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Huntington’s disease (HD) is a neurodegenerative disorder caused by the expansion of the polyglutamine (polyQ) segment in the exon 1 of the huntingtin (HttEx1) protein. This polyQ expansion leads to protein misfolding and the formation of β-sheet-rich fibrillar aggregates. Several studies have shown that these protein deposits can cause cytotoxicity, suggesting the development of small molecule aggregation inhibitors as potential modulators of HD pathogenesis. This requires a molecular understanding of the impacts of such modulators on the interplay of aggregation, polymorphism and toxic gain-of-function. Here, we study how a polyphenol modulates the HttEx1 aggregation mechanism at sub-stoichiometric ratios. Moreover, we examine how the disrupted aggregation process impacts the protein’s misfolded structure and neurotoxic properties. We combine measurements of aggregation kinetics, electron microscopy, solid-state NMR, cytometry, and cytotoxicity assays. A notable delay of protein aggregation was observed even at sub-stoichiometric ratios of curcumin relative to the HttEx1 protein. Mechanistically, extension of the lag phase indicates an impact on the primary nucleation process that underpins the complex HttEx1 aggregation pathway, with an apparent role for β-hairpin formation. Remarkably, the deposits formed (more slowly) in presence of inhibitor displayed reduced toxicity in cultured neuronal cells, seemingly derived from their modulated structures. Thus, curcumin has a multifaceted effect based on delaying the fibril formation, while also changing the toxic properties of formed fibrils. Our findings highlight the ability of small molecule inhibitors to modulate the protein misfolding landscape, with potential implications for treatment strategies in HD and other protein deposition disorders.Significance StatementHuntington’s disease is an incurable inherited neurodegenerative disorder in which the mutated protein undergoes misfolding and forms fibrillar aggregates. The inhibition of these pathogenic processes represents a major challenge, which also requires an understanding of the complex aggregation mechanism. We observe how small molecule inhibitors can delay formation of toxic deposits by perturbing the aggregation kinetics. Crucially, we find that the modulated aggregation process yields less-toxic fibrils with a different molecular structure. This highlights how inhibition of aggregation process can have unexpected and unintended consequences by yielding novel fibril polymorphs with different biological properties. Analogous multifaceted effects should be considered in the design and testing of aggregation modulators, as they offer both risks and unexpected opportunities as new treatment modalities.

Hyaluronic acid (HA) is a naturally occurring polysaccharide that is abundant in the extracellular matrix (ECM) of all vertebrate cells. HA-based hydrogels have attracted great interest for biomedical applications due to their high viscoelasticity and biocompatibility. In both ECM and hydrogel applications, high molecular weight (HMW)-HA can absorb a large amount of water to yield matrices with a high level of structural integrity. To understand the molecular underpinnings of structural and functional properties of HA-containing hydrogels, few techniques are available. Nuclear magnetic resonance (NMR) spectroscopy is a powerful tool for such studies, e.g.13C NMR measurements can reveal the structural and dynamical features of (HMW) HA. However, a major obstacle to13C NMR is the low natural abundance of13C, necessitating the generation of HMW-HA that is enriched with13C isotopes. Here we present a convenient method to obtain13C- and15N-enriched HMW-HA in good yield fromStreptococcus equisubsp. zooepidemicus. The labeled HMW-HA has been characterized by solution and magic angle spinning (MAS) solid-state NMR spectroscopy, as well as other methods. These results will open new ways to study the structure and dynamics of HMW-HA-based hydrogels, and interactions of HMW-HA with proteins and other ECM components, using advanced NMR techniques.

International audience; We show that multidimensional solid-state NMR 13C–13C correlation spectra of biomolecular assemblies and microcrystalline organic molecules can be acquired at natural isotopic abundance with only milligram quantities of sample. These experiments combine fast Magic Angle Spinning of the sample, low-power dipolar recoupling, and dynamic nuclear polarization performed with AsymPol biradicals, a recently introduced family of polarizing agents. Such experiments are essential for structural characterization as they provide short- and long-range distance information. This approach is demonstrated on diverse sample types, including polyglutamine fibrils implicated in Huntington's disease and microcrystalline ampicillin, a small antibiotic molecule.

Alginate hydrogels are versatile self-assembling biocompatible materials with diverse biomedical and food industrial applications, which includes uses in encapsulation, (drug) delivery and tissue engineering. Hydrogel formation requires cross-linking, which for alginates is often done with calcium ions that engage in specific interactions with the polysaccharide carboxylic acid groups. Water molecules also hydrate these alginate groups and fill macropores within the hydrogels, with implications for both mechanical properties and cargo encapsulation. Understanding these aspects of hydrogels requires the observation and characterization of the hydrogel waters, how they engage the alginate, and fill the macropores. Here we employed solid-state NMR (ssNMR) spectroscopy to detect and study water molecules in re-hydrated alginate hydrogels. 1H, 2H, and 13C magic angle spinning (MAS) NMR and relaxation measurements were combined to observe both water and alginate. Two different water phases were detected that vary upon gradual (re)hydration of the alginate hydrogels. These water pools differ in their chemical shifts and NMR relaxation properties, reflecting hydration waters directly associated with the carbohydrate polymers alongside dynamic waters in the macropores. Thus, the ssNMR detects the water-filled macropore water pools and how they vary upon calcium cross-linking. We also observe how calcium cross-linking selectively immobilizes the α-guluronate monosaccharides, but leaves the β-mannuronate units more flexible and prone to selective re-hydration. Thus, these ssNMR experiments can be used to probe cross-linking and hydration of alginate hydrogels, with implications for our understanding of design parameters that tune their performance in (drug) delivery and other food industrial applications.

Huntington's disease is a progressive neurodegenerative disease caused by expansion of the polyglutamine domain in the first exon of huntingtin (HttEx1). The extent of expansion correlates with disease progression and formation of amyloid-like protein deposits within the brain. The latter display polymorphism at the microscopic level, both in cerebral tissue and in vitro. Such polymorphism can dramatically influence cytotoxicity, leading to much interest in the conditions and mechanisms that dictate the formation of polymorphs. We examine conditions that govern HttEx1 polymorphism in vitro, including concentration and the role of the non-polyglutamine flanking domains. Using electron microscopy, we observe polymorphs that differ in width and tendency for higher-order bundling. Strikingly, aggregation yields different polymorphs at low and high concentrations. Narrow filaments dominate at low concentrations that may be more relevant in vivo. We dissect the role of N- and C-terminal flanking domains using protein with the former (httNT or N17) largely removed. The truncated protein is generated by trypsin cleavage of soluble HttEx1 fusion protein, which we analyze in some detail. Dye binding and solid-state NMR studies reveal changes in fibril surface characteristics and flanking domain mobility. Higher-order interactions appear facilitated by the C-terminal tail, while the polyglutamine forms an amyloid core resembling those of other polyglutamine deposits. Fibril-surface-mediated branching, previously attributed to secondary nucleation, is reduced in absence of httNT. A new model for the architecture of the HttEx1 filaments is presented and discussed in context of the assembly mechanism and biological activity.

In structural studies of immobilized, aggregated and self-assembled biomolecules, solid-state NMR (ssNMR) spectroscopy can provide valuable high-resolution structural information. Among the structural restraints provided by magic angle spinning (MAS) ssNMR the canonical focus is on inter-atomic distance measurements. In the current review, we examine the utility of ssNMR measurements of angular constraints, as a complement to distance-based structure determination. The focus is on direct measurements of angular restraints via the judicious recoupling of multiple anisotropic ssNMR parameters, such as dipolar couplings and chemical shift anisotropies. Recent applications are highlighted, with a focus on studies of nanocrystalline polypeptides, aggregated peptides and proteins, receptor-substrate interactions, and small molecule interactions with amyloid protein fibrils. The review also examines considerations of when and where ssNMR torsion angle experiments are (most) effective, and discusses challenges and opportunities for future applications.

Mitochondria form tubular networks that undergo coordinated cycles of fission and fusion. Emerging evidence suggests that a direct yet unresolved interaction of the mechanoenzymatic GTPase dynamin-related protein 1 (Drp1) with mitochondrial outer membrane-localized cardiolipin (CL), externalized under stress conditions including mitophagy, catalyzes essential mitochondrial hyperfragmentation. Here, using a comprehensive set of structural, biophysical, and cell biological tools, we have uncovered a CL-binding motif (CBM) conserved between the Drp1 variable domain (VD) and the unrelated ADP/ATP carrier (AAC/ANT) that intercalates into the membrane core to effect specific CL interactions. CBM mutations that weaken VD-CL interactions manifestly impair Drp1-dependent fission under stress conditions and induce "donut" mitochondria formation. Importantly, VD membrane insertion and GTP-dependent conformational rearrangements mediate only transient CL nonbilayer topological forays and high local membrane constriction, indicating that Drp1-CL interactions alone are insufficient for fission. Our studies establish the structural and mechanistic bases of Drp1-CL interactions in stress-induced mitochondrial fission., The research in this study was supported by NIH R01 Grant GM121583 awarded to R.R. P.C.A.v.d.W. was supported by NIH R01 Grant GM112678, and J.C.B. was supported by NIH T32 Grant GM088119 and the Achievement Rewards for College Scientists Foundation. NIH R01 Grant GM112491 to M.B. supported his and M.M.’s contributions, while NIH R01 Grant NS088192 supported X.Q., Y.S., and R.W. A.S. was supported by Spain Ministry of Science and Innovation/National Research Agency/European Regional Development Fund Grant PGC2018-099971-B-I00 and EUR2019-103830, and Basque Government Grant IT1270-19. J.M.M.G. acknowledges the predoctoral fellowship from the University of the Basque Country. The 750-MHz ssNMR instrument was funded by NIH Grant S10RR033477.

Cardiolipin (CL) is a mitochondrial anionic lipid that plays important roles in the regulation and signaling of mitochondrial apoptosis. CL peroxidation catalyzed by the assembly of CL-cytochrome c (cyt c) complexes at the inner mitochondrial membrane is a critical checkpoint. The structural changes in the protein, associated with peroxidase activation by CL and different anionic lipids, are not known at a molecular level. To better understand these peripheral protein-lipid interactions, we compare how phosphatidylglycerol (PG) and CL lipids trigger cyt c peroxidase activation, and correlate functional differences to structural and motional changes in membrane-associated cyt c. Structural and motional studies of the bound protein are enabled by magic angle spinning solid state NMR spectroscopy, while lipid peroxidase activity is assayed by mass spectrometry. PG binding results in a surface-bound state that preserves a nativelike fold, which nonetheless allows for significant peroxidase activity, though at a lower level than binding its native substrate CL. Lipid-specific differences in peroxidase activation are found to correlate to corresponding differences in lipid-induced protein mobility, affecting specific protein segments. The dynamics of omega loops C and D are upregulated by CL binding, in a way that is remarkably controlled by the protein:lipid stoichiometry. In contrast to complete chemical denaturation, membrane-induced protein destabilization reflects a destabilization of select cyt c foldons, while the energetically most stable helices are preserved. Our studies illuminate the interplay of protein and lipid dynamics in the creation of lipid peroxidase-active proteolipid complexes implicated in early stages of mitochondrial apoptosis.

Cardiolipin (CL) is a mitochondrial anionic lipid that plays important roles in the regulation and signaling of mitochondrial apoptosis. CL peroxidation catalyzed by the assembly of CL-cytochrome c (cyt c) complexes at the inner mitochondrial membrane is a critical checkpoint. The structural changes in the protein, associated with peroxidase activation by CL and different anionic lipids, are not known at a molecular level. To better understand these peripheral protein-lipid interactions, we compare how phosphatidylglycerol (PG) and CL lipids trigger cyt c peroxidase activation, and correlate functional differences to structural and motional changes in membrane-associated cyt c. Structural and motional studies of the bound protein are enabled by magic angle spinning solid state NMR spectroscopy, while lipid peroxidase activity is assayed by mass spectrometry. PG binding results in a surface-bound state that preserves a nativelike fold, which nonetheless allows for significant peroxidase activity, though at a lower level than binding its native substrate CL. Lipid-specific differences in peroxidase activation are found to correlate to corresponding differences in lipid-induced protein mobility, affecting specific protein segments. The dynamics of omega loops C and D are upregulated by CL binding, in a way that is remarkably controlled by the protein:lipid stoichiometry. In contrast to complete chemical denaturation, membrane-induced protein destabilization reflects a destabilization of select cyt c foldons, while the energetically most stable helices are preserved. Our studies illuminate the interplay of protein and lipid dynamics in the creation of lipid peroxidase-active proteolipid complexes implicated in early stages of mitochondrial apoptosis.

The Hsp40/Hsp70 chaperone families combine versatile folding capacity with high substrate specificity, which is mainly facilitated by Hsp40s. The structure and function of many Hsp40s remain poorly understood, particularly oligomeric Hsp40s that suppress protein aggregation. Here, we used a combination of biochemical and structural approaches to shed light on the domain interactions of the Hsp40 DnaJB8, and how they may influence recruitment of partner Hsp70s. We identify an interaction between the J-Domain (JD) and C-terminal domain (CTD) of DnaJB8 that sequesters the JD surface, preventing Hsp70 interaction. We propose a model for DnaJB8-Hsp70 recruitment, whereby the JD-CTD interaction of DnaJB8 acts as a reversible switch that can control the binding of Hsp70. These findings suggest that the evolutionarily conserved CTD of DnaJB8 is a regulatory element of chaperone activity in the proteostasis network., The Hsp70/Hsp40 system plays an important role in maintaining cellular proteostasis but so far it is not well understood how Hsp70 proteins are recruited to specific Hsp40 co-chaperones. Here, the authors combine biochemical and biophysical approaches to characterise the oligomeric mammalian Hsp40 DnaJB8. They identify an intra-oligomer DnaJB8 interaction between the N-terminal J-Domain and the C-terminal domain that occludes the J-Domain surface that binds Hsp70 and propose a model for DnaJB8-Hsp70 recruitment.

Hydrogels find application in many areas of technology and research due to their ability to combine responsiveness and robustness. A detailed understanding of their molecular structure and dynamics (which ultimately underpin their functional properties) is needed for their design to be optimized and these hydrogels to be exploited effectively. In this review, we shed light on the unique capabilities of solid-state NMR spectroscopy to reveal this information in molecular detail. We review recent literature on the advancements in solid-state NMR techniques in resolving the structure, degree of grafting, molecular organization, water-biopolymer interactions and internal dynamical behavior of hydrogels. Among various solid-state NMR techniques, 13C cross polarization (CP) magic angle spinning (MAS) NMR is examined for its ability to probe the hydrogel and its trapped solvent. Although widely applicable to many types of polymeric and supramolecular hydrogels, the current review focuses on polysaccharide-based hydrogels.

International audience; A pathological hallmark of Huntington's disease (HD) is the formation of neuronal protein deposits containing mutant huntingtin fragments with expanded polyglutamine (polyQ) domains. Prior studies have shown the strengths of solid-state NMR (ssNMR) to probe the atomic structure of such aggregates, but have required in vitro isotopic labeling. Herein, we present an approach for the structural fingerprinting of fibrils through ssNMR at natural isotopic abundance (NA). These methods will enable the spectroscopic fingerprinting of unlabeled (e.g., ex vivo) protein aggregates and the extraction of valuable new long-range 13 C− 13 C distance constraints

Solid-state nuclear magnetic resonance (ssNMR) spectroscopy enables the structural characterization of a diverse array of biological assemblies that include amyloid fibrils, non-amyloid aggregates, membrane-associated proteins and viral capsids. Such biological samples feature functionally relevant molecular dynamics, which often affect different parts of the sample in different ways. Solid-state NMR experiments' sensitivity to dynamics represents a double-edged sword. On the one hand, it offers a chance to measure dynamics in great detail. On the other hand, certain types of motion lead to signal loss and experimental inefficiencies that at first glance interfere with the application of ssNMR to overly dynamic proteins. Dynamics-based spectral editing (DYSE) ssNMR methods leverage motion-dependent signal losses to simplify spectra and enable the study of substructures with particular motional properties.

Various recent developments in solid-state nuclear magnetic resonance (ssNMR) spectroscopy have enabled an array of new insights regarding the structure, dynamics, and interactions of biomolecules. In the ever more integrated world of structural biology, ssNMR studies provide structural and dynamic information that is complementary to the data accessible by other means. ssNMR enables the study of samples lacking a crystalline lattice, featuring static as well as dynamic disorder, and does so independent of higher-order symmetry. The present study surveys recent applications of biomolecular ssNMR and examines how this technique is increasingly integrated with other structural biology techniques, such as (cryo) electron microscopy, solution-state NMR, and X-ray crystallography. Traditional ssNMR targets include lipid bilayer membranes and membrane proteins in a lipid bilayer environment. Another classic application has been in the area of protein misfolding and aggregation disorders, where ssNMR has provided essential structural data on oligomers and amyloid fibril aggregates. More recently, the application of ssNMR has expanded to a growing array of biological assemblies, ranging from non-amyloid protein aggregates, protein–protein complexes, viral capsids, and many others. Across these areas, multidimensional magic angle spinning (MAS) ssNMR has, in the last decade, revealed three-dimensional structures, including many that had been inaccessible by other structural biology techniques. Equally important insights in structural and molecular biology derive from the ability of MAS ssNMR to probe information beyond comprehensive protein structures, such as dynamics, solvent exposure, protein–protein interfaces, and substrate–enzyme interactions.

Apolipoprotein A-I (apoA-I) shares with other exchangeable apolipoproteins a high level of structural plasticity. In the lipid-free state, the apolipoprotein amphipathic α-helices interact intra- and intermolecularly, providing structural stabilization by self-association. We have reported that lipid-free apoA-I becomes amyloidogenic upon physiologically relevant (myeloperoxidase-mediated) Met oxidation. In this study, we established that Met oxidation promotes amyloidogenesis by reducing the stability of apoA-I monomers and irreversibly disrupting self-association. The oxidized apoA-I monomers also exhibited increased cellular cholesterol release capacity and stronger association with macrophages, compared to nonoxidized apoA-I. Of physiologic relevance, preformed oxidized apoA-I amyloid fibrils induced amyloid formation in nonoxidized apoA-I. This process was enhanced when self-association of nonoxidized apoA-I was disrupted by thermal treatment. Solid state NMR analysis revealed that aggregates formed by seeded nonoxidized apoA-I were structurally similar to those formed by the oxidized protein, featuring a β-structure-rich amyloid fold alongside α-helices retained from the native state. In atherosclerotic lesions, the conditions that promote apoA-I amyloid formation are readily available: myeloperoxidase, active oxygen species, low pH, and high concentration of lipid-free apoA-I. Our results suggest that even partial Met oxidation of apoA-I can nucleate amyloidogenesis, thus sequestering and inactivating otherwise antiatherogenic and HDL-forming apoA-I.—Witkowski, A., Chan, G. K. L., Boatz, J. C., Li, N. J., Inoue, A. P., Wong, J. C., van der Wel, P. C. A., Cavigiolio, G. Methionine oxidized apolipoprotein A-I at the crossroads of HDL biogenesis and amyloid formation.

The aggregation of proteins and peptides into a variety of insoluble, and often non-native, aggregated states plays a central role in many devastating diseases. Analogous processes undermine the efficacy of polypeptide-based biological pharmaceuticals, but are also being leveraged in the design of biologically inspired self-assembling materials. This Trends article surveys the essential contributions made by recent solid-state NMR (ssNMR) studies to our understanding of the structural features of polypeptide aggregates, and how such findings are informing our thinking about the molecular mechanisms of misfolding and aggregation. A central focus is on disease-related amyloid fibrils and oligomers involved in neurodegenerative diseases such as Alzheimer’s, Parkinson’s and Huntington’s disease. SSNMR-enabled structural and dynamics-based findings are surveyed, along with a number of resulting emerging themes that appear common to different amyloidogenic proteins, such as their compact alternating short-β-strand/β-arc amyloid core architecture. Concepts, methods, future prospects and challenges are discussed.

Multiplication-stimulating activity (MSA) produced by Buffalo rat liver cells (BRL-3A) in culture is related to the somatomedin family of growth regulatory polypeptides. MSA will stimulate glucose transport and DNA synthesis in normal chicken embryo fibroblasts (CEF) at concentrations of 10-200 ng/ml. MSA found in BRL-3A-conditioned medium, like the somatomedins in serum, does not exist as the free hormone but is bound to a specific high molecular weight carrier protein. In this report we demonstrate that purified MSA carrier protein (MCP) inhibits the biological activity of MSA on CEF as measured by the stimulation of glucose transport and DNA synthesis. In addition, purified MCP competitively inhibits the binding of 125I-labeled MSA to these cells. In control experiments in which insulin was used as the mitogenic agent, MCP had no effect on these biological responses. These results indicate that the inhibitory effect of MCP is the result of specific interaction with MSA and support the hypothesis that cells may be unresponsive to somatomedins bound to their serum carrier proteins.

The lipid bilayer typical of hydrated biological membranes is characterized by a liquid-crystalline, highly dynamic state. Upon cooling or dehydration, these membranes undergo a cooperative transition to a rigidified, more-ordered, gel phase. This characteristic phase transition is of significant biological and biophysical interest, for instance in studies of freezing-tolerant organisms. Magic-angle-spinning (MAS) solid-state NMR (ssNMR) spectroscopy allows for the detection and characterization of the phase transitions over a wide temperature range. In this study we employ MAS 1H NMR to probe the phase transitions of both solvent molecules and different hydrated phospholipids, including tetraoleoyl cardiolipin (TOCL) and several phosphatidylcholine lipid species. The employed MAS NMR sample conditions cause a previously noted substantial reduction in the freezing point of the solvent phase. The effect on the solvent is caused by confinement of the aqueous solvent in the small and densely packed MAS NMR samples. In this study we report and examine how the freezing point depression also impacts the lipid phase transition, causing a ssNMR-observed reduction in the lipids' melting temperature (Tm). The molecular underpinnings of this phenomenon are discussed and compared with previous studies of membrane-associated water phases and the impact of membrane-protective cryoprotectants.

Polyglutamine expansion within the exon1 of huntingtin leads to protein misfolding, aggregation, and cytotoxicity in Huntington's disease. This incurable neurodegenerative disease is the most prevalent member of a family of CAG repeat expansion disorders. Although mature exon1 fibrils are viable candidates for the toxic species, their molecular structure and how they form have remained poorly understood. Using advanced magic angle spinning solid-state NMR, we directly probe the structure of the rigid core that is at the heart of huntingtin exon1 fibrils and other polyglutamine aggregates, via measurements of long-range intramolecular and intermolecular contacts, backbone and side-chain torsion angles, relaxation measurements, and calculations of chemical shifts. These experiments reveal the presence of β-hairpin-containing β-sheets that are connected through interdigitating extended side chains. Despite dramatic differences in aggregation behavior, huntingtin exon1 fibrils and other polyglutamine-based aggregates contain identical β-strand-based cores. Prior structural models, derived from X-ray fiber diffraction and computational analyses, are shown to be inconsistent with the solid-state NMR results. Internally, the polyglutamine amyloid fibrils are coassembled from differently structured monomers, which we describe as a type of "intrinsic" polymorphism. A stochastic polyglutamine-specific aggregation mechanism is introduced to explain this phenomenon. We show that the aggregation of mutant huntingtin exon1 proceeds via an intramolecular collapse of the expanded polyglutamine domain and discuss the implications of this observation for our understanding of its misfolding and aggregation mechanisms.

The peroxidation of cardiolipins by reactive oxygen species (ROS), which is regulated and enhanced by cytochrome c (cyt c), is a critical signaling event in mitochondrial apoptosis. We probe the molecular underpinnings of this mitochondrial death signal through structural and functional studies of horse heart cyt c binding to mixed-lipid membranes containing cardiolipin with mono- and polyunsaturated acyl chains. Lipidomics reveal the selective oxidation of polyunsaturated fatty acid (PUFA) cardiolipin, while multidimensional solid state NMR (ssNMR) probes the structure and dynamics of the membrane and the peripherally bound protein. The hydrophilic milieu at the membrane interface stabilizes a native-like fold, but also leads to localized flexibility at the membrane-interacting protein face. PUFA CL acts as both a preferred substrate and a dynamic regulator by impacting the dynamics of the cyt c N70-I85 omega loop, which covers the heme cavity.

The cellular process of intrinsic apoptosis relies on the peroxidation of mitochondrial lipids as a critical molecular signal. Lipid peroxidation is connected to increases in mitochondrial reactive oxygen species, but there is also a required role for mitochondrial cytochrome c (cyt-c). In apoptotic mitochondria, cyt-c gains a new function as a lipid peroxidase that catalyzes the reactive oxygen species-mediated chemical modification of the mitochondrial lipid cardiolipin (CL). This peroxidase activity is caused by a conformational change in the protein, resulting from interactions between cyt-c and CL. The nature of the conformational change and how it causes this gain-of-function remain uncertain. Via a combination of functional, structural, and biophysical experiments we investigate the structure and peroxidase activity of cyt-c in its membrane-bound state. We reconstituted cyt-c with CL-containing lipid vesicles, and determined the increase in peroxidase activity resulting from membrane binding. We combined these assays of CL-induced proapoptotic activity with structural and dynamic studies of the membrane-bound protein via solid-state NMR and optical spectroscopy. Multidimensional magic angle spinning (MAS) solid-state NMR of uniformly 13C,15N-labeled protein was used to detect site-specific conformational changes in oxidized and reduced horse heart cyt-c bound to CL-containing lipid bilayers. MAS NMR and Fourier transform infrared measurements show that the peripherally membrane-bound cyt-c experiences significant dynamics, but also retains most or all of its secondary structure. Moreover, in two-dimensional and three-dimensional MAS NMR spectra the CL-bound cyt-c displays a spectral resolution, and thus structural homogeneity, that is inconsistent with extensive membrane-induced unfolding. Cyt-c is found to interact primarily with the membrane interface, without significantly disrupting the lipid bilayer. Thus, membrane binding results in cyt-c gaining the increased peroxidase activity that represents its pivotal proapoptotic function, but we do not observe evidence for large-scale unfolding or penetration into the membrane core.

Amyloid fibrils are highly ordered protein aggregates associated with more than 40 human diseases. The exact conditions in which the fibrils are grown determine many types of reported fibril polymorphism, including different twist patterns. Twist-based polymorphs display unique mechanical properties in vitro and the relevance of twist polymorphism in amyloid diseases has been suggested. We present transmission electron microscopy images of Aβ42-derived (amyloid β) fibrils, which are associated with Alzheimer's disease, demonstrating the presence of twist variability even within a single long fibril. To better understand the molecular underpinnings of twist polymorphism, we present a structural and thermodynamics analysis of molecular dynamics simulations of the twisting of β-sheet protofilaments of a well-characterized cross-β model: the GNNQQNY peptide from the yeast prion Sup35. The results show that a protofilament model of GNNQQNY is able to adopt twist angles from -11 degrees on the left-handed side to +8 degrees on the right-handed side in response to various external conditions while keeping an unchanged peptide structure. The potential of mean force (PMF) of this cross-β structure upon twisting revealed that only ~2 kB¬T per peptide are needed to stabilize a straight conformation with respect to the left-handed free energy minimum. The PMF also shows that the canonical structural core of β-sheets, i.e. the hydrogen-bonded backbone β-strands, favours the straight conformation. However, the concerted effects of the side chains contribute to twisting, which provides a rationale to correlate polypeptide sequence, environmental growth conditions and number of protofilaments in a fibril with twist polymorphisms.

Polyglutamine expansion in the huntingtin protein is the primary genetic cause of Huntington's disease (HD). Fragments coinciding with mutant huntingtin exon1 aggregate in vivo and induce HD-like pathology in mouse models. The resulting aggregates can have different structures that affect their biochemical behaviour and cytotoxic activity. Here we report our studies of the structure and functional characteristics of multiple mutant htt exon1 fibrils by complementary techniques, including infrared and solid-state NMR spectroscopies. Magic-angle-spinning NMR reveals that fibrillar exon1 has a partly mobile α-helix in its aggregation-accelerating N terminus, and semi-rigid polyproline II helices in the proline-rich flanking domain (PRD). The polyglutamine-proximal portions of these domains are immobilized and clustered, limiting access to aggregation-modulating antibodies. The polymorphic fibrils differ in their flanking domains rather than the polyglutamine amyloid structure. They are effective at seeding polyglutamine aggregation and exhibit cytotoxic effects when applied to neuronal cells., Huntington's disease is caused by a polyglutamine stretch expansion in the first exon of huntingtin. Here, the authors use infrared spectroscopy and solid-state NMR and show that polymorphic huntingtin exon1 fibres differ in their flanking regions but not their core polyglutamine amyloid structures.

Cataracts cause vision loss through the large-scale aggregation of eye lens proteins as a result of ageing or congenital mutations. The development of new treatments is hindered by uncertainty about the nature of the aggregates and their mechanism of formation. We describe the structure and morphology of aggregates formed by the P23T human γD-crystallin mutant associated with congenital cataracts. At physiological pH, the protein forms aggregates that look amorphous and disordered by electron microscopy, reminiscent of the reported formation of amorphous deposits by other crystallin mutants. Surprisingly, solid-state NMR reveals that these amorphous deposits have a high degree of structural homogeneity at the atomic level and that the aggregated protein retains a native-like conformation, with no evidence for large-scale misfolding. Non-physiological destabilizing conditions used in many in vitro aggregation studies are shown to yield qualitatively different, highly misfolded amyloid-like fibrils., Aggregation of eye lens proteins leads to cataracts, a major cause of blindness. Here the authors use solid state NMR to probe the structure of γD-crystallin eye lens proteins aggregates, which are found to retain a native-like conformation.

Polyglutamine (polyQ) amyloid fibrils are observed in disease tissue and have been implicated as toxic agents responsible for neurodegeneration in expanded CAG repeat diseases like Huntington’s disease (HD). Despite intensive efforts, the mechanism of amyloid toxicity remains unknown. As a novel approach to probing polyQ toxicity, we investigate here how some cellular and physical properties of polyQ amyloid vary with the chirality of the glutamine residues in the polyQ. We challenged PC12 cells with small amyloid fibrils composed of either L- or D-polyQ peptides and found that D-fibrils are as cytotoxic as L-fibrils. We also found using fluorescence microscopy that both aggregates effectively seed the aggregation of cell-produced L-polyQ proteins, suggesting a surprising lack of stereochemical restriction in seeded elongation of polyQ amyloid. To investigate this effect further, we studied chemically synthesized D- and L-polyQ in vitro. We found that, as expected, D-polyQ monomers are not recognized by proteins that recognize L-polyQ monomers. However, amyloid fibrils prepared from D-polyQ peptides can efficiently seed the aggregation of L-polyQ monomers in vitro, and vice versa. This result is consistent with our cell results on polyQ recruitment, but is inconsistent with previous literature reports on the chiral specificity of amyloid seeding. This chiral cross-seeding can be rationalized by a model for seeded elongation featuring a “rippled β-sheet” interface between seed fibril and docked monomers of opposite chirality. The lack of chiral discrimination in polyQ amyloid cytotoxicity is consistent with several toxicity mechanisms, including recruitment of cellular polyQ proteins.

Structural measurements in magic-angle-spinning (MAS) solid-state NMR rely heavily on (13)C-(13)C distance measurements. Broadbanded recoupling methods are used to generate many cross-peaks, but have complex polarization transfer mechanisms that limit the precision of distance constraints and can suffer from weak intensities for distant peaks due to relaxation, the broad distribution of polarization, as well as dipolar truncation. Frequency-selective methods that feature narrow-banded recoupling can reduce these effects. Indeed, rotational resonance (R(2)) experiments have found application in many different biological systems, where they have afforded improved precision and accuracy. Unfortunately, a highly selective transfer mechanism also leads to few cross-peaks in the resulting spectra, which complicates the extraction of multiple constraints. R(2)-width (R(2)W) measurements that scan a range of MAS rates to probe the R(2) matching conditions of one or more sites can improve precision, and also permit multiple simultaneous distance measurements. However, multidimensional R(2)W can be very time-consuming. Here, we present an approach that facilitates the acquisition of 2D-like spectra based on a series of 1D R(2)W experiments, by taking advantage of the chemical shift information encoded in the MAS rates where matching occurs. This yields a more time-efficient experiment with many of the benefits of more conventional multidimensional R(2)W measurements. The obtained spectra reveal long-distance (13)C-(13)C cross-peaks resulting from R(2)-mediated polarization transfer. This experiment also enables the efficient setup and targeted implementation of traditional R(2) or R(2)W experiments. Analogous applications may extend to other variable-MAS and frequency-selective solid-state NMR experiments.

Candidates for the toxic molecular species in the expanded polyglutamine (polyQ) repeat diseases range from various types of aggregates to "misfolded" monomers. One way to vet these candidates is to develop mutants that restrict conformational landscapes. Previously, we inserted two self-complementary β-hairpin enhancing motifs into a short polyQ sequence to generate a mutant, here called "βHP," that exhibits greatly improved amyloid nucleation without measurably enhancing β-structure in the monomer ensemble. We extend these studies here by introducing single-backbone H-bond impairing modifications αN-methyl Gln or l-Pro at key positions within βHP. Modifications predicted to allow formation of a fully H-bonded β-hairpin at the fibril edge while interfering with H-bonding to the next incoming monomer exhibit poor amyloid formation and act as potent inhibitors in trans of simple polyQ peptide aggregation. In contrast, a modification that disrupts intra-β-hairpin H-bonding within βHP, while also aggregating poorly, is ineffective at inhibiting amyloid formation in trans. The inhibitors constitute a dynamic version of the edge-protection negative design strategy used in protein evolution to limit unwanted protein aggregation. Our data support a model in which polyQ peptides containing strong β-hairpin encouraging motifs only rarely form β-hairpin conformations in the monomer ensemble, but nonetheless take on such conformations at key steps during amyloid formation. The results provide insights into polyQ solution structure and fibril formation while also suggesting an approach to the design of inhibitors of polyQ amyloid growth that focuses on conformational requirements for fibril and nucleus elongation.

A number of recent advances in the field of magic-angle-spinning (MAS) solid-state NMR have enabled its application to a range of biological systems of ever increasing complexity. To retain biological relevance, these samples are increasingly studied in a hydrated state. At the same time, experimental feasibility requires the sample preparation process to attain a high sample concentration within the final MAS rotor. We discuss these considerations, and how they have led to a number of different approaches to MAS NMR sample preparation. We describe our experience of how custom-made (or commercially available) ultracentrifugal devices can facilitate a simple, fast and reliable sample preparation process. A number of groups have since adopted such tools, in some cases to prepare samples for sedimentation-style MAS NMR experiments. Here we argue for a more widespread adoption of their use for routine MAS NMR sample preparation.

Chiral nanoparticle assemblies are an interesting class of materials whose chiroptical properties make them attractive for a variety of applications. Here, C18-(PEPAu(M-ox))2 (PEPAu(M-ox) = AYSSGAPPM(ox)PPF) is shown to direct the assembly of single-helical gold nanoparticle superstructures that exhibit exceptionally strong chiroptical activity at the plasmon frequency with absolute g-factor values up to 0.04. Transmission electron microscopy (TEM) and cryogenic electron tomography (cryo-ET) results indicate that the single helices have a periodic pitch of approximately 100 nm and consist of oblong gold nanoparticles. The morphology and assembled structure of C18-(PEPAu(M-ox))2 are studied using TEM, atomic force microscopy (AFM), Fourier transform infrared (FTIR) spectroscopy, circular dichroism (CD) spectroscopy, X-ray diffraction (XRD), and solid-state nuclear magnetic resonance (ssNMR) spectroscopy. TEM and AFM reveal that C18-(PEPAu(M-ox))2 assembles into linear amyloid-like 1D helical ribbons having structural parameters that correlate to those of the single-helical gold nanoparticle superstructures. FTIR, CD, XRD, and ssNMR indicate the presence of cross-β and polyproline II secondary structures. A molecular assembly model is presented that takes into account all experimental observations and that supports the single-helical nanoparticle assembly architecture. This model provides the basis for the design of future nanoparticle assemblies having programmable structures and properties.

Since experimental measurements of NMR chemical shifts provide time and ensemble averaged values, we investigated how these effects should be included when chemical shifts are computed using density functional theory (DFT). We measured the chemical shifts of the N-formyl-L-methionyl-L-leucyl-L-phenylalanine-OMe (MLF) peptide in the solid state, and then used the X-ray structure to calculate the (13)C chemical shifts using the gauge including projector augmented wave (GIPAW) method, which accounts for the periodic nature of the crystal structure, obtaining an overall accuracy of 4.2 ppm. In order to understand the origin of the difference between experimental and calculated chemical shifts, we carried out first-principles molecular dynamics simulations to characterize the molecular motion of the MLF peptide on the picosecond time scale. We found that (13)C chemical shifts experience very rapid fluctuations of more than 20 ppm that are averaged out over less than 200 fs. Taking account of these fluctuations in the calculation of the chemical shifts resulted in an accuracy of 3.3 ppm. To investigate the effects of averaging over longer time scales we sampled the rotameric states populated by the MLF peptides in the solid state by performing a total of 5 micros classical molecular dynamics simulations. By averaging the chemical shifts over these rotameric states, we increased the accuracy of the chemical shift calculations to 3.0 ppm, with less than 1 ppm error in 10 out of 22 cases. These results suggests that better DFT-based predictions of chemical shifts of peptides and proteins will be achieved by developing improved computational strategies capable of taking into account the averaging process up to the millisecond time scale on which the chemical shift measurements report.

The formation of amyloid-like fibrils is characteristic of various diseases, but the underlying mechanism and the factors that determine whether, when, and how proteins form amyloid, remain uncertain. Certain mechanisms have been proposed based on the three-dimensional or runaway domain swapping, inspired by the fact that some proteins show an apparent correlation between the ability to form domain-swapped dimers and a tendency to form fibrillar aggregates. Intramolecular β-sheet contacts present in the monomeric state could constitute intermolecular β-sheets in the dimeric and fibrillar states. One example is an amyloid-forming mutant of the immunoglobulin binding domain B1 of streptococcal protein G, which in its native conformation consists of a four-stranded β-sheet and one α-helix. Under native conditions this mutant adopts a domain-swapped dimer, and it also forms amyloid-like fibrils, seemingly in correlation to its domain-swapping ability. We employ magic angle spinning solid-state NMR and other methods to examine key structural features of these fibrils. Our results reveal a highly rigid fibril structure that lacks mobile domains and indicate a parallel in-register β-sheet structure and a general loss of native conformation within the mature fibrils. This observation contrasts with predictions that native structure, and in particular intermolecular β-strand interactions seen in the dimeric state, may be preserved in “domain-swapping” fibrils. We discuss these observations in light of recent work on related amyloid-forming proteins that have been argued to follow similar mechanisms and how this may have implications for the role of domain-swapping propensities for amyloid formation.

We present a de novo high-resolution structure of the peptide Alanyl-Prolyl-Glycine using a combination of sensitive solid-state NMR techniques that each yield precise structural constraints. High-quality (13)C-(13)C distance constraints are extracted by fitting rotational resonance width (R(2)W) experiments using Multimode Multipole Floquet Theory and experimental chemical shift anisotropy (CSA) orientations. In this strategy, a structure is first calculated using DANTE-REDOR and torsion angle measurements and the resulting relative CSA orientations are used as an input parameter in the (13)C-(13)C distance calculations. Finally, a refined structure is calculated using all the constraints. We investigate the effect of different structural constraints on structure quality, as determined by comparison to the crystal structure and also self-consistency of the calculated structures. Inclusion of all or subsets of these constraints into CNS calculations resulted in high-quality structures (0.02A backbone RMSD using all 11 constraints).

We used solid-state deuterium NMR spectroscopy and geometric analysis of labeled alanines to investigate the structure and orientation of a designed synthetic hydrophobic, membrane-spanning alpha-helical peptide that is anchored within phosphatidylcholine (PC) bilayers using both Trp and Lys side chains near the membrane/water interface. The 23-amino-acid peptide consists of an alternating Leu/Ala core sequence that is expected to be alpha-helical, flanked by aromatic and then cationic anchors at both ends of the peptide: acetyl-GKALW(LA)(6)LWLAKA-amide (KWALP23). The geometric analysis of labeled alanines method was elaborated to permit the incorporation and assignment of multiple alanine labels within a single synthetic peptide. Peptides were incorporated into oriented bilayers of dilauroyl- (di-C12:0-), dimyristoyl- (di-C14:0-), or dioleoyl- (di-C18:1c-) PC. In the C12:0 and C14:0 lipids, the (2)H-NMR quadrupolar splittings for the set of six core alanines could not be fit to a canonical undistorted alpha-helix. Rather, we found that a model containing a helical distortion, such as a localized discontinuity or "kink" near the peptide and bilayer center, could fit the data for KWALP23 in these shorter lipids. The suggestion of helix distortion was confirmed by (2)H-NMR spectra for KWALP23 in which Leu(8) was changed to deuterated Ala(8). Further analysis involving reexamination of earlier data led to a similar conclusion that acetyl-GWW(LA)(8)LWWA-amide (WALP23) is distorted in dilauroyl-PC, allowing significant improvement in the fitting of the (2)H-NMR results. In contrast, WALP23 and KWALP23 are well represented as undistorted alpha-helices in dioleoyl-PC, suggesting that the distortion could be a response to hydrophobic mismatch between peptide and lipids.

Dynamic nuclear polarization (DNP) permits a approximately 10(2)-10(3) enhancement of the nuclear spin polarization and therefore increases sensitivity in nuclear magnetic resonance (NMR) experiments. Here, we demonstrate the efficient transfer of DNP-enhanced (1)H polarization from an aqueous, radical-containing solvent matrix into peptide crystals via (1)H-(1)H spin diffusion across the matrix-crystal interface. The samples consist of nanocrystals of the amyloid-forming peptide GNNQQNY(7-13), derived from the yeast prion protein Sup35p, dispersed in a glycerol-water matrix containing a biradical polarizing agent, TOTAPOL. These crystals have an average width of 100-200 nm, and their known crystal structure suggests that the size of the biradical precludes its penetration into the crystal lattice; therefore, intimate contact of the molecules in the nanocrystal core with the polarizing agent is unlikely. This is supported by the observed differences between the time-dependent growth of the enhanced polarization in the solvent versus the nanocrystals. Nevertheless, DNP-enhanced magic-angle spinning (MAS) spectra recorded at 5 T and 90 K exhibit an average signal enhancement epsilon approximately 120. This is slightly lower than the DNP enhancement of the solvent mixture surrounding the crystals (epsilon approximately 160), and we show that it is consistent with spin diffusion across the solvent-matrix interface. In particular, we correlate the expected DNP enhancement to several properties of the sample, such as crystal size, the nuclear T(1), and the average (1)H-(1)H spin diffusion constant. The enhanced (1)H polarization was subsequently transferred to (13)C and (15)N via cross-polarization, and allowed rapid acquisition of two-dimensional (13)C-(13)C correlation data.

N Solid state CP/MAS NMR spectroscopy has been used to study a dinitrogen-derived terminal nitride of molybdenum, 15 NMo(N( t Bu)Ar)3 (Ar = 3,5-(CH3)2C6H3). A number of Lewis acid adducts, including X3E-NMo(N( t Bu)Ar)3 (X =F , E =B ; X = Cl, E = B, Al, Ga, In; X = Br, E = Al; X = I, E = Al) and Cl2E-NMo(N( t Bu)Ar)3 (E = Ge, Sn), were prepared by the combi- nation of 15 NMo(N( t Bu)Ar)3 with 1 equiv. of Lewis acid. A series of cationic imido complexes, (RNMo(N( t Bu)Ar)3)X was prepared by the reaction of electrophiles, RX (R = CH3, X = I; R = PhC(O) or Me3Si, X = OTf (OTf = SO3CF3)), with NMo(N( t Bu)Ar)3. Deprotonation of (CH3NMo(N( t Bu)Ar)3)I by LiN(SiMe3)2 afforded the ketimide complex H2CNMo(N( t Bu)Ar)3, which has been shown to undergo a reaction with neat CH3I to form (CH3CH2NMo(N( t Bu)Ar)3)I. 15 N solid state CP/MAS NMR spectroscopy was employed in the characterization of each complex. Complementary density functional theory (DFT) studies of 15 NMo(N( t- Bu)Ar)3 and derivatives enabled a detailed examination of the experimental solid state NMR parameters in terms of electronic struc- ture at the labeled N-atom. Computational analysis demonstrated that significant paramagnetic contributions to the perpendicular components of the chemical shielding tensor (d11 and d22) were responsible for the huge span of the tensor measured for 15 NMo(N( t- Bu)Ar)3 (X = 1187 ppm). Perturbation of the electronic structure in 15 NMo(N( t Bu)Ar)3 upon coordination of a Lewis acid or for- mation of a cationic imido complex was attributed to stabilization of a r-symmetric orbital. An upfield shift in the perpendicular components of the chemical shift tensor results from the reduced paramagnetic contribution to these tensor components upon increasing the energy gap between the magnetically coupled occupied and virtual orbitals (eoccevir). � 2004 Elsevier Ltd. All rights reserved.

Cytochrome c (cyt-c) plays a key role in activating intrinsic apoptosis, a closely regulated cell death mechanism. Complete understanding of this process has implications for treatment of neurodegenerative diseases like Huntington's disease, as well as cancer. A pivotal signaling event in this apoptotic pathway is the peroxidation of the mitochondrial lipid cardiolipin (CL) by cyt-c. Cyt-c gains CL-specific lipid peroxidase activity by binding to CL during apoptosis, but exactly what structural changes lead to its proapoptotic activity have remained elusive. We have performed both structural and functional measurements to determine changes in structure and dynamics of cyt-c that accompany the activation of its peroxidase function upon binding to CL. Specifically, we used FTIR and multidimensional magic-angle-spinning (MAS) solid-state NMR (ssNMR) on samples mimicking proapoptotic conditions that trigger cyt-c's peroxidase activity. Comparison to FTIR and solution NMR data for the soluble protein revealed that cyt-c did not experience major unfolding upon membrane binding. We probed the protein-lipid interactions via ssNMR on both the protein and the lipids, and additionally used fluorescence quenching measurements of resonance energy transfer between the cyt-c heme and fluorescently labeled lipids. We conclude that increases in peroxidase activity of peripherally CL-bound cyt-c do not result from extensive unfolding of cyt-c. We instead propose an alternative mechanism in which localized structural changes, rather than global unfolding, in the CL-bound protein may be enough to allow for significant lipid peroxidation function to arise.

31P NMR spectroscopy was used to investigate the effects of transmembrane alpha-helical peptides with different flanking residues on the phase behavior of phosphatidylethanolamine and phosphatidylethanolamine/phosphatidylglycerol (molar ratio 7:3) model membranes. It was found that tryptophan-flanked (WALP) peptides and lysine-flanked (KALP) peptides both promote formation of nonlamellar phases in these lipid systems in a mismatch-dependent manner. Based on this mismatch dependence, it was concluded that the effective hydrophobic length of KALP peptides is considerably shorter than that of the corresponding WALP peptides. Peptides with other positively charged residues showed very similar effects as KALP. The results suggest that the peptides have a well-defined effective hydrophobic length, which is different for charged and aromatic flanking residues, but which is independent of the precise chemical nature of the side chain. Strikingly, the effective length of KALP peptides in the lipid systems investigated here is much smaller than that previously found for the same peptides in phosphatidylcholine. This suggests that snorkeling of lysine side chains, as proposed to occur in phosphatidylcholine, does not occur in lipid systems that are prone to form nonlamellar phases by themselves. This suggestion was supported by using peptides with shortened lysine side chains and by investigating the effects of mixtures of WALP and KALP peptides. The lipid dependency of the snorkeling behavior is explained by considering the free energy cost of snorkeling in relation to the free energy cost of the formation of nonlamellar phases.

In Huntington's disease, expansion of a polyglutamine (polyQ) domain in the huntingtin (htt) protein leads to misfolding and aggregation. There is much interest in the molecular features that distinguish monomeric, oligomeric, and fibrillar species that populate the aggregation pathway and likely differ in cytotoxicity. The mechanism and rate of aggregation are greatly affected by the domains flanking the polyQ segment within exon 1 of htt. A "protective" C-terminal proline-rich flanking domain inhibits aggregation by inducing polyproline II structure (PPII) within an extended portion of polyQ. The N-terminal flanking segment (htt(NT)) adopts an α-helical structure as it drives aggregation, helps stabilize oligomers and fibrils, and is seemingly integral to their supramolecular assembly. Via solid-state nuclear magnetic resonance (ssNMR), we probe how, in the mature fibrils, the htt flanking domains impact the polyQ domain and in particular the localization of the β-structured amyloid core. Using residue-specific and uniformly labeled samples, we find that the amyloid core occupies most of the polyQ domain but ends just prior to the prolines. We probe the structural and dynamical features of the remarkably abrupt β-sheet to PPII transition and discuss the potential connections to certain htt-binding proteins. We also examine the htt(NT) α-helix outside the polyQ amyloid core. Despite its presumed structural and demonstrated stabilizing roles in the fibrils, quantitative ssNMR measurements of residue-specific dynamics show that it undergoes distinct solvent-coupled motion. This dynamical feature seems reminiscent of molten-globule-like α-helix-rich features attributed to the nonfibrillar oligomeric species of various amyloidogenic proteins.

Integral membrane proteins are of great biomedical and biological interest but are challenging targets for structural biology. One of the challenges is the characterization of membrane protein interactions with a proper lipid bilayer that mimics their native environment. The membrane environment can directly affect the structure of integral membrane proteins and can also be crucial for optimal activity. Solid-state NMR is in a unique position to probe the protein–lipid interplay, by providing insight into both structure and dynamics of the protein as well as the lipids. Under native conditions, biological membranes are characterized by a dynamic and disordered ‘liquid crystalline’ structure. Solid-state NMR experiments are affected by dynamics, at times in negative ways, but the dynamics can also be leveraged to gain valuable information, including orientational constraints. This article discusses approaches to probe integral membrane protein interactions with lipids, and the role of lipid dynamics, through the use of static as well as magic-angle-spinning solid-state NMR. Keywords: solid-state NMR; membrane proteins; molecular dynamics; protein structure; order parameters; relaxation; oriented samples; magic-angle-spinning NMR

The structure and function of biological membranes can be expected to be sensitive to the extent of hydrophobic matching between the length of the membrane-spanning part of intrinsic membrane proteins and the hydrophobic thickness of the lipid bilayer. To gain insight into the consequences of hydrophobic mismatch on a molecular level, we have carried out systematic studies on well-defined peptidehpid complexes, using artifical transmembrane peptides, anchored to the membrane by tryptophan residues. It is shown that hydrophobic mismatch can result in a dramatic change in lipid organization, and that the presence of interfacially localized aromatic amino acid residues is important for determining the exact consequences of hydrophobic mismatch.

More than 20 human diseases are associated with amyloid fibril formation, one of which is Huntington's Disease (HD). HD, which poses a risk for ∼200,000 Americans, is a neurodegenerative disease that degrades cognitive and motor functions and is one of ten disorders to feature polyglutamine (polyQ) domain expansion. The self-aggregation of expanded polyQ segments in the huntingtin protein (htt) leads to fibril formation. Flanking regions of the polyQ domain affect the kinetics of this aggregation. As protein aggregation appears to correlate to disease onset, we aim to understand the structure of the mature fibrils, a critical step toward resolving structural changes along the fibril formation pathway.Fibrils of site-specific isotopically labeled peptides mimicking polyQ-containing N-terminal fragments of the htt protein were studied by magic-angle-spinning (MAS) solid-state NMR (ssNMR). We probed the conformation of both the polyQ amyloid core and its flanking regions by multidimensional MAS ssNMR, finding an a-helical conformation for residues in the very N-terminus (httNT), which is known to initiate the aggregation process. Structural models of the aggregation pathway and fibril were assessed based on structural data, relaxation times, and order parameters from MAS ssNMR measurements. Fibrils featuring mutations in the linker region connecting the httNT helix to the amyloid core were also investigated. Earlier animal studies have shown these mutations to reduce toxicity, and EM shows significant changes in fibril morphology. The site-specific effects of these and other mutations, on both the httNT and the polyQ core, were explored through structural and dynamics measurements. The characterization of the mutants, which mimic post-translational Ser phosphorylation, suggests biophysical connections between the stability and molecular structure of species along the aggregation pathway and the observed changes in toxicity.

The recognition and binding of specific lipid species is critical for the cellular targeting, folding and activity of various membrane-associated or peripherally bound proteins. These interactions facilitate localization of the protein in question in the proper membrane in the cell. In certain cases, binding to cholesterol is thought to play a key role in the targeting of membrane-associated proteins to cholesterol-rich membranes, and to laterally-segregated cholesterol-enriched domains within the plasma membrane. Previous studies have identified a cholesterol-binding motif that may be found in numerous proteins. This relatively short cholesterol recognition/interaction amino acid consensus (CRAC) is thought to enable tight cholesterol binding, both in a protein-context and in isolation. Experimental challenges have limited the availability of structural data on the molecular or biophysical nature of this interaction. Using solid-state NMR (ssNMR), we explore the structural features of this domain as found in the cholesterol-binding protein caveolin 1. The use of ssNMR permits the study of this cholesterol-binding domain in cholesterol-rich lipid bilayers designed to emulate native caveolar membranes. Site-specific structural constraints were obtained for the bound protein fragment, revealing a transition from an extended, β-stranded conformation to an α-helical structure near the central Tyr residue characteristic of CRAC motifs – consistent with certain pre-existing models of CRAC motif structure. Furthermore, complementary static and magic-angle-spinning (MAS) NMR measurements reveal the effects on lipid dynamics and indicate immersion of the polypeptide into the membrane hydrophobic core. These structural and motional data on both partners in this polypeptide-membrane interaction provide an unprecedented site-specific perspective on a cholesterol-binding CRAC motif.

The conformational preferences of polyglutamine (polyQ) sequences are of major interest because of their central importance in the expanded CAG repeat diseases that include Huntington's disease. Here, we explore the response of various biophysical parameters to the introduction of β-hairpin motifs within polyQ sequences. These motifs (tryptophan zipper, disulfide, d-Pro-Gly, Coulombic attraction, l-Pro-Gly) enhance formation rates and stabilities of amyloid fibrils with degrees of effectiveness well correlated with their known abilities to enhance β-hairpin formation in other peptides. These changes led to decreases in the critical nucleus for amyloid formation from a value of n=4 for a simple, unbroken Q23 sequence to approximate unitary n values for similar length polyQs containing β-hairpin motifs. At the same time, the morphologies, secondary structures, and bioactivities of the resulting fibrils were essentially unchanged from simple polyQ aggregates. In particular, the signature pattern of solid-state NMR (13)C Gln resonances that appears to be unique to polyQ amyloid is replicated exactly in fibrils from a β-hairpin polyQ. Importantly, while β-hairpin motifs do produce enhancements in the equilibrium constant for nucleation in aggregation reactions, these Kn values remain quite low (~10(-)(10)) and there is no evidence for significant enhancement of β-structure within the monomer ensemble. The results indicate an important role for β-turns in the nucleation mechanism and structure of polyQ amyloid and have implications for the nature of the toxic species in expanded CAG repeat diseases.