Improved Detection and Quantification of Cyclopropane Fatty Acids via Homonuclear Decoupling Double Irradiation NMR Methods.

Over the years, NMR spectroscopy has become a powerful analytical tool for the identification and quantification of a variety of natural compounds in a broad range of food matrices. Furthermore, NMR can be useful for characterizing food matrices in terms of quality and authenticity, also allowing for the identification of counterfeits. Although NMR requires minimal sample preparation, this technique suffers from low intrinsic sensitivity relative to complementary techniques; thus, the detection of adulterants or markers for authenticity at low concentrations remains challenging. Here, we present a strategy to overcome this limitation by the introduction of a simple band-selective homonuclear decoupling sequence that consists of double irradiation on 1H during NMR signal acquisition. The utility of the proposed method is tested on dihydrosterculic acid (DHSA), one of the cyclopropane fatty acids (CPFAs) shown to be a powerful molecular marker for authentication of milk products. A quantitative description of how the proposed NMR scheme allows sensitivity enhancement yet accurate quantification of DHSA is provided.

Applications of Solution NMR Spectroscopy in Quality Assessment and Authentication of Bovine Milk.

Nuclear magnetic resonance (NMR) spectroscopy is emerging as a promising technique for the analysis of bovine milk, primarily due to its non-destructive nature, minimal sample preparation requirements, and comprehensive approach to untargeted milk analysis. These inherent strengths of NMR make it a formidable complementary tool to mass spectrometry-based techniques in milk metabolomic studies. This review aims to provide a comprehensive overview of the applications of NMR techniques in the quality assessment and authentication of bovine milk. It will focus on the experimental setup and data processing techniques that contribute to achieving accurate and highly reproducible results. The review will also highlight key studies that have utilized commonly used NMR methodologies in milk analysis, covering a wide range of application fields. These applications include determining milk animal species and feeding regimes, as well as assessing milk nutritional quality and authenticity. By providing an overview of the diverse applications of NMR in milk analysis, this review aims to demonstrate the versatility and significance of NMR spectroscopy as an invaluable tool for milk and dairy metabolomics research and hence, for assessing the quality and authenticity of bovine milk.

Global Dynamics of a Protein on the Surface of Anisotropic Lipid Nanoparticles Derived from Relaxation-Based NMR Spectroscopy.

The global motions of ubiquitin, a model protein, on the surface of anisotropically tumbling 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1'-rac-glycerol) (POPG):1,2-dihexanoyl-sn-glycero-3-phosphocholine (DHPC) bicelles are described. The shapes of POPG:DHPC bicelles prepared with high molar ratios q of POPG to DHPC can be approximated by prolate ellipsoids, with the ratio of ellipsoid dimensions and dimensions themselves increasing with higher values of q. Adaptation of the nuclear magnetic resonance (NMR) relaxation-based approach that we previously developed for interactions of ubiquitin with spherical POPG liposomes (Ceccon, A. J. Am. Chem. Soc. 2016, 138, 5789-5792) allowed us to quantitatively analyze the variation in lifetime line broadening of NMR signals (ΔR2) measured for ubiquitin in the presence of q = 2 POPG:DHPC bicelles and the associated transverse spin relaxation rates (R2,B) of bicelle-bound ubiquitin. Ubiquitin, transiently bound to POPG:DHPC bicelles, undergoes internal rotation about an axis orthogonal to the surface of the bicelle and perpendicular to the principal axis of its rotational diffusion tensor on the low microsecond time scale (∼3 µs), while the rotation axis itself wobbles in a cone on a submicrosecond time scale (≤ 500 ns).

Quantitative NMR analysis of the kinetics of prenucleation oligomerization and aggregation of pathogenic huntingtin exon-1 protein.

The N-terminal region of the huntingtin protein, encoded by exon-1 (httex1) and containing an expanded polyglutamine tract, forms fibrils that accumulate in neuronal inclusion bodies, resulting in Huntington's disease. We previously showed that reversible formation of a sparsely populated tetramer of the N-terminal amphiphilic domain, comprising a dimer of dimers in a four-helix bundle configuration, occurs on the microsecond timescale and is an essential prerequisite for subsequent nucleation and fibril formation that takes place orders of magnitude slower on a timescale of hours. For pathogenic httex1, such as httex1Q35 with 35 glutamines, NMR signals decay too rapidly to permit measurement of time-intensive exchange-based experiments. Here, we show that quantitative analysis of both the kinetics and mechanism of prenucleation tetramerization and aggregation can be obtained simultaneously from a series of 1H-15N band-selective optimized flip-angle short-transient heteronuclear multiple quantum coherence (SOFAST-HMQC) correlation spectra. The equilibria and kinetics of tetramerization are derived from the time dependence of the 15N chemical shifts and 1H-15N cross-peak volume/intensity ratios, while the kinetics of irreversible fibril formation are afforded by the decay curves of 1H-15N cross-peak intensities and volumes. Analysis of data on httex1Q35 over a series of concentrations ranging from 200 to 750 µM and containing variable (7 to 20%) amounts of the Met7O sulfoxide species, which does not tetramerize, shows that aggregation of native httex1Q35 proceeds via fourth-order primary nucleation, consistent with the critical role of prenucleation tetramerization, coupled with first-order secondary nucleation. The Met7O sulfoxide species does not nucleate but is still incorporated into fibrils by elongation.

NMR methods for exploring 'dark' states in ligand binding and protein-protein interactions.

A survey, primarily based on work in the authors' laboratory during the last 10 years, is provided of recent developments in NMR studies of exchange processes involving protein-ligand and protein-protein interactions. We start with a brief overview of the theoretical background of Dark state Exchange Saturation Transfer (DEST) and lifetime line-broadening (ΔR2) NMR methodology. Some limitations of the DEST/ΔR2 methodology in applications to molecular systems with intermediate molecular weights are discussed, along with the means of overcoming these limitations with the help of closely related exchange NMR techniques, such as the measurements of Carr-Purcell-Meiboom-Gill (CPMG) relaxation dispersion, exchange-induced chemical shifts or rapidly-relaxing components of relaxation decays. Some theoretical underpinnings of the quantitative description of global dynamics of proteins on the surface of very high molecular weight particles (nanoparticles) are discussed. Subsequently, several applications of DEST/ΔR2 methodology are described from a methodological perspective with an emphasis on providing examples of how kinetic and relaxation parameters for exchanging systems can be reliably extracted from NMR data for each particular model of exchange. Among exchanging systems that are not associated with high molecular weight species, we describe several exchange NMR-based studies that focus on kinetic modelling of transient pre-nucleation oligomerization of huntingtin peptides that precedes aggregation and fibril formation.

Quantitative Exchange NMR-Based Analysis of Huntingtin-SH3 Interactions Suggests an Allosteric Mechanism of Inhibition of Huntingtin Aggregation.

Huntingtin polypeptides (httex1), encoded by exon 1 of the htt gene and containing an expanded polyglutamine tract, form fibrils that accumulate within neuronal inclusion bodies, resulting in the fatal neurodegenerative condition known as Huntington's disease. Httex1 comprises three regions: a 16-residue N-terminal amphiphilic domain (NT), a polyglutamine tract of variable length (Qn), and a polyproline-rich domain containing two polyproline tracts. The NT region of httex1 undergoes prenucleation transient oligomerization on the sub-millisecond time scale, resulting in a productive tetramer that promotes self-association and nucleation of the polyglutamine tracts. Here we show that binding of Fyn SH3, a small intracellular proline-binding domain, to the first polyproline tract of httex1Q35 inhibits fibril formation by both NMR and a thioflavin T fluorescence assay. The interaction of Fyn SH3 with httex1Q7 was investigated using NMR experiments designed to probe kinetics and equilibria at atomic resolution, including relaxation dispersion, and concentration-dependent exchange-induced chemical shifts and transverse relaxation in the rotating frame. Sub-millisecond exchange between four species is demonstrated: two major states comprising free (P) and SH3-bound (PL) monomeric httex1Q7, and two sparsely populated dimers in which either both subunits (P2L2) or only a single subunit (P2L) is bound to SH3. Binding of SH3 increases the helical propensity of the NT domain, resulting in a 25-fold stabilization of the P2L2 dimer relative to the unliganded P2 dimer. The P2L2 dimer, in contrast to P2, does not undergo any detectable oligomerization to a tetramer, thereby explaining the allosteric inhibition of httex1 fibril formation by Fyn SH3.

Probing Side-Chain Dynamics in Proteins by NMR Relaxation of Isolated 13C Magnetization Modes in 13CH3 Methyl Groups.

The dynamics of methyl-bearing side chains in proteins were probed by 13C relaxation measurements of a number of 13C magnetization modes in selectively 13CH3-labeled methyl groups of proteins. We first show how 13C magnetization modes in a 13CH3 spin-system can be isolated using acute-angle 1H radio-frequency pulses. The parameters of methyl-axis dynamics, a measure of methyl-axis ordering (Saxis2) and the correlation time of fast local methyl-axis motions (τf), derived from 13C relaxation in 13CH3 groups are compared with their counterparts obtained from 13C relaxation in 13CHD2 methyl isotopomers. We show that in high-molecular-weight proteins, excellent correlations are obtained between the [13CHD2]-derived Saxis2 values and those extracted from relaxation of the 13C magnetization of the I = 1/2 manifold in 13CH3 methyls. In smaller proteins, a certain degree of anticorrelation is observed between the Saxis2 and τf values obtained from 13C relaxation of the I = 1/2 manifold magnetization in 13CH3 methyls. These parameters can be partially decorrelated by inclusion in the analysis of relaxation data of the I = 3/2 manifold 13C magnetization.

Kinetics of Fast Tetramerization of the Huntingtin Exon 1 Protein Probed by Concentration-Dependent On-Resonance R1ρ Measurements.

An approach for the quantitative description of the kinetics of very fast exchange processes (τex < 50-100 µs) associated with transient, reversible protein oligomerization, is presented. We show that on-resonance 15N-R1ρ measurements conducted as a function of protein concentration at several spin-lock radio frequency field strengths are indispensable for unambiguous determination of the rate constants for interconversion between monomeric and higher order oligomeric species. The approach is experimentally demonstrated on the study of fast, reversible tetramerization of the full-length Huntingtin exon 1 protein, httex1, responsible for Huntington's disease. Incorporation of concentration-dependent 15N-R2,eff data, obtained from on-resonance R1ρ measurements performed at three spin-lock field strengths, into analysis of the kinetic scheme describing reversible tetramerization of httex1 allowed us to uniquely determine the rate constants of interconversion between the various species. This approach serves as a valuable complement to the existing array of NMR techniques for studying early, transient oligomerization events in protein aggregation pathways.

Abrogation of prenucleation, transient oligomerization of the Huntingtin exon 1 protein by human profilin I.

Human profilin I reduces aggregation and concomitant toxicity of the polyglutamine-containing N-terminal region of the huntingtin protein encoded by exon 1 (httex1) and responsible for Huntington's disease. Here, we investigate the interaction of profilin with httex1 using NMR techniques designed to quantitatively analyze the kinetics and equilibria of chemical exchange at atomic resolution, including relaxation dispersion, exchange-induced shifts, and lifetime line broadening. We first show that the presence of two polyproline tracts in httex1, absent from a shorter huntingtin variant studied previously, modulates the kinetics of the transient branched oligomerization pathway that precedes nucleation, resulting in an increase in the populations of the on-pathway helical coiled-coil dimeric and tetrameric species (τex ≤ 50 to 70 µs), while leaving the population of the off-pathway (nonproductive) dimeric species largely unaffected (τex ∼750 µs). Next, we show that the affinity of a single molecule of profilin to the polyproline tracts is in the micromolar range (Kdiss ∼ 17 and ∼ 31 µM), but binding of a second molecule of profilin is negatively cooperative, with the affinity reduced ∼11-fold. The lifetime of a 1:1 complex of httex1 with profilin, determined using a shorter huntingtin variant containing only a single polyproline tract, is shown to be on the submillisecond timescale (τex ∼ 600 µs and Kdiss ∼ 50 µM). Finally, we demonstrate that, in stable profilin-httex1 complexes, the productive oligomerization pathway, leading to the formation of helical coiled-coil httex1 tetramers, is completely abolished, and only the pathway resulting in "nonproductive" dimers remains active, thereby providing a mechanistic basis for how profilin reduces aggregation and toxicity of httex1.

Optimized NMR Experiments for the Isolation of I=1/2 Manifold Transitions in Methyl Groups of Proteins.

Optimized NMR experiments are developed for isolating magnetization belonging to the I=1/2 manifolds of 13 CH3 methyl groups in proteins, enabling the manipulation of the magnetization of a 13 CH3 moiety as if it were an AX (1 H-13 C) spin-system. These experiments result in the same 'simplification' of a 13 CH3 spin-system that would be obtained from the production of {13 CHD2 }-methyl-labeled protein samples. The sensitivity of I=1/2 manifold-selection experiments is a factor of approximately 2 less than that of the corresponding experiments acquired on {13 CHD2 }-labeled methyl groups. The methodology described here is primarily intended for small-to-medium sized proteins, where the losses in sensitivity associated with the isolation of I=1/2 manifold transitions can be tolerated. Several NMR applications that benefit from simplification of the 13 CH3 (AX3 ) spin-systems are described, with an emphasis on the measurements of methyl 1 H-13 C residual dipolar couplings in a {13 CH3 }-methyl-labeled deletion mutant of the human chaperone DNAJB6b, where modulation of NMR signal intensities due to evolution of methyl 1 H-13 C scalar and dipolar couplings follows a simple cosine function characteristic of an AX (1 H-13 C) spin-system, significantly simplifying data analysis.

Probing initial transient oligomerization events facilitating Huntingtin fibril nucleation at atomic resolution by relaxation-based NMR.

The N-terminal region of the huntingtin protein, encoded by exon-1, comprises an amphiphilic domain (httNT), a polyglutamine (Q n ) tract, and a proline-rich sequence. Polyglutamine expansion results in an aggregation-prone protein responsible for Huntington's disease. Here, we study the earliest events involved in oligomerization of a minimalistic construct, httNTQ7, which remains largely monomeric over a sufficiently long period of time to permit detailed quantitative NMR analysis of the kinetics and structure of sparsely populated [Formula: see text] oligomeric states, yet still eventually forms fibrils. Global fitting of concentration-dependent relaxation dispersion, transverse relaxation in the rotating frame, and exchange-induced chemical shift data reveals a bifurcated assembly mechanism in which the NMR observable monomeric species either self-associates to form a productive dimer (τex ∼ 30 µs, Kdiss ∼ 0.1 M) that goes on to form a tetramer ([Formula: see text] µs; Kdiss ∼ 22 µM), or exchanges with a "nonproductive" dimer that does not oligomerize further (τex ∼ 400 µs; Kdiss ∼ 0.3 M). The excited state backbone chemical shifts are indicative of a contiguous helix (residues 3-17) in the productive dimer/tetramer, with only partial helical character in the nonproductive dimer. A structural model of the productive dimer/tetramer was obtained by simulated annealing driven by intermolecular paramagnetic relaxation enhancement data. The tetramer comprises a D2 symmetric dimer of dimers with largely hydrophobic packing between the helical subunits. The structural model, validated by EPR distance measurements, illuminates the role of the httNT domain in the earliest stages of prenucleation and oligomerization, before fibril formation.

TiO2 Nanoparticles Catalyze Oxidation of Huntingtin Exon 1-Derived Peptides Impeding Aggregation: A Quantitative NMR Study of Binding and Kinetics.

Polyglutamine expansion within the N-terminal region of the huntingtin protein results in the formation of intracellular aggregates responsible for Huntington's disease, a fatal neurodegenerative condition. The interaction between TiO2 nanoparticles and huntingtin peptides comprising the N-terminal amphiphilic domain without (httNT) or with (httNTQ10) a ten-residue C-terminal polyglutamine tract, is investigated by NMR spectroscopy. TiO2 nanoparticles decrease aggregation of httNTQ10 by catalyzing the oxidation of Met7 to a sulfoxide, resulting in an aggregation-incompetent peptide. The oxidation agent is hydrogen peroxide generated on the surface of the TiO2 nanoparticles either by UV irradiation or at low steady-state levels in the dark. The binding kinetics of nonaggregating httNT to TiO2 nanoparticles is characterized by quantitative analysis of 15N dark state exchange saturation transfer and lifetime line broadening NMR data. Binding involves a sparsely populated intermediate that experiences hindered rotational diffusion relative to the free state. Catalysis of methionine oxidation within the N-terminal domain of the huntingtin protein may potentially provide a strategy for delaying the onset of Huntington's disease.

Decorrelating Kinetic and Relaxation Parameters in Exchange Saturation Transfer NMR: A Case Study of N-Terminal Huntingtin Peptides Binding to Unilamellar Lipid Vesicles.

Dark state exchange saturation transfer (DEST) and lifetime line-broadening (Δ R2, the difference in the measured transverse relaxation rates for the observable species in the presence and absence of exchange with a species characterized by very large intrinsic transverse relaxation rates) have proven to be powerful NMR tools for studying exchange phenomena between a NMR visible species and a high-molecular weight, "dark", NMR invisible state. However, in the exchange regime, where the transverse spin relaxation rates in the bound state ( R2bound) are smaller than the strength of the DEST saturation radio frequency field, typically corresponding to systems below ∼6 MDa, the combination of DEST and Δ R2 data, while sufficient to define the apparent association rate constant, cannot unambiguously determine the population of the bound state pB and R2bound values independently. We show that the latter exchange and relaxation parameters can be decorrelated by the measurement of the maximal value of the contribution of the fast-relaxing magnetization component to the total NMR signal, Cfastmax, an observable that is directly proportional to pB. When integrated into the analysis of DEST/Δ R2 data, Cfastmax provides an indispensable source of information for quantitative studies of exchange involving high-molecular-weight dark states. We demonstrate the utility of this approach by investigating the binding kinetics of two huntingtin exon-1-derived peptides to small unilamellar lipid vesicles (SUV), ∼ 31 nm in diameter and 4.3 MDa in molecular weight. The interaction of the N-terminal amphiphilic domain of huntingtin exon-1 with membrane surfaces promotes polyglutamine-mediated aggregation and, as such, is thought to play a role in the etiology of Huntington's disease, an autosomal dominant fatal neurodegenerative condition. The first peptide comprises the 16-residue N-terminal amphiphilic domain (httNT) alone, while the second contains an additional seven residue polyglutamine tract at the C-terminus (httNTQ7). At a peptide-to-lipid molar ratio of 1:4, the population of peptide bound to the SUV surface is substantial, ∼ 7-8%, while exchange between the free and SUV-bound peptide is slow on the relaxation time-scale ( kex ∼ 200 s-1). The last two C-terminal residues of httNT and the last 9 of httNTQ7 remain flexible in the SUV-bound form due to transient detachment from the lipid surface that occurs on a time-scale several-fold faster than binding.

Interaction of Huntingtin Exon-1 Peptides with Lipid-Based Micellar Nanoparticles Probed by Solution NMR and Q-Band Pulsed EPR.

Lipid-based micellar nanoparticles promote aggregation of huntingtin exon-1 peptides. Here we characterize the interaction of two such peptides, httNTQ 7 and httNTQ 10 comprising the N-terminal amphiphilic domain of huntingtin followed by 7 and 10 glutamine repeats, respectively, with 8 nm lipid micelles using NMR chemical exchange saturation transfer (CEST), circular dichroism and pulsed Q-band EPR. Exchange between free and micelle-bound httNTQ  n peptides occurs on the millisecond time scale with a KD ∼ 0.5-1 mM. Upon binding micelles, residues 1-15 adopt a helical conformation. Oxidation of Met7 to a sulfoxide reduces the binding affinity for micelles ∼3-4-fold and increases the length of the helix by a further two residues. A structure of the bound monomer unit is calculated from the backbone chemical shifts of the micelle-bound state obtained from CEST. Pulsed Q-band EPR shows that a monomer-dimer equilibrium exists on the surface of the micelles and that the two helices of the dimer adopt a parallel orientation, thereby bringing two disordered polyQ tails into close proximity which may promote aggregation upon dissociation from the micelle surface.

Probing the Binding Modes of a Multidomain Protein to Lipid-based Nanoparticles by Relaxation-based NMR.

The interactions of two model multidomain proteins-covalently linked diubiquitins, Ub2-with lipid-based nanoparticles have been quantitatively probed by the measurements of NMR lifetime line broadening, ΔR2. By combined analysis of ΔR2 profiles arising from interactions with liposomes of varying sizes, an approach recently developed for the characterization of interactions of monoubiquitin with liposomes, we determine how the parameters of exchange (liposome binding) and dynamics of each individual domain of Ub2 on the surface of liposomes change when the domains are covalently attached to one another by a flexible linker. Two different covalent linkages were used: K63-linked and K48-linked Ub2. The possibility of three distinct modes of binding of Ub2 to liposomes requires the introduction of simple but important modifications to the strategy of analysis originally developed for monoubiquitin.

Towards interpretation of intermolecular paramagnetic relaxation enhancement outside the fast exchange limit.

In an exchanging system between major and minor species, the transverse paramagnetic relaxation enhancement rate observed on the resonances of the major species (Γ 2 (app) ) is dependent upon the exchange regime between the species. Quantitative analysis of PRE data in such systems typically assumes that the overall exchange rate k ex between the species is fast on the PRE time scale (k ex â‰« Γ2). Recently, we have characterized the kinetics of binding of the model protein ubiquitin to large (LUV) and small (SUV) unilamellar lipid-based nanoparticles or liposomes (Ceccon A, Tugarinov V, Bax A, Clore GM (2016). J Am Chem Soc 138:5789-5792). Building upon these results and taking advantage of a strong paramagnetic agent with an isotropic g-tensor, Gd(3+), we were able to measure intermolecular methyl carbon and proton PREs between paramagnetically-tagged liposomes and ubiquitin. In the limit of fast exchange (k ex â‰« Γ2) the ratio of the apparent proton to carbon methyl PREs, ((1)Hm-Γ 2 (app) )/((13)Cm-Γ 2 (app) ), is equal to the square of the ratio of the gyromagnetic ratios of the two nuclei, (γΗ/γC)(2). However, outside the fast exchange regime, under intermediate exchange conditions (e.g. when Γ2 is comparable in magnitude to k ex) the ((1)Hm-Γ 2 (app) )/((13)Cm-Γ 2 (app) ) ratio provides a reliable measure of the 'true' methyl PREs.

Global Dynamics and Exchange Kinetics of a Protein on the Surface of Nanoparticles Revealed by Relaxation-Based Solution NMR Spectroscopy.

The global motions and exchange kinetics of a model protein, ubiquitin, bound to the surface of negatively charged lipid-based nanoparticles (liposomes) are derived from combined analysis of exchange lifetime broadening arising from binding to nanoparticles of differing size. The relative contributions of residence time and rotational tumbling to the total effective correlation time of the bound protein are modulated by nanoparticle size, thereby permitting the various motional and exchange parameters to be determined. The residence time of ubiquitin bound to the surface of both large and small unilamellar liposomes is ∼20 µs. Bound ubiquitin undergoes internal rotation about an axis approximately perpendicular to the lipid surface on a low microsecond time scale (∼2 µs), while simultaneously wobbling in a cone of semiangle 30-55° centered about the internal rotation axis on the nanosecond time scale. The binding interface of ubiquitin with liposomes is mapped by intermolecular paramagnetic relaxation enhancement using Gd(3+)-tagged vesicles, to a predominantly positively charged surface orthogonal to the internal rotation axis.

Transient Interactions of a Cytosolic Protein with Macromolecular and Vesicular Cosolutes: Unspecific and Specific Effects.

Cytosolic proteins do not occur as isolated but are exposed to many interactions within a crowded cellular environment. We investigated the associations between a test cytosolic protein, human ileal bile acid binding protein (IBABP), and model cosolutes mimicking macromolecular and lipid membrane intracellular components. Using fluorescence spectroscopy, heteronuclear NMR, and molecular dynamics, we found that IBABP associated weakly with anionic lipid vesicles and experienced transient unspecific contacts with albumin. Localized dynamic perturbations were observed even in the case of apparent unspecific binding. IBABP and ubiquitin did not display mutually attractive forces, whereas IBABP associated specifically with lysozyme. A structural model of the IBABP-lysozyme complex was obtained by data-driven docking simulation. Presumably, all the interactions shown here contribute to modulating functional communication of a protein in its native environment.

Are they in or out? The elusive interaction between Qtracker ® 800 vascular labels and brain endothelial cells.

AIM: Qtracker(®)800 Vascular labels (Qtracker(®)800) are promising biomedical tools for high-resolution vasculature imaging; their effects on mouse and human endothelia, however, are still unknown. MATERIALS & METHODS: Qtracker(®)800 were injected in Balb/c mice, and brain endothelium uptake was investigated by transmission electron microscopy 3-h post injection. We then investigated, in vitro, the effects of Qtracker(®)800 exposure on mouse and human endothelial cells by calcium imaging. RESULTS: Transmission electron microscopy images showed nanoparticle accumulation in mouse brain endothelia. A subset of mouse and human endothelial cells generated intracellular calcium transients in response to Qtracker(®)800. CONCLUSION: Qtracker(®)800 nanoparticles elicit endothelial functional responses, which prompts biomedical safety evaluations and may bias the interpretation of experimental studies involving vascular imaging.

Polyhydroxylated [60]fullerene binds specifically to functional recognition sites on a monomeric and a dimeric ubiquitin.

The use of nanoparticles (NPs) in biomedical applications requires an in-depth understanding of the mechanisms by which NPs interact with biomolecules. NPs associating with proteins may interfere with protein-protein interactions and affect cellular communication pathways, however the impact of NPs on biomolecular recognition remains poorly characterized. In this respect, particularly relevant is the study of NP-induced functional perturbations of proteins implicated in the regulation of key biochemical pathways. Ubiquitin (Ub) is a prototypical protein post-translational modifier playing a central role in numerous essential biological processes. To contribute to the understanding of the interactions between this universally distributed biomacromolecule and NPs, we investigated the adsorption of polyhydroxylated [60]fullerene on monomeric Ub and on a minimal polyubiquitin chain in vitro at atomic resolution. Site-resolved chemical shift and intensity perturbations of Ub's NMR signals, together with (15)N spin relaxation rate changes, exchange saturation transfer effects, and fluorescence quenching data were consistent with the reversible formation of soluble aggregates incorporating fullerenol clusters. The specific interaction epitopes were identified, coincident with functional recognition sites in a monomeric and lysine48-linked dimeric Ub. Fullerenol appeared to target the open state of the dynamic structure of a dimeric Ub according to a conformational selection mechanism. Importantly, the protein-NP association prevented the enzyme-catalyzed synthesis of polyubiquitin chains. Our findings provide an experiment-based insight into protein/fullerenol recognition, with implications in functional biomolecular communication, including regulatory protein turnover, and for the opportunity of therapeutic intervention in Ub-dependent cellular pathways.