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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.

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.

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.

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.

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).

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.

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.

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.

For several different proteins an apparent correlation has been observed between the propensity for dimerization by domain-swapping and the ability to aggregate into amyloid-like fibrils. Examples include the disease-related proteins β 2-microglobulin and transthyretin. This has led to proposals that the amyloid-formation pathway may feature extensive domain swapping. One possible consequence of such an aggregation pathway is that the resulting fibrils would incorporate structural elements that resemble the domain-swapped forms of the protein and, thus, reflect certain native-like structures or domain-interactions. In magic angle spinning solid-state NMR-based and other structural studies of such amyloid fibrils, it appears that many of these proteins form fibrils that are not native-like. Several fibrils, instead, have an in-register, parallel conformation, which is a common amyloid structural motif and is seen, for instance, in various prion fibrils. Such a lack of native structure in the fibrils suggests that the apparent connection between domain-swapping ability and amyloid-formation may be more subtle or complex than may be presumed at first glance.

Several human diseases are associated with the formation of amyloid aggregates, but experimental characterization of these amyloid fibrils and their oligomeric precursors has remained challenging. Experimental and computational analysis of simpler model systems has therefore been necessary, for instance, on the peptide fragment GNNQQNY7−13 of yeast prion protein Sup35p. Expanding on a previous publication, we report here a detailed structural characterization of GNNQQNY fibrils using magic angle spinning (MAS) NMR. On the basis of additional chemical shift assignments we confirm the coexistence of three distinct peptide conformations within the fibrillar samples, as reflected in substantial chemical shift differences. Backbone torsion angle measurements indicate that the basic structure of these coexisting conformers is an extended β-sheet. We structurally characterize a previously identified localized distortion of the β-strand backbone specific to one of the conformers. Intermolecular contacts are consistent with each of the conformers being present in its own parallel and in-register sheet. Overall the MAS NMR data indicate a substantial difference between the structure of the fibrillar and crystalline forms of these peptides, with a clearly increased complexity in the GNNQQNY fibril structure. These experimental data can provide guidance for future work, both experimental and theoretical, and provide insights into the distinction between fibril growth and crystal formation.

Dynamic nuclear polarization (DNP) utilizes the inherently larger polarization of electrons to enhance the sensitivity of conventional solid-state NMR experiments at low temperature. Recent advances in instrumentation development and sample preparation have transformed this field and have opened up new opportunities for its application to biological systems. Here, we present DNP-enhanced [superscript 13]C–[superscript 13]C and [superscript 15]N–[superscript 13]C correlation experiments on GNNQQNY nanocrystals and amyloid fibrils acquired at 9.4 T and 100 K and demonstrate that DNP can be used to obtain assignments and site-specific structural information very efficiently. We investigate the influence of temperature on the resolution, molecular conformation, structural integrity and dynamics in these two systems. In addition, we assess the low-temperature performance of two commonly used solid-state NMR experiments, proton-driven spin diffusion (PDSD) and transferred echo double resonance (TEDOR), and discuss their potential as tools for measurement of structurally relevant distances at low temperature in combination with DNP., National Institutes of Health (U.S.) (Grant EB002804), National Institutes of Health (U.S.) (Grant EB003151), National Institutes of Health (U.S.) (Grant EB002026)

Rotational resonance width (R(2)W) magic-angle spinning (MAS) NMR experiments are performed to measure (13)C-(13)C distances in the hydrophobic core of the microcrystalline model protein G(Beta1). Such inter-residue distances are of particular value in NMR structure determinations. The experiments are done at a Larmor frequency of 750 MHz (1)H where the contribution of (13)C chemical shift anisotropy (CSA) to the R(2) transfer mechanism is significant. To minimize line broadening in the 2D spectra, we employ a combination of even/odd isotopic labeling with [1,3-(13)C] glycerol, and J-decoupling in the indirect dimension. This results in high-precision distance measurements between aromatic side chains of three tyrosine residues and distant methyl groups in the hydrophobic core of the protein. Even in the absence of information on the relative orientation of the shift tensors, we obtain relatively high precision data, which can be further improved by additional constraints on the tensor orientations.

We describe a cryogenic sample exchange system that dramatically improves the efficiency of magic angle spinning (MAS) dynamic nuclear polarization (DNP) experiments by reducing the time required to change samples and by improving long-term instrument stability. Changing samples in conventional cryogenic MAS DNP/NMR experiments involves warming the probe to room temperature, detaching all cryogenic, RF, and microwave connections, removing the probe from the magnet, replacing the sample, and reversing all the previous steps, with the entire cycle requiring a few hours. The sample exchange system described here—which relies on an eject pipe attached to the front of the MAS stator and a vacuum jacketed dewar with a bellowed hole—circumvents these procedures. To demonstrate the excellent sensitivity, resolution, and stability achieved with this quadruple resonance sample exchange probe, we have performed high precision distance measurements on the active site of the membrane protein bacteriorhodopsin. We also include a spectrum of the tripeptide N-f-MLF-OH at 100 K which shows 30 Hz linewidths., National Institute for Biomedical Imaging and Bioengineering (U.S.) (Grant EB-002804), National Institute for Biomedical Imaging and Bioengineering (U.S.) (Grant EB-001960), National Institute for Biomedical Imaging and Bioengineering (U.S.) (Grant EB-001035), National Institute for Biomedical Imaging and Bioengineering (U.S.) (Grant EB-002026), National Institute for Biomedical Imaging and Bioengineering (U.S.) (Grant EB-003151), National Science Foundation (U.S.). Graduate Research Fellowship Program

At reduced temperatures, proteins and other biomolecules are generally found to exhibit dynamic as well as structural transitions. This includes a so-called protein glass transition that is universally observed in systems cooled between 200 and 230 K, and which is generally attributed to interactions between hydrating solvent molecules and protein side chains. However, there is also experimental and theoretical evidence for a low-temperature transition in the intrinsic dynamics of the protein itself, absent any solvent. Here, we use low-temperature solid-state NMR to examine site-specific fluctuations in atomic structure and dynamics in the absence of solvents. In particular, we employ magic angle spinning NMR to examine a structural phase transition associated with dynamic processes in a solvent-free polypeptide, N-f-MLF-OH, lattice at temperatures as low as 90 K. This transition is characterized by the appearance of an extra set of lines in 1D (15)N spectra as well as additional cross peaks in 2D (13)C-(13)C and (13)C-(15)N spectra. Interestingly, the gradual, temperature-dependent appearance of the new spectral component is not accompanied by the line broadening typical of dynamic transitions. A direct comparison between the spectra of N-f-MLF-OH and the analog N-f-MLF-OMe, which does not display this transition, indicates a correlation of the structural transition to the temperature dependent motion of the aromatic phenylalanine side chain. Several quantitative solid state NMR experiments were employed to provide site-specific measurements of structural and motional features of the observed transition.

The tryptophans of integral membrane proteins have been suggested to play specific roles as "interfacial anchors", based on their preference for a location near the lipid head groups. Still, the underlying mechanism behind this behavior remains unclear. NMR experiments can provide an important tool to study this interaction in an actual bilayer environment. Here solid-state deuterium nuclear magnetic resonance was used to study the tryptophans in membrane-spanning model peptides from the WALP family (acetyl-GWW(LA)nWWA-ethanolamide with n = 5 and 6.5) in samples of mechanically aligned dimyristoylphosphatidylcholine (DMPC) bilayers. The data indicate that the tryptophans near the C-terminal end of the peptide display a significantly different behavior from those near the N-terminus. This is reflected prominently in a large difference in the motion experienced by the indoles at either end of the peptide, highlighting the directionality of the helix. Nevertheless, our observations indicate high levels of motional freedom for all tryptophans in these membrane spanning domains that exceed the dynamics for the helix itself. These observations signify that steric and dynamic features of the polypeptide context modulate the tryptophan anchoring in the membrane interface. Measurements of WALP19 in the ether-linked DMPC analogue ditetradecylphosphatidylcholine (missing the lipid carbonyls) show very similar Trp dynamics and suggest similar orientations for some or all of the tryptophans. This suggests that the lipid acyl chain carbonyls play at most a minor role in the anchoring interaction between these Trp residues and the DMPC interfacial region.

Sup35p is a prion protein found in yeast that contains a prion-forming domain characterized by a repetitive sequence rich in Gln, Asn, Tyr, and Gly amino acid residues. The peptide GNNQQNY7-13 is one of the shortest segments of this domain found to form amyloid fibrils, in a fashion similar to the protein itself. Upon dissolution in water, GNNQQNY displays a concentration-dependent polymorphism, forming monoclinic and orthorhombic crystals at low concentrations and amyloid fibrils at higher concentrations. We prepared nanocrystals of both space groups as well as fibril samples that reproducibly contain three (coexisting) structural forms and examined the specimens with magic angle spinning (MAS) solid-state nuclear magnetic resonance. 13C and 15N MAS spectra of both nanocrystals and fibrils reveal narrow resonances indicative of a high level of microscopic sample homogeneity that permitted resonance assignments of all five species. We observed variations in chemical shift among the three dominant forms of the fibrils which were indicated by the presence of three distinct, self-consistent sets of correlated NMR signals. Similarly, the monoclinic and orthorhombic crystals exhibit chemical shifts that differ from one another and from the fibrils. Collectively, the chemical shift data suggest that the peptide assumes five conformations in the crystals and fibrils that differ from one another in subtle but distinct ways. This includes variations in the mobility of the aromatic Tyr ring. The data also suggest that various structures assumed by the peptide may be correlated to the "steric zipper" observed in the monoclinic crystals.

We have used experimental deuterium NMR spectra from labeled tryptophans in membrane-spanning gramicidin A (gA)(1) channels to refine the geometry of the indole ring and, specifically, the C2-(2)H bond direction. By using partial exchange in a cold organic acid, we were able to selectively deuterate ring positions C2 and C5 and, thereby, define unambiguous spectral assignments. In a backbone-independent analysis, the assigned spectra from four distinct labeled tryptophans were used to assess the geometry of the planar indole ring. We found that the C2-(2)H bond makes an angle of about 6 degrees with respect to the normal to the indole ring bridge, and the experimental geometry was confirmed by density functional calculations using a 6-311G** basis set. The precisely determined ring geometry and the experimental spectra in turn are the foundation for calculations of the orientation of each tryptophan indole ring, with respect to the bilayer membrane normal, and of a principal order parameter S(zz) for each ring. The results have general significance for revising the tryptophan ring geometry that is used in protein molecular modeling, as well as for the analysis of tryptophan ring orientations in membrane-spanning proteins. The experimental precision in the definition of the indole ring geometry demonstrates yet another practical application emanating from fundamental research on the robust gramicidin channel.

In humans, transport of metabolites through the outer mitochondrial membrane is controlled by a 283 residue, 31 kDa pore-forming protein, the voltage-dependent anion-selective channel (VDAC). In addition to providing a main pathway for metabolite trafficking through the outer membrane, VDAC is postulated to play a critical role in cell apoptosis. VDAC interacts with the Bcl-2 family of pro-apoptotic and anti-apoptotic proteins that control the permeability of the outer membrane to apoptotic signals including the release of cytochrome c.Recently, the solution NMR structure of human VDAC-1 (hVDAC1) reconstituted in detergent micelles has been reported by two groups, revealing a 19-stranded β-barrel with a short α helix at the N terminus. While these structures are consistent with previous sequence analysis and biophysical studies, there are indications that interactions of the protein with a lipid bilayer are essential for its proper structure as well as function.Accordingly, we have undertaken studies on hVDAC1 reconstituted in DMPC bilayers with magic angle spinning (MAS) solid state NMR (SSNMR). hVDAC1 forms structurally homogeneous two-dimensional microcrystals as judged by high resolution 13C/15N MAS spectra. In addition, uniformly 13C and 15N labeled hVDAC1 in 2D DMPC cyrstals exhibit well resolved multidimensional MAS spectra that allow group assignments, structural analysis, and comparison of chemical shifts with the solution spectra.

Solid state NMR allows the site-specific characterization of structure and dynamics in a variety of immobilized biomolecules, and thus allows a unique structural view on amyloid fibrils. These fibrils are common to various human disorders and appear to share a number of characteristic features, both in terms of their structure and formation. In the hope of delineating the biophysical details of the fibrillization process and the fibrils themselves, various groups have focused on the experimental and theoretical study of small peptide fragments of amyloid-forming proteins. One prominent system is the GNNQQNY7-13 fragment of the yeast prion protein Sup35p, since it was found to form not just amyloid-like fibrils, but also seemingly amyloid-like microcrystals. X-ray diffraction based structures from the latter have inspired numerous theoretical analyses and generalizations regarding the biophysics and structures of amyloid fibrils.We have instead applied biological solid state NMR methods to characterize the GNNQQNY fibrillar aggregates. Magic angle spinning (MAS) solid state NMR was used for various structural measurements, aimed at both the intramolecular as well as intermolecular structural motifs of the fibrils (as well as the crystalline aggregates). Our studies have revealed a remarkable complexity in these fibrils, despite the relatively small size of the peptide building blocks. This is in marked contrast with the rigid and homogeneous nature of the crystalline structures, as revealed by X-ray crystallography and solid state NMR. These observations provide further insights into the structure of the fibrils of this peptide model system and should also be of importance as input to numerous theoretical studies that rely on the crystal structure data.

Solid-state NMR methods employing (2)H NMR and geometric analysis of labeled alanines (GALA) were used to study the structure and orientation of the transmembrane alpha-helical peptide acetyl-GWW(LA)(8)LWWA-amide (WALP23) in phosphatidylcholine (PC) bilayers of varying thickness. In all lipids the peptide was found to adopt a transmembrane alpha-helical conformation. A small tilt angle of 4.5 degrees was observed in di-18:1-PC, which has a hydrophobic bilayer thickness that approximately matches the hydrophobic length of the peptide. This tilt angle increased slightly but systematically with increasing positive mismatch to 8.2 degrees in di-C12:0-PC, the shortest lipid used. This small increase in tilt angle is insufficient to significantly change the effective hydrophobic length of the peptide and thereby to compensate for the increasing hydrophobic mismatch, suggesting that tilt of these peptides in a lipid bilayer is energetically unfavorable. The tilt and also the orientation around the peptide axis were found to be very similar to the values previously reported for a shorter WALP19 peptide (GWW(LA)(6)LWWA). As also observed in this previous study, the peptide rotates rapidly around the bilayer normal, but not around its helix axis. Here we show that these properties allow application of the GALA method not only to macroscopically aligned samples but also to randomly oriented samples, which has important practical advantages. A minimum of four labeled alanine residues in the hydrophobic transmembrane sequence was found to be required to obtain accurate tilt values using the GALA method.