Perfluorinated Iridium Catalyst for Signal Amplification by Reversible Exchange Provides Metal-Free Aqueous Hyperpolarized [1-13C]-Pyruvate.

Hyperpolarized (HP) carbon-13 [13C] enables the specific investigation of dynamic metabolic and physiologic processes via in vivo MRI-based molecular imaging. As the leading HP metabolic agent, [1-13C]pyruvate plays a pivotal role due to its rapid tissue uptake and central role in cellular energetics. Dissolution dynamic nuclear polarization (d-DNP) is considered the gold standard method for the production of HP metabolic probes; however, development of a faster, less expensive technique could accelerate the translation of metabolic imaging via HP MRI to routine clinical use. Signal Amplification by Reversible Exchange in SHield Enabled Alignment Transfer (SABRE-SHEATH) achieves rapid hyperpolarization by using parahydrogen (p-H2) as the source of nuclear spin order. Currently, SABRE is clinically limited due to the toxicity of the iridium catalyst, which is crucial to the SABRE process. To mitigate Ir contamination, we introduce a novel iteration of the SABRE catalyst, incorporating bis(polyfluoroalkylated) imidazolium salts. This novel perfluorinated SABRE catalyst retained polarization properties while exhibiting an enhanced hydrophobicity. This modification allows the easy removal of the perfluorinated SABRE catalyst from HP [1-13C]-pyruvate after polarization in an aqueous solution, using the ReD-SABRE protocol. The residual Ir content after removal was measured via ICP-MS at 177 ppb, which is the lowest reported to date for pyruvate and is sufficiently safe for use in clinical investigations. Further improvement is anticipated once automated processes for delivery and recovery are initiated. SABRE-SHEATH using the perfluorinated SABRE catalyst can become an attractive low-cost alternative to d-DNP to prepare biocompatible HP [1-13C]-pyruvate formulations for in vivo applications in next-generation molecular imaging modalities.

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.

Probing the Interaction of Huntingtin Exon-1 Polypeptides with the Chaperonin Nanomachine GroEL.

Huntington's disease arises from polyQ expansion within the exon-1 region of huntingtin (httex1 ), resulting in an aggregation-prone protein that accumulates in neuronal inclusion bodies. We investigate the interaction of various httex1 constructs with the bacterial analog (GroEL) of the human chaperonin Hsp60. Using fluorescence spectroscopy and electron and atomic force microscopy, we show that GroEL inhibits fibril formation. The binding kinetics of httex1 constructs with intact GroEL and a mini-chaperone comprising the apical domain is characterized by relaxation-based NMR measurements. The lifetimes of the complexes range from 100 to 400 µs with equilibrium dissociation constants (KD ) of ∼1-2 mM. The binding interface is formed by the N-terminal amphiphilic region of httex1 (which adopts a partially helical conformation) and the H and I helices of the GroEL apical domain. Sequestration of monomeric httex1 by GroEL likely increases the critical concentration required for fibrillization.

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.

Detergent-type membrane fragmentation by MSI-78, MSI-367, MSI-594, and MSI-843 antimicrobial peptides and inhibition by cholesterol: a solid-state nuclear magnetic resonance study.

Multidrug resistance against the existing antibiotics is becoming a global threat, and any potential drug that can be designed using cationic antimicrobial peptides (AMP) could be an alternate solution to alleviate this existing problem. The mechanism of action of killing bacteria by an AMP differs drastically in comparison to that of small molecule antibiotics. The main target of AMPs is to interact with the lipid bilayer of the cell membrane and disrupt it to kill bacteria. Consequently, the modes of membrane interaction that lead to the selectivity of an AMP are very important to understand. Here, we have used different membrane compositions, such as negatively charged, zwitterionic, or mixed large unilamellar vesicles (LUVs), to study the interaction of four different synthetically designed cationic, linear antimicrobial peptides: MSI-78 (commercially known as pexiganan), MSI-367, MSI-594, and MSI-843. Our solid-state nuclear magnetic resonance (NMR) experiments confirmed that the MSI peptides fragmented LUVs through a detergent-like carpet mechanism depending on the amino acid sequence of the MSI peptide and/or the membrane composition of LUVs. Interestingly, the fragmented lipid aggregates such as SUVs or micelles are sufficiently small to produce an isotropic peak in the (31)P NMR spectrum. These fragmented lipid aggregates contain only MSI peptides bestowed with lipid molecules as confirmed by NMR in conjunction with circular dichroism spectroscopy. Our results also demonstrate that cholesterol, which is present only in the eukaryotic cell membrane, inhibits the MSI-induced fragmentation of LUVs, suggesting that the MSI peptides can discriminate the bacteria and the eukaryotic cell membranes, and this selectivity could be used for further development of novel antibiotics.

Differences between amyloid-ß aggregation in solution and on the membrane: insights into elucidation of the mechanistic details of Alzheimer's disease.

The association of the amyloid-ß (Aß) peptide with cellular membranes is hypothesized to be the underlying phenomenon of neurotoxicity in Alzheimer's disease. Misfolding of proteins and peptides, as is the case with Aß, follows a progression from a monomeric state, through intermediates, ending at long, unbranched amyloid fibers. This tutorial review offers a perspective on the association of toxic Aß structures with membranes as well as details of membrane-associated mechanisms of toxicity.

Membrane disordering is not sufficient for membrane permeabilization by islet amyloid polypeptide: studies of IAPP(20-29) fragments.

A key factor in the development of type II diabetes is the loss of insulin-producing beta-cells. Human islet amyloid polypeptide protein (human-IAPP) is believed to play a crucial role in this process by forming small aggregates that exhibit toxicity by disrupting the cell membrane. The actual mechanism of membrane disruption is complex and appears to involve an early component before fiber formation and a later component associated with fiber formation on the membrane. By comparing the peptide-lipid interactions derived from solid-state NMR experiments of two IAPP fragments that cause membrane disordering to IAPP derived peptides known to cause significant early membrane permeabilization, we show here that membrane disordering is not likely to be sufficient by itself to cause the early membrane permeabilization observed by IAPP, and may play a lesser role in IAPP membrane disruption than expected.

Two-step mechanism of membrane disruption by Aß through membrane fragmentation and pore formation.

Disruption of cell membranes by Aß is believed to be one of the key components of Aß toxicity. However, the mechanism by which this occurs is not fully understood. Here, we demonstrate that membrane disruption by Aß occurs by a two-step process, with the initial formation of ion-selective pores followed by nonspecific fragmentation of the lipid membrane during amyloid fiber formation. Immediately after the addition of freshly dissolved Aß(1-40), defects form on the membrane that share many of the properties of Aß channels originally reported from single-channel electrical recording, such as cation selectivity and the ability to be blockaded by zinc. By contrast, subsequent amyloid fiber formation on the surface of the membrane fragments the membrane in a way that is not cation selective and cannot be stopped by zinc ions. Moreover, we observed that the presence of ganglioside enhances both the initial pore formation and the fiber-dependent membrane fragmentation process. Whereas pore formation by freshly dissolved Aß(1-40) is weakly observed in the absence of gangliosides, fiber-dependent membrane fragmentation can only be observed in their presence. These results provide insights into the toxicity of Aß and may aid in the design of specific compounds to alleviate the neurodegeneration of Alzheimer's disease.

Probing structural features of Alzheimer's amyloid-ß pores in bilayers using site-specific amino acid substitutions.

A current hypothesis for the pathology of Alzheimer's disease (AD) proposes that amyloid-ß (Aß) peptides induce uncontrolled, neurotoxic ion flux across cellular membranes. The mechanism of ion flux is not fully understood because no experiment-based Aß channel structures at atomic resolution are currently available (only a few polymorphic states have been predicted by computational models). Structural models and experimental evidence lend support to the view that the Aß channel is an assembly of loosely associated mobile ß-sheet subunits. Here, using planar lipid bilayers and molecular dynamics (MD) simulations, we show that amino acid substitutions can be used to infer which residues are essential for channel structure. We created two Aß(1-42) peptides with point mutations: F19P and F20C. The substitution of Phe19 with Pro inhibited channel conductance. MD simulation suggests a collapsed pore of F19P channels at the lower bilayer leaflet. The kinks at the Pro residues in the pore-lining ß-strands induce blockage of the solvated pore by the N-termini of the chains. The cysteine mutant is capable of forming channels, and the conductance behavior of F20C channels is similar to that of the wild type. Overall, the mutational analysis of the channel activity performed in this work tests the proposition that the channels consist of a ß-sheet rich organization, with the charged/polar central strand containing the mutation sites lining the pore, and the C-terminal strands facing the hydrophobic lipid tails. A detailed understanding of channel formation and its structure should aid studies of drug design aiming to control unregulated Aß-dependent ion fluxes.

Molecular Details of a Salt Bridge and Its Role in Insulin Fibrillation by NMR and Raman Spectroscopic Analysis.

Insulin, a simple polypeptide hormone with huge biological importance, has long been known to self-assemble in vitro and form amyloid-like fibrillar aggregates. Utilizing high-resolution NMR, Raman spectroscopy, and computational analysis, we demonstrate that the fluctuation of the carboxyl terminal (C-ter) residues of the insulin B-chain plays a key role in the growth phase of insulin aggregation. By comparing the insulin sourced from bovine, human, and the modified glargine (GI), we observed reduced aggregation propensity in the GI variant, resulting from two additional Arg residues at its C-ter. NMR analysis showed atomic contacts and residue-specific interactions, particularly the salt bridge and H-bond formed among the C-ter residues Arg31B, Lys29B, and Glu4A. These inter-residue interactions were reflected in strong nuclear Overhauser effects among Arg31BδH-Glu4AδH and Lys29BδHs-Glu4AδH in GI, as well as the associated downfield chemical shift of several A-chain amino terminal (N-ter) residues. The two additional Arg residues of GI, Arg31B and Arg32B, enhanced the stability of the GI native structure by strengthening the Arg31B, Lys29B, and Glu4A salt bridge, thus reducing extensive thermal distortion and fluctuation of the terminal residues. The high stability of the salt bridge retards tertiary collapse, a crucial biochemical event for oligomerization and subsequent fibril formation. Circular dichroism and Raman spectroscopic measurement also suggest slow structural distortion in the early phase of the aggregation of GI because of the restricted mobility of the C-ter residues as explained by NMR. In addition, the structural and dynamic parameters derived from molecular dynamics simulations of insulin variants highlight the role of residue-specific contacts in aggregation and amyloid-like fibril formation.

Probing transient non-native states in amyloid beta fiber elongation by NMR.

Using NMR to probe transient binding of Aß1-40 monomers to fibers, we find partially bound conformations with the highest degree of interaction near F19-K28 and a lesser degree of interaction near the C-terminus (L34-G37). This represents a shift away from the KLVFFA recognition sequence (residues 16-21) currently used for inhibitor design.

Lipopolysaccharide from Gut Microbiota Modulates α-Synuclein Aggregation and Alters Its Biological Function.

Altered intestinal permeability has been correlated with Parkinson's pathophysiology in the enteric nervous system, before manifestations in the central nervous system (CNS). The inflammatory endotoxin or lipopolysaccharide (LPS) released by gut bacteria is known to modulate α-synuclein amyloidogenesis through the formation of intermediate nucleating species. Here, biophysical techniques in conjunction with microscopic images revealed the molecular interaction between lipopolysaccharide and α-synuclein that induce rapid nucleation events. This heteromolecular interaction stabilizes the α-helical intermediates in the α-synuclein aggregation pathway. Multitude NMR studies probed the residues involved in the LPS-binding structural motif that modulates the nucleating forms, affecting the cellular internalization and associated cytotoxicity. Collectively, our data characterizes this heteromolecular interaction associated with an alternative pathway in Parkinson's disease progression.

Preparation of Stable Amyloid-ß Oligomers Without Perturbative Methods.

Soluble amyloid-ß (Aß) oligomers have become a focal point in the study of Alzheimer's disease due to their ability to elicit cytotoxicity. A number of recent studies have concentrated on the structural characterization of soluble Aß oligomers to gain insight into their mechanism of toxicity. Consequently, providing reproducible protocols for the preparation of such oligomers is of utmost importance. The method presented in this chapter details a protocol for preparing an Aß oligomer, with a primarily disordered secondary structure, without the need for chemical modification or amino acid substitution. Due to the stability of these disordered Aß oligomers and the reproducibility with which they form, they are amenable for biophysical and high-resolution structural characterization.

A blend of two resveratrol derivatives abolishes hIAPP amyloid growth and membrane damage.

Type II diabetes mellitus (T2DM) is characterized by the presence of amyloid deposits of the human islet amyloid polypeptide (hIAPP) in pancreatic ß-cells. A wealth of data supports the hypothesis that hIAPP's toxicity is related to an abnormal interaction of amyloids with islet cell membranes. Thus, many studies aimed at finding effective therapies for T2DM focus on the design of molecules that are able to inhibit hIAPP's amyloid growth and the related membrane damage as well. Based on this view and inspired by its known anti-amyloid properties, we have functionalized resveratrol with a phosphoryl moiety (4'-O-PR) that improves its solubility and pharmacological properties. A second resveratrol derivative has also been obtained by conjugating resveratrol with a dimyristoylphosphatidyl moiety (4'-DMPR). The use of both compounds resulted in abolishing both amyloid growth and amyloid mediated POPC/POPS membrane damage in tube tests. We propose that a mixture of a water-soluble anti-aggregating compound and its lipid-anchored derivative may be employed as a general strategy to prevent and/or to halt amyloid-related membrane damage.

High-resolution NMR characterization of low abundance oligomers of amyloid-ß without purification.

Alzheimer's disease is characterized by the misfolding and self-assembly of the amyloidogenic protein amyloid-ß (Aß). The aggregation of Aß leads to diverse oligomeric states, each of which may be potential targets for intervention. Obtaining insight into Aß oligomers at the atomic level has been a major challenge to most techniques. Here, we use magic angle spinning recoupling (1)H-(1)H NMR experiments to overcome many of these limitations. Using (1)H-(1)H dipolar couplings as a NMR spectral filter to remove both high and low molecular weight species, we provide atomic-level characterization of a non-fibrillar aggregation product of the Aß1-40 peptide using non-frozen samples without isotopic labeling. Importantly, this spectral filter allows the detection of the specific oligomer signal without a separate purification procedure. In comparison to other solid-state NMR techniques, the experiment is extraordinarily selective and sensitive. A resolved 2D spectra could be acquired of a small population of oligomers (6 micrograms, 7% of the total) amongst a much larger population of monomers and fibers (93% of the total). By coupling real-time (1)H-(1)H NMR experiments with other biophysical measurements, we show that a stable, primarily disordered Aß1-40 oligomer 5-15 nm in diameter can form and coexist in parallel with the well-known cross-ß-sheet fibrils.

Atomic force microscopy and MD simulations reveal pore-like structures of all-D-enantiomer of Alzheimer's ß-amyloid peptide: relevance to the ion channel mechanism of AD pathology.

Alzheimer's disease (AD) is a protein misfolding disease characterized by a buildup of ß-amyloid (Aß) peptide as senile plaques, uncontrolled neurodegeneration, and memory loss. AD pathology is linked to the destabilization of cellular ionic homeostasis and involves Aß peptide-plasma membrane interactions. In principle, there are two possible ways through which disturbance of the ionic homeostasis can take place: directly, where the Aß peptide either inserts into the membrane and creates ion-conductive pores or destabilizes the membrane organization, or, indirectly, where the Aß peptide interacts with existing cell membrane receptors. To distinguish between these two possible types of Aß-membrane interactions, we took advantage of the biochemical tenet that ligand-receptor interactions are stereospecific; L-amino acid peptides, but not their D-counterparts, bind to cell membrane receptors. However, with respect to the ion channel-mediated mechanism, like L-amino acids, D-amino acid peptides will also form ion channel-like structures. Using atomic force microscopy (AFM), we imaged the structures of both D- and L-enantiomers of the full length Aß(1-42) when reconstituted in lipid bilayers. AFM imaging shows that both L- and D-Aß isomers form similar channel-like structures. Molecular dynamics (MD) simulations support the AFM imaged 3D structures. Previously, we have shown that D-Aß(1-42) channels conduct ions similarly to their L- counterparts. Taken together, our results support the direct mechanism of Aß ion channel-mediated destabilization of ionic homeostasis rather than the indirect mechanism through Aß interaction with membrane receptors.

All-d-Enantiomer of ß-Amyloid Peptide Forms Ion Channels in Lipid Bilayers.

Alzheimer's disease (AD) is the most common type of senile dementia in aging populations. Amyloid ß (Aß)-mediated dysregulation of ionic homeostasis is the prevailing underlying mechanism leading to synaptic degeneration and neuronal death. Aß-dependent ionic dysregulation most likely occurs either directly via unregulated ionic transport through the membrane or indirectly via Aß binding to cell membrane receptors and subsequent opening of existing ion channels or transporters. Receptor binding is expected to involve a high degree of stereospecificity. Here, we investigated whether an Aß peptide enantiomer, whose entire sequence consists of d-amino acids, can form ion-conducting channels; these channels can directly mediate Aß effects even in the absence of receptor-peptide interactions. Using complementary approaches of planar lipid bilayer (PLB) electrophysiological recordings and molecular dynamics (MD) simulations, we show that the d-Aß isomer exhibits ion conductance behavior in the bilayer indistinguishable from that described earlier for the l-Aß isomer. The d isomer forms channel-like pores with heterogeneous ionic conductance similar to the l-Aß isomer channels, and the d-isomer channel conductance is blocked by Zn(2+), a known blocker of l-Aß isomer channels. MD simulations further verify formation of ß-barrel-like Aß channels with d- and l-isomers, illustrating that both d- and l-Aß barrels can conduct cations. The calculated values of the single-channel conductance are approximately in the range of the experimental values. These findings are in agreement with amyloids forming Ca(2+) leaking, unregulated channels in AD, and suggest that Aß toxicity is mediated through a receptor-independent, nonstereoselective mechanism.