Curcumin is the active ingredient of the turmeric powder, a natural spice used for generations in traditional medicines. Curcumin's broad spectrum of anti-oxidant, anti-carcinogenic, antimutagenic, and anti-inflammatory properties makes it particularly interesting for the development of pharmaceutical compounds. Due to curcumin's various effects on the function of numerous unrelated membrane proteins, it has been suggested that it affects the properties of the bilayer itself. However, a detailed atomic-level study of the interaction of curcumin with membranes has not been attempted. A combination of solid-state NMR and differential scanning calorimetry experiments shows curcumin has a strong effect on membrane structure at low concentrations. Curcumin inserts deep into the membrane in a transbilayer orientation, anchored by hydrogen bonding to the phosphate group of lipids in a manner analogous to cholesterol. Like cholesterol, curcumin induces segmental ordering in the membrane. Analysis of the concentration dependence of the order parameter profile derived from NMR results suggests curcumin forms higher order oligomeric structures in the membrane that span and likely thin the bilayer. Curcumin promotes the formation of the highly curved inverted hexagonal phase which may influence exocytotic and membrane fusion processes within the cell. The experiments outlined here show promise for understanding the action of other drugs such as capsaicin in which drug-induced alterations of membrane structure have strong pharmacological effects.Turmeric powder prepared from the turmeric plant has been in use for centuries in the traditional medicine of China and India for treating wounds, infections, and other skin problems. 1 The active component of turmeric powder, curcumin (Fig. 1 for the structure of curcumin), has a surprising array of anti-oxidant, anti-cancer, anti-mutagenic, antibiotic, antiviral, anti-fungal, anti-amyloid, anti-diabetic, and anti-inflammatory properties. 2-9 Because of its numerous pharmaceutical effects and its inherent non-toxicity, curcumin has been the focus of many recent biochemical investigations, primarily reporting on its medicinal properties and interactions with specific proteins. Despite intense interest in the physiological effects of curcumin, a general mechanism for its action has not been identified.Studies of curcumin have shown that it influences structurally unrelated membrane proteins across several signaling pathways. 10 For a small minority of these proteins, specific binding of curcumin to the protein has been detected with a binding constant typically in the nanomolar

The death of insulin-producing β-cells is a key step in the pathogenesis of type 2 diabetes. The amyloidogenic peptide Islet Amyloid Polypeptide (IAPP, also known as amylin) has been shown to disrupt β-cell membranes leading to β-cell death. Despite the strong evidence linking IAPP to the destruction of β-cell membrane integrity and cell death, the mechanism of IAPP toxicity is poorly understood. In particular, the effect of IAPP on the bilayer structure has largely been uncharacterized. In this study, we have determined the effect of the amyloidogenic and toxic hIAPP 1-37 peptide and the non-toxic and non-amyloidogenic rIAPP 1-37 peptide on membranes by a combination of DSC and solid-state NMR spectroscopy. We also characterized the toxic but largely non-amyloidogenic rIAPP 1-19 and hIAPP 1-19 fragments. DSC shows that both amyloidogenic (hIAPP 1-37 ) and largely non-amyloidogenic (hIAPP 1-19 and rIAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] ) toxic versions of the peptide strongly favor the formation of negative curvature in lipid bilayers, while the non-toxic full-length rat IAPP 1-37 peptide does not. This result was confirmed by solid-state NMR spectroscopy which shows that in bicelles composed of regions of high curvature and low curvature, non-toxic rIAPP 1-37 binds to the regions of low curvature while toxic rIAPP [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] binds to regions of high curvature. Similarly, solid-state NMR spectroscopy shows that the toxic rIAPP 1-19 peptide significantly disrupts the lipid bilayer structure, whereas the non-toxic rIAPP1-37 does not have a significant effect. These results indicate IAPP may induce the formation of pores by the induction of excess membrane curvature and can be used to guide the design of compounds that can prevent the cell-toxicity of IAPP. This mechanism may be important to understand the toxicity of other amyloidogenic proteins. Our solid-state NMR results also demonstrate the possibility of using bicelles to measure the affinity of biomolecules for negatively or positively curved regions of the membrane, which we believe will be useful in a variety of biochemical and biophysical investigations related to the cell membrane.

Poly(amidoamine) (PAMAM) dendrimer nanobiotechnology shows great promise in targeted drug delivery and gene therapy. Because of the involvement of cell membrane lipids with the pharmacological activity of dendrimer nanomedicines, the interactions between dendrimers and lipids are of particular relevance to the pharmaceutical applications of dendrimers. In this study, solid-state NMR was used to obtain a molecular image of the complex of generation 5 PAMAM dendrimer with the lipid bilayer. Using 1H radio frequency driven dipolar recoupling (RFDR) and 1H magic angle spinning (MAS) nuclear Overhauser effect spectroscopy (NOESY) techniques, we show that dendrimers are thermodynamically stable when inserted into zwitterionic lipid bilayers. 14N and 31P NMR experiments on static samples and measurements of the mobility of C–H bonds using a 2D proton detected local field protocol under MAS corroborate these results. The localization of dendrimers in the hydrophobic core of lipid bilayers restricts the motion of bilayer lipid tails, with the smaller G5 dendrimer having more of an effect than the larger G7 dendrimer. Fragmentation of the membrane does not occur at low dendrimer concentrations in zwitterionic membranes. Because these results show that the amphipathic dendrimer molecule can be stably incorporated in the interior of the bilayer (as opposed to electrostatic binding at the surface), they are expected to be useful in the design of dendrimer-based nanobiotechnologies.

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Many neurodegenerative diseases are characterized by misfolding and aggregation of an expanded polyglutamine tract (polyQ). Huntington's Disease, caused by expansion of the polyQ tract in exon 1 of the Huntingtin protein (Htt), is associated with aggregation and neuronal toxicity. Despite recent structural progress in understanding the structures of amyloid fibrils, little is known about the solution states of Htt in general, and about molecular details of their transition from soluble to aggregation-prone conformations in particular. This is an important question, given the increasing realization that toxicity may reside in soluble conformers. This study presents an approach that combines NMR with computational methods to elucidate the structural conformations of Htt Exon 1 in solution. Of particular focus was Htt's N17 domain sited N-terminal to the polyQ tract, which is key to enhancing aggregation and modulate Htt toxicity. Such in-depth structural study of Htt presents a number of unique challenges: the long homopolymeric polyQ tract contains nearly identical residues, exon 1 displays a high degree of conformational flexibility leading to a scaling of the NMR chemical shift dispersion, and a large portion of the backbone amide groups are solvent-exposed leading to fast hydrogen exchange and causing extensive line broadening. To deal with these problems, NMR assignment was achieved on a minimal Htt exon 1, comprising the N17 domain, a polyQ tract of 17 glutamines, and a short hexameric polyProline region that does not contribute to the spectrum. A pH titration method enhanced this polypeptide's solubility and, with the aid of ≤5D NMR, permitted the full assignment of N17 and the entire polyQ tract. Structural predictions were then derived using the experimental chemical shifts of the Htt peptide at low and neutral pH, together with various different computational approaches. All these methods concurred in indicating that low-pH protonation stabilizes a soluble conformation where a helical region of N17 propagates into the polyQ region, while at neutral pH both N17 and the polyQ become largely unstructured-thereby suggesting a mechanism for how N17 regulates Htt aggregation.

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Structural biology of membrane proteins has rapidly evolved into a new frontier of science. Although solving the structure of a membrane protein with atomic-level resolution is still a major challenge, separated local field (SLF) NMR spectroscopy has become an invaluable tool in obtaining structural images of membrane proteins under physiological conditions. Recent studies have demonstrated the use of rotating-frame SLF techniques to accurately measure strong heteronuclear dipolar couplings between directly bonded nuclei. However, in these experiments, all weak dipolar couplings are suppressed. On the other hand, weak heteronuclear dipolar couplings can be measured using laboratory-frame SLF experiments, but only at the expense of spectral resolution for strongly dipolar coupled spins. In the present study, we implemented 2D PELF (proton-evolved local-field) pulse sequences using either COMPOZER-CP (composite zero degree cross-polarization) or WIM (windowless isotropic mixing) for magnetization transfer. These PELF sequences can be used for the measurement of a broad range of heteronuclear dipolar couplings, allowing for a complete mapping of protein dynamics in a lipid bilayer environment. Experimental results from magnetically-aligned bicelles containing uniformly 15 N-labeled cytochrome b 5 are presented and theoretical analyses of the new PELF sequences are reported. Our results suggest that the PELF based experimental approaches will have a profound impact on solid-state NMR spectroscopy of membrane proteins and other membrane-associated molecules in magnetically-aligned bicelles.

Since the pioneering works of Carr-Purcell and Meiboom-Gill [Carr HY, Purcell EM (1954) Phys Rev 94:630; Meiboom S, Gill D (1985) Rev Sci Instrum 29:688], trains of π-pulses have featured amongst the main tools of quantum control. Echo trains find widespread use in nuclear magnetic resonance spectroscopy (NMR) and imaging (MRI), thanks to their ability to free the evolution of a spin-1/2 from several sources of decoherence. Spin echoes have also been researched in dynamic decoupling scenarios, for prolonging the lifetimes of quantum states or coherences. Inspired by this search we introduce a family of spin-echo sequences, which can still detect site-specific interactions like the chemical shift. This is achieved thanks to the presence of weak environmental fluctuations of common occurrence in high-field NMR-such as homonuclear spinspin couplings or chemical/biochemical exchanges. Both intuitive and rigorous derivations of the resulting "selective dynamical recoupling" sequences are provided. Applications of these novel experiments are given for a variety of NMR scenarios including determinations of shift effects under inhomogeneities overwhelming individual chemical identities, and model-free characterizations of chemically exchanging partners.chemical exchange | dynamic decoupling | magnetic field inhomogeneity | magnetic resonance | quantum control T he unprecedented scope of applications achieved by contemporary magnetic resonance reflects the degree of control that can be imparted on the spins' evolution. Using judicious combinations of multiple-pulse sequences one can tailor Hamiltonians that highlight a variety of interactions. When coupled to the long-lived coherences typical of spin-1/2 nuclei, this enables probing matter over a broad range of conditions and scenarios -from nanomaterials to rocks under the oceans; from proteins to human metabolism and disease (1-4). Besides its wide scope of applications, NMR awakes constant interest as a benchmark for other fields of physics; in particular, the quantum information community has adopted a number of NMR paradigms as tools of its own (5-16). The most prominent example among these is the spin-echo sequence, proposed over half a century ago for removing inhomogeneous broadenings (17)(18)(19). When extrapolated to trains of π-pulses spaced by a constant delay (Fig. 1A), the resulting CPMG sequences eventually became associated to a long list of landmark measurements including T 2 determinations, structural-oriented experiments, diffusion measurements, ex situ investigations in inhomogeneous fields, kinetic chemical and biophysical determinations, spin decoupling, and single scan MRI (1-4). Overall, the extremely wide range of useful measurements that this single sequence enabled is truly remarkable.Further CPMG generalizations have been proposed in recent years, aimed at improving the dynamical decoupling (DD) between a spin and its surroundings (5-9). In cases where environmental fluctuations are dominated by low-frequency components having a sharp cutoff...