due to the exposure of phosphatidylserine. Once Thymosin α1's N-terminus has been inserted into the membrane, the rest may interact with nearby proteins and/or receptors acting as effectors and causing a biological signaling cascade, thus exerting thymosin α1's pleiotropy.

Probing membrane protein structure using water polarization transfer solid-state NMR

Water plays an essential role in the structure and function of proteins, lipid membranes and other biological macromolecules. Solid-state NMR heteronuclear-detected 1H polarization transfer from water to biomolecules is a versatile approach for studying water–protein, water–membrane, and water–carbohydrate interactions in biology. We review radiofrequency pulse sequences for measuring water polarization transfer to biomolecules, the mechanisms of polarization transfer, and the application of this method to various biological systems. Three polarization transfer mechanisms, chemical exchange, spin diffusion and NOE, manifest themselves at different temperatures, magic-angle-spinning frequencies, and pulse irradiations. Chemical exchange is ubiquitous in all systems examined so far, and spin diffusion plays the key role in polarization transfer within the macromolecule. Tightly bound water molecules with long residence times are rare in proteins at ambient temperature. The water polarization-transfer technique has been used to study the hydration of microcrystalline proteins, lipid membranes, and plant cell wall polysaccharides, and to derive atomic-resolution details of the kinetics and mechanism of ion conduction in channels and pumps. Using this approach, we have measured the water polarization transfer to the transmembrane domain of the influenza M2 protein to obtain information on the structure of this tetrameric proton channel. At short mixing times, the polarization transfer rates are site-specific and depend on the pH, labile protons, sidechain conformation, as well as the radial position of the residues in this four-helix bundle. Despite the multiple dependences, the initial transfer rates reflect the periodic nature of the residue positions from the water-filled pore, thus this technique provides a way of gleaning secondary structure information, helix tilt angle, and the oligomeric structure of membrane proteins.

Understanding of Protein-Ligand Interactions with INPHARMA

The major impediment in application of structure based drug design is insufficient information about interaction between small molecule and its target protein. Although crystallographic and protein based NMR methods provide superb input to the protein – ligand interaction, the inability to obtain crystals for many proteins of interest in parallel with a 50 kDa cut off for standard NMR techniques, highlights a need for alternative techniques. The newly established INPHARMA (Interligand NOEs for Pharmacophore Mapping) method fills this gap. The INPHARMA enables pharmacophore mapping by means of NOE transfer from one ligand to another via protein protons in their common binding site. The presence of INPHARMA between two compounds is indicative of their relative orientation, and if the binding site of one of them is known, information on the binding modes of both compounds can be obtained.

Determination of the binding epitope of RGD-peptidomimetics to αvβ3 and αIIbβ3 integrin-rich intact cells by NMR and computational studies

NMR experiments (transferred NOE and Saturation Transfer Difference) were used to shed light on the binding epitope of RGD peptidomimetics 1-3 with integrins αvβ3 and α(IIb)β3, expressed on the membrane of ECV304 bladder cancer cells and human platelets, respectively. The NMR results were supported by docking calculations of 1-3 in the active sites of αvβ3 and α(IIb)β3 integrin receptors and were compared to the results of competitive αvβ3 receptor binding assays and competitive ECV304 cell adhesion experiments. While cis RGD ligand 1 interacts mainly with the α integrin subunit through its basic guanidine group, trans RGD ligands 2 and 3 are able to interact with both the α and β integrin subunits via an electrostatic clamp.

NMR Spectroscopic Analysis Reveals Extensive Binding Interactions of Complex Xyloglucan Oligosaccharides with the Cellvibrio japonicus Glycoside Hydrolase Family 31 α‐Xylosidase

The study of the interaction of glycoside hydrolases with their substrates is fundamental to diverse applications in medicine, food and feed production, and biomass-resource utilization. Recent molecular modeling of the α-xylosidase CjXyl31A from the soil saprophyte Cellvibrio japonicus, together with protein crystallography and enzyme-kinetic analysis, has suggested that an appended PA14 protein domain, unique among glycoside hydrolase family 31 members, may confer specificity for large oligosaccharide fragments of the ubiquitous plant polysaccharide xyloglucan (J. Larsbrink, A. Izumi, F.M. Ibatullin, A. Nakhai, H.J. Gilbert, G.J. Davies, H. Brumer, Biochem. J. 2011, 436, 567-580). In the present study, a combination of NMR spectroscopic techniques, including saturation transfer difference (STD) and transfer NOE (TR-NOE) spectroscopy, was used to reveal extensive interactions between CjXyl31A active-site variants and xyloglucan hexa- and heptasaccharides. The data specifically indicate that the enzyme recognizes the entire cello-tetraosyl backbone of the substrate and product in positive enzyme subsites and makes further significant interactions with internal pendant α-(1→6)-linked xylosyl units. As such, the present analysis provides an important rationalization of previous kinetic data on CjXyl31A and unique insight into the role of the PA14 domain, which was not otherwise obtainable by protein crystallography.

A foldamer-dendrimer conjugate neutralizes synaptotoxic β-amyloid oligomers.

Background and AimsUnnatural self-organizing biomimetic polymers (foldamers) emerged as promising materials for biomolecule recognition and inhibition. Our goal was to construct multivalent foldamer-dendrimer conjugates which wrap the synaptotoxic β-amyloid (Aβ) oligomers with high affinity through their helical foldamer tentacles. Oligomeric Aβ species play pivotal role in Alzheimer's disease, therefore recognition and direct inhibition of this undruggable target is a great current challenge.Methods and ResultsShort helical β-peptide foldamers with designed secondary structures and side chain chemistry patterns were applied as potential recognition segments and their binding to the target was tested with NMR methods (saturation transfer difference and transferred-nuclear Overhauser effect). Helices exhibiting binding in the µM region were coupled to a tetravalent G0-PAMAM dendrimer. In vitro biophysical (isothermal titration calorimetry, dynamic light scattering, transmission electron microscopy and size-exclusion chromatography) and biochemical tests (ELISA and dot blot) indicated the tight binding between the foldamer conjugates and the Aβ oligomers. Moreover, a selective low nM interaction with the low molecular weight fraction of the Aβ oligomers was found. Ex vivo electrophysiological experiments revealed that the new material rescues the long-term potentiation from the toxic Aβ oligomers in mouse hippocampal slices at submicromolar concentration.ConclusionsThe combination of the foldamer methodology, the fragment-based approach and the multivalent design offers a pathway to unnatural protein mimetics that are capable of specific molecular recognition, and has already resulted in an inhibitor for an extremely difficult target.

Atomic-level structure characterization of an ultrafast folding mini-protein denatured state.

Atomic-level analyses of non-native protein ensembles constitute an important aspect of protein folding studies to reach a more complete understanding of how proteins attain their native form exhibiting biological activity. Previously, formation of hydrophobic clusters in the 6 M urea-denatured state of an ultrafast folding mini-protein known as TC5b from both photo-CIDNP NOE transfer studies and FCS measurements was observed. Here, we elucidate the structural properties of this mini-protein denatured in 6 M urea performing 15N NMR relaxation studies together with a thorough NOE analysis. Even though our results demonstrate that no elements of secondary structure persist in the denatured state, the heterogeneous distribution of R 2 rate constants together with observing pronounced heteronuclear NOEs along the peptide backbone reveals specific regions of urea-denatured TC5b exhibiting a high degree of structural rigidity more frequently observed for native proteins. The data are complemented with studies on two TC5b point mutants to verify the importance of hydrophobic interactions for fast folding. Our results corroborate earlier findings of a hydrophobic cluster present in urea-denatured TC5b comprising both native and non-native contacts underscoring their importance for ultra rapid folding. The data assist in finding ways of interpreting the effects of pre-existing native and/or non-native interactions on the ultrafast folding of proteins; a fact, which might have to be considered when defining the starting conditions for molecular dynamics simulation studies of protein folding.

Measurement of 1H–15N and 1H–13C residual dipolar couplings in nucleic acids from TROSY intensities

Analogous to the recently introduced ARTSY method for measurement of one-bond (1)H-(15)N residual dipolar couplings (RDCs) in large perdeuterated proteins, we introduce methods for measurement of base (13)C-(1)H and (15)N-(1)H RDCs in protonated nucleic acids. Measurements are based on quantitative analysis of intensities in (1)H-(15)N and (13)C-(1)H TROSY-HSQC spectra, and are illustrated for a 71-nucleotide adenine riboswitch. Results compare favorably with those of conventional frequency-based measurements in terms of completeness and convenience of use. The ARTSY method derives the size of the coupling from the ratio of intensities observed in two TROSY-HSQC spectra recorded with different dephasing delays, thereby minimizing potential resonance overlap problems. Precision of the RDC measurements is limited by the signal-to-noise ratio, S/N, achievable in the 2D TROSY-HSQC reference spectrum, and is approximately given by 30/(S/N) Hz for (15)N-(1)H and 65/(S/N) Hz for (13)C-(1)H. The signal-to-noise ratio of both (1)H-(15)N and (1)H-(13)C spectra greatly benefits when water magnetization during the experiments is not perturbed, such that rapid magnetization transfer from bulk water to the nucleic acid, mediated by rapid amino and hydroxyl hydrogen exchange coupled with (1)H-(1)H NOE transfer, allows for fast repetition of the experiment. RDCs in the mutated helix 1 of the riboswitch are compatible with nucleotide-specifically modeled, idealized A-form geometry and a static orientation relative to the helix 2/3 pair, which differs by ca 6° relative to the X-ray structure of the native riboswitch.

Measuring rapid hydrogen exchange in the homodimeric 36 kDa HIV‐1 integrase catalytic core domain

AbstractMeasurements of rapid hydrogen exchange (HX) of water with protein amide sites contain valuable information on protein structure and function, but current NMR methods for measuring HX rates are limited in their applicability to large protein systems. An alternate method for measuring rapid HX is presented that is well‐suited for larger proteins, and we apply the method to the deuterated, homodimeric 36 kDa HIV‐1 integrase catalytic core domain (CCD). Using long mixing times for water‐amide magnetization exchange at multiple pH values, HX rates spanning more than four orders of magnitude were measured, as well as NOE cross‐relaxation rates to nearby exchangeable protons. HX protection factors for the CCD are found to be large (>104) for residues along the dimer interface, but much smaller in many other regions. Notably, the catalytic helix (residues 152‐167) exhibits low HX protection at both ends, indicative of fraying at both termini as opposed to just the N‐terminal end, as originally thought. Residues in the LEDGF/p75 binding pocket also show marginal stability, with protection factors in the 10–100 range (∼1.4–2.7 kcal/mol). Additionally, elevated NOE cross‐relaxation rates are identified and, as expected, correspond to proximity of the amide proton to a rapidly exchanging proton, typically from an OH side chain. Indirect NOE transfer between H2O and the amide proton of I141, a residue in the partially disordered active site of the enzyme, suggests its proximity to the side chain of S147, an interaction seen in the DNA‐bound form for a homologous integrase.

NMR spectroscopy and computational analysis of interaction between Serratia marcescens chitinase B and a dipeptide derived from natural-product cyclopentapeptide chitinase inhibitor argifin

The dipeptide N-acetyl-Arg{N(omega)-(N-methylcarbamoyl)}-N-methyl-Phe(2), which is a part of the natural-product cyclopentapeptide chitinase inhibitor argifin (1), inhibits chitinase B from Serratia marcescens (SmChiB) with a half-maximal inhibitory concentration (IC(50)) of 3.7 microM. Despite the relatively small size of 2, its inhibitory activity is comparable with that of 1 (IC(50)=6.4 microM). To elucidate the basis for this interesting phenomenon, we investigated the interaction between 2 and SmChiB using a combination of nuclear magnetic resonance spectroscopy and computational methods. The transferred nuclear Overhauser effect (TRNOE) experiment obtained structural information on the SmChiB-bound conformation of 2. The binding mode of 2 and SmChiB was modeled by the novel molecular-docking approach proposed in our laboratory, which can explicitly consider water-mediated hydrogen-bonding interactions in protein-ligand interfaces. The SmChiB-bound conformation of 2 in the resulting model satisfied all proton-proton distance constraints derived from the TRNOE experiment, indicating that our model structure of the 2-SmChiB complex is reasonable. A molecular dynamics (MD) simulation examined the stability of the resultant complex structure and suggested that 2 binds to SmChiB in a similar fashion to the binding mode observed for N(omega)-(N-methylcarbamoyl)-Arg(1) and N-methyl-Phe(2) of 1 in the crystal structure of the argifin-SmChiB complex. Finally, the binding free energies of 1 and 2 with SmChiB were estimated by the molecular mechanics Poisson-Boltzmann surface area (MM-PBSA) method using the MD trajectory. The MM-PBSA calculation suggested that both 1 and 2 bind to SmChiB with similar affinities, which is consistent with their experimental IC(50) values. Energetic analysis revealed that the van der Waals interaction of 2 with SmChiB is much less than that of 1, but is completely compensated by the more favorable contribution of solute entropy and the total electrostatic component. The improved total electrostatic component was derived from more favorable electrostatic interactions. Therefore, we conclude that dipeptide 2 was also better optimized against SmChiB than 1 in an electrostatic point of view.

Precise structural determination of weakly binding peptides by utilizing dihedral angle constraints

Structural determination of target-bound conformations of peptides is of primary importance for the optimization of peptide ligands and peptide-mimetic design. In the structural determination of weakly binding ligands, transferred nuclear Overhauser effect (TrNOE) methods have been widely used. However, not many distance constraints can be obtained from small peptide ligands by TrNOE, especially for peptides bound to a target molecule in an extended conformation. Therefore, for precise structural determination of weakly binding peptides, additional structural constraints are required. Here, we present a strategy to systematically introduce dihedral angle constraints obtained from multiple transferred cross-correlated relaxation experiments and demonstrate precise structures of weakly binding peptides. As a result, we could determine the bioactive conformations of phage-derived peptide ligands and define their core binding motifs.

NMR and Molecular Dynamics Study of the Binding Mode of Naphthalene-N-sulfonyl-d-glutamic Acid Derivatives: Novel MurD Ligase Inhibitors

The presented series of naphthalene-N-sulfonyl-D-glutamic acid derivatives are novel MurD ligase inhibitors with moderate affinity that occupy the D-Glu binding site. We performed an NMR study including transfer NOE to determine the ligand bound conformation, as well as saturation transfer difference experiments to obtain ligand epitope maps. The difference in overall appearance of the epitope maps highlights the importance of hydrophobic interactions and shows the segments of molecular structure that are responsible for them. Transfer NOE experiments indicate the conformational flexibility of bound ligands, which were then further examined by unrestrained molecular dynamics calculations. The results revealed the differing degrees of ligand flexibility and their effect on particular ligand-enzyme contacts. Conformational flexibility not evident in the crystal structures may have an effect on ligand-binding site adaptability, and this is probably one of the important reasons for the only moderate activity of novel derivatives.

In Situ Observation of Rapid Ligand Exchange in Colloidal Nanocrystal Suspensions Using Transfer NOE Nuclear Magnetic Resonance Spectroscopy

Recently, solution NMR-based approaches have been developed that represent useful new tools for the in situ characterization of the capping ligand in colloidal nanocrystal dispersions. So far, this development has focused mainly on tightly bound ligands (no exchange) or ligands in slow exchange with the nanocrystal surface. In such systems, the ligand can be identified and its amount and interaction quantified via 1D (1)H NMR, (1)H-(13)C HSQC, and DOSY spectra. Here, we explore the case where capping ligands are in fast exchange with the nanocrystal surface. Using dodecylamine-stabilized CdTe (Q-CdTe|DDA) and octylamine-stabilized ZnO (Q-ZnO|OctA) nanoparticles, we first show that the NMR methods developed so far fail to evidence the bound ligand when the effect of the latter on the exchange-averaged parameters is marginalized by an excess of free ligand. Next, transfer NOE spectroscopy, a well-established technique in biomolecular NMR, is introduced to demonstrate and characterize the interaction of a ligand with the nanocrystal surface. Using Q-PbSe nanocrystals capped with oleic acids as a reference system, we show that bound and free ligands have strongly different NOE spectra wherein only bound ligands develop strong and negative NOEs. For the Q-CdTe|DDA system, transfer NOE spectra show a similar rapid appearance of strong, negative NOEs, thereby unambiguously demonstrating that DDA molecules spend time at the nanocrystal surface. In the case of Q-ZnO|OctA, where a more complex mixture is analyzed, transfer NOE spectroscopy allows distinguishing capping from noncapping molecules, thereby demonstrating the screening potential offered by this technique for colloidal quantum dot dispersions.

Ligand based NMR methods for drug discovery

NMR has a long history in drug discovery and hit-to-lead optimization. Compared to many other methods NMR has the advantage of combining structural and functional parameters to characterize protein inhibitor interactions. NMR methods used in this context can be split into two categories; protein based experiments using isotopically labelled protein samples and a broad range of ligand based methods. Recently, there has been a strong emphasis on so-called ligand-based methods which offer a broad range of options to determine binding epitopes. Ligand-based methods are attractive because they are broadly applicable, impose few constraints on the composition of the target protein and don't require isotopic labeling of the protein or ligands. Such experiments include diffusion experiments, saturation transfer difference (STD-NMR), NOE pumping, waterLOGSY, SALMON, transferred-NOE and INPHARMA. Ligand-based NMR methods have been employed in screening and in lead optimization. One key advantage arises from their capability to pick up specific interactions for compounds of relatively low affinity and their ability to provide limited structural information without any need of crystallization or isotopic labeling.