Oriented-sample NMR Research Articles

Aligned peptoid-based macrodiscs for structural studies of membrane proteins by oriented-sample NMR

Development of a robust, uniform, and magnetically orientable lipid mimetic will undoubtedly advance solid-state NMR of macroscopically aligned membrane proteins. Here, we report on a novel lipid membrane mimetic based on peptoid belts. The peptoids, composed of 15 residues, were synthesized by alternating N-(2-phenethyl)glycine with N-(2-carboxyethyl)glycine residues at a 2:1 molar ratio. The chemically synthesized peptoids possess a much lower degree of polydispersity versus styrene-maleic acid polymers, thus yielding uniform discs. Moreover, the peptoid oligomers are more flexible and do not require a specific folding, unlike lipoproteins, in order to wrap around the hydrophobic membrane core. The NMR spectra measured for the membrane-bound form of Pf1 coat protein incorporated in this new lipid mimetics demonstrate a higher order parameter and uniform linewidths compared with the conventional bicelles and peptide-based macrodiscs. Importantly, unlike bicelles, the peptoid-based macrodiscs are detergent free.

Life During Wartime: A Personal Recollection of the Circa 1990 Prestegard Lab and Its Contributions to Membrane Biophysics.

A subjective account is presented of challenges and excitement of being a postdoctoral trainee in the lab of James H. Prestegard at Yale University in New Haven, Connecticut from 1989 to 1991. This includes accounts of the early development of bicelles and of oriented sample NMR results that contributed to our modern understanding of the properties of the water-lipid interface of disordered phase biological membranes.

Nanopore-Confined Bilayers: A Model of Biomembranes with Defined Curvature and a Tool for Oriented Sample Magnetic Resonance

It is well established that biological function of cellular membranes is determined by both membrane proteins and the membrane biochemical composition. Moreover, the membrane shape/local curvature may also play a role in modulating membrane properties and the proteins’ function. While specific biomembrane compositions are more readily replicated in biophysical bench experiments, formation of bilayers of specific shapes and curvature represents a more challenging task. Macroscopic alignment of such membranes – an important prerequisite of high resolution magnetic resonance and other studies – is even more difficult especially over a broad range of experimental conditions such pH, ionic strength, and temperature. Here we describe methods for forming self-assembled lipid nanotubular bilayers inside cylindrical nanopores composed of anodic aluminum oxide (AAO). Such hybrid nanostructures, named lipid nanotube arrays, represent a new type of substrate-supported and macroscopically-aligned lipid bilayers of defined curvature that have many attractive features for both membrane biophysics and structure-function protein studies by spectroscopic techniques. Optical properties of AAO allow for assessing the integrity of membrane protein complexes by UV-vis while high density of the deposited lipids and proteins enable examination by other biophysical methods, including DSC, QCM, and magnetic resonance. The latter studies have shown that the individual lipids in such nanopore-confined structures maintain fast uniaxial diffusion and a high degree of macroscopic alignment. The macroscopic alignment enables detailed studies of effects of lipid composition on structure of integral membrane proteins by solid state oriented sample NMR, EPR and DEER. Accessibility of either both or mainly inner leaflet of the nanotubular bilayers to water-soluble species provides for studies of protein, peptides, and drug binding. Supported by DE-FG02-02ER15354 to AIS.

Sensitivity enhancement for membrane proteins reconstituted in parallel and perpendicular oriented bicelles obtained by using repetitive cross-polarization and membrane-incorporated free radicals.

Multidimensional separated local-field and spin-exchange experiments employed by oriented-sample solid-state NMR are essential for structure determination and spectroscopic assignment of membrane proteins reconstituted in macroscopically aligned lipid bilayers. However, these experiments typically require a large number of scans in order to establish interspin correlations. Here we have shown that a combination of optimized repetitive cross polarization (REP-CP) and membrane-embedded free radicals allows one to enhance the signal-to-noise ratio by factors 2.4-3.0 in the case of Pf1 coat protein reconstituted in magnetically aligned bicelles with their normals being either parallel or perpendicular to the main magnetic field. Notably, spectral resolution is not affected at the 2:1 radical-to-protein ratio. Spectroscopic assignment of Pf1 coat protein in the parallel bicelles has been established as an illustration of the method. The proposed methodology will advance applications of oriented-sample NMR technique when applied to samples containing smaller quantities of proteins and three-dimensional experiments.

Interactions of Antibacterial Peptides with Nanotubular Lipid Bilayers: Binding Kinetics and Distortions of the Bilayer Structure

Interactions of peptides with membranes are central for understanding key cellular processes involving membrane proteins such as folding, signaling, transport, energy conversion, and immune response. These interactions also determine efficiency of antibacterial and cytotoxic peptides in disrupting the barrier function of cellular membranes. We employed macroscopically aligned tubular lipid bilayers confined inside cylindrical nanopores of anodic aluminum oxide (AAO) as a versatile nanotechnology platform to study membrane interactions of antibacterial peptides of melittin and alamecitin. Kinetics of peptide binding to lipid nanotubes and the eventual lipid removal/lysis (for melittin) were observed by quartz crystal microbalance (QCM) using a crystal with an in-house fabricated nanostructured surface while the structural changes in bilayers were monitored by solid state oriented sample NMR. Specifically, using 78 nm nanopores we have achieved exceptionally narrow (100-140 Hz or <1 ppm) 31P resonances of the lipid phosphate groups of macroscopically aligned nanotubular bilayers that indicated <1-2o mosaic spread in the lipid alignment in the absence of bioreactive peptides. 31P resonances broadened and shifted towards the isotropic values upon interacting with melittin with some lysis of lipids observed after several hours of exposure. The binding and lipid lysis from bilayers confined in essentially the same nanopores albeit at much smaller lipid quantity were further quantified by real-time QCM for bilayers of various lipid compositions. Taken together, the data relate peptide binding and lysis kinetics to structural changes in lipid bilayers, thus, shedding the light on the underlying multistep mechanism. The main advantage of the lipid nanotube AAO platform is in its versatile applicability to various biophysical methods such QCM and NMR under essentially the same environmental conditions such as pH, ionic strength, temperature, etc. and exceptionally broad range of lipid bilayer compositions.

Assignment of oriented sample NMR resonances from a three transmembrane helix protein

Oriented sample solid state NMR techniques have been routinely employed to determine the structures of membrane proteins with one or two transmembrane helices. For larger proteins the technique has been limited by spectral resolution and lack of assignment strategies. Here, a strategy for resonance assignment is devised and applied to a three transmembrane helix protein. Sequence specific assignments for all labeled transmembrane amino acid sites are obtained, which provide a set of orientational restraints and helix orientations in the bilayer. Our experiments expand the utility of solid state NMR in membrane protein structure characterization to three transmembrane helix proteins and represent a straightforward strategy for routinely characterizing multiple transmembrane helix protein structures.

Dynamic and Contrasting Information by Oriented-Sample Solid-State NMR Spectroscopy of Membrane Proteins

Oriented-sample NMR (OS NMR) has emerged as a powerful technique for structure determination of membrane proteins in their native-like lipid environment. We have developed a model relating OS NMR lineshapes to uniaxial ordering (mosaic spread) and rotational diffusion of the protein within the membrane. The model is exemplified by 15N NMR spectra of Pf1 coat protein in both magnetically aligned phage and reconstituted in oriented bicelles. In the case of Pf1 phage, the lineshapes are dominated by static uniaxial disorder; whereas fast rotational diffusion of the protein is responsible for the motional line narrowing in perpendicularly oriented bicelles. From the analysis of 15N NMR linewidths, rotational diffusion coefficient can be estimated. Since the value of the diffusion coefficient is ultimately related to the overall protein size, information about oligomerization states is potentially obtainable. Second, the use of various membrane-embedded radicals allows one to both dramatically speed up data acquisition, on the one hand, and obtain contrasting information for membrane-embedded proteins, on the other. While membrane-bound paramagnetic species drastically affect the T1Z relaxation times (at 2:1 molar ratio relative to the protein), the transverse T2 relaxation is only slightly affected. 5-DOXYL stearic acid, TEMPOL, and CAT-1 radicals exhibit different partitioning within the membrane, and result in differential paramagnetic effect on the spectral peaks arising from different residues of Pf1 protein in bicelles. This allows one to obtain contrasting information about the location of the residues relative to the membrane. As was shown by EPR, TEMPOL partitions itself equally in and out of the membrane, and almost uniformly affects all residues within the bilayer. By contrast, 5-DOXYL stearic acid affects mostly the residues below the interfacial region, while CAT-1 affects the residues located within the polar head groups.

Sensitivity enhancement and contrasting information provided by free radicals in oriented-sample NMR of bicelle-reconstituted membrane proteins

Elucidating structure and topology of membrane proteins (MPs) is essential for unveiling functionality of these important biological constituents. Oriented-sample solid-state NMR (OS-NMR) is capable of providing such information on MPs under nearly physiological conditions. However, two dimensional OS-NMR experiments can take several days to complete due to long longitudinal relaxation times combined with the large number of scans to achieve sufficient signal sensitivity in biological samples. Here, free radicals 5-DOXYL stearic acid, TEMPOL, and CAT-1 were added to uniformly 15N-labeled Pf1 coat protein reconstituted in DMPC/DHPC bicelles, and their effect on the longitudinal relaxation times (T1Z) was investigated. The dramatically shortened T1Z’s allowed for the signal gain per unit time to be used for either: (i) up to a threefold reduction of the total experimental time at 99% magnetization recovery or (ii) obtaining up to 74% signal enhancement between the control and radical samples during constant experimental time at “optimal” relaxation delays. In addition, through OS-NMR and high-field EPR studies, free radicals were able to provide positional constraints in the bicelle system, which provide a description of the location of each residue in Pf1 coat protein within the bicellar membranes. This information can be useful in the determination of oligomerization states and immersion depths of larger membrane proteins.

Lipid bilayer preparations of membrane proteins for oriented and magic-angle spinning solid-state NMR samples

Solid-state NMR spectroscopy has been used successfully for characterizing the structure and dynamics of membrane proteins as well as their interactions with other proteins in lipid bilayers. Such an environment is often necessary for achieving native-like structures. Sample preparation is the key to this success. Here we present a detailed description of a robust protocol that results in high-quality membrane protein samples for both magic-angle spinning and oriented-sample solid-state NMR. The procedure is demonstrated using two proteins: CrgA (two transmembrane helices) and Rv1861 (three transmembrane helices), both from Mycobacterium tuberculosis. The success of this procedure relies on two points. First, for samples for both types of NMR experiment, the reconstitution of the protein from a detergent environment to an environment in which it is incorporated into liposomes results in 'complete' removal of detergent. Second, for the oriented samples, proper dehydration followed by rehydration of the proteoliposomes is essential. By using this protocol, proteoliposome samples for magic-angle spinning NMR and uniformly aligned samples (orientational mosaicity of <1°) for oriented-sample NMR can be obtained within 10 d.

A spectroscopic assignment technique for membrane proteins reconstituted in magnetically aligned bicelles

Oriented-sample NMR (OS-NMR) has emerged as a powerful tool for the structure determination of membrane proteins in their physiological environments. However, the traditional spectroscopic assignment method in OS NMR that uses the "shotgun" approach, though effective, is quite labor- and time-consuming as it is based on the preparation of multiple selectively labeled samples. Here we demonstrate that, by using a combination of the spin exchange under mismatched Hartmann-Hahn conditions and a recent sensitivity-enhancement REP-CP sequence, spectroscopic assignment of solid-state NMR spectra of Pf1 coat protein reconstituted in magnetically aligned bicelles can be significantly improved. This method yields a two-dimensional spin-exchanged version of the SAMPI4 spectrum correlating the (15)N chemical shift and (15)N-(1)H dipolar couplings, as well as spin-correlations between the (i, i ± 1) amide sites. Combining the spin-exchanged SAMPI4 spectrum with the original SAMPI4 experiment makes it possible to establish sequential assignments, and this technique is generally applicable to other uniaxially aligned membrane proteins. Inclusion of an (15)N-(15)N correlation spectrum into the assignment process helps establish correlations between the peaks in crowded or ambiguous spectral regions of the spin-exchanged SAMPI4 experiment. Notably, unlike the traditional method, only a uniformly labeled protein sample is required for spectroscopic assignment with perhaps only a few selectively labeled "seed" spectra. Simulations for the magnetization transfer between the dilute spins under mismatched Hartmann Hahn conditions for various B (1) fields have also been performed. The results adequately describe the optimal conditions for establishing the cross peaks, thus eliminating the need for lengthy experimental optimizations.

Structure determination in “shiftless” solid state NMR of oriented protein samples

An efficient formalism for calculating protein structures from oriented-sample NMR data in the torsion-angle space is presented. Angular anisotropies of the NMR observables are treated by utilizing an irreducible spherical basis of rotations. An intermediate rotational transformation is introduced that greatly speeds up structural fitting by rendering the dependence on the torsion angles Φ and Ψ in a purely diagonal form. Back-calculation of the simulated solid-state NMR spectra of protein G involving 15N chemical shift anisotropy (CSA), and 1H– 15N and 1H α– 13C α dipolar couplings was performed by taking into account non-planarity of the peptide linkages and experimental uncertainty. Even a relatively small (to within 1 ppm) random variation in the CSA values arising from uncertainties in the tensor parameters yields the RMSD’s of the back-calculated structures of more than 10 Å. Therefore, the 15N CSA has been substituted with heteronuclear dipolar couplings which are derived from the highly conserved bond lengths and bond angles associated with the amino-acid covalent geometry. Using the additional 13C α– 15N and 13C′– 15N dipolar couplings makes it possible to calculate protein structures entirely from “shiftless” solid-state NMR data. With the simulated “experimental” uncertainty of 15 Hz for protein G and 120 Hz for a helical hairpin derived from bacteriorhodopsin, back-calculation of the synthetic dipolar NMR spectra yielded a converged set of solutions. The use of distance restraints dramatically improves structural convergence even if larger experimental uncertainties are assumed.

Magic angle spinning and static oriented sample NMR studies of the relaxation in the rotating frame of membrane peptides

To demonstrate the influence of motions with medium-to-slow correlation times (milliseconds to nanoseconds), a systematic study of the spin-lattice relaxation in the rotating frame was conducted for several nuclei (H1, C13, and N15) in small membrane polypeptides, either in oriented or magic angle spinning samples. This study not only assesses the validity of some motional models, but also characterizes the magnetization relaxation rates crucial for the design of polarization transfer experiments. It was found that relaxation time constant (T1ρ) values on the order of 10−3–10−2s for backbone nuclei and their dependence on sample orientation are consistent with the model of transmembrane polypeptides undergoing axial diffusion (τc∼10−8–10−7s) and small amplitude off-axis reorientation (τc∼10−6–10−5s).

Facile Acquisition and Assignment of Oriented Sample NMR Spectra for Bilayer Surface-Associated Proteins

Facile Acquisition and Assignment of Oriented Sample NMR Spectra for Bilayer Surface-Associated Proteins

Qualitative comparison of the bilayer-associated structures of diacylglycerol and a fluorinated analog based upon oriented sample NMR data

sn-1,2-Dimyristoylglycerol (DMDAG) and sn-1,2-dimyristoyl-3-fluoropropanediol (DMFPD) were synthesized in carbonyl 13C-labeled and acyl chain perdeuterated forms. These compounds were reconstituted at low levels into both randomly dispersed dimyristoylphosphatidylcholine (DMPC) bilayers and magnetically orientable DMPC media. Samples were subjected to NMR analysis, leading to a substantial body of 2H quadrupolar splitting, 13C- 13C and 13C- 19F dipolar coupling and 13C chemical shift anisotropy data for both compounds. A number of measurements were also made for DMPC. The data acquired from magnetically oriented samples were found to be undesirably affected by the presence of artifacts related to the experimental use of the oriented lipid media. However, it was possible to draw a number of qualitative conclusions from the data and to correct the data so that they may ultimately prove useful in quantitative structural analyses of bilayer-associated DMDAG and DMFPD. Comparison of the DMDAG data with corresponding measurements for DMPC suggests a high degree of similarity between the two compounds within bilayers composed primarily of L α phase phosphatidylcholine, consistent with previous work (S.O. Smith et al. (1992) Biochemistry 31, 11660–11664). Comparison of the data for DMDAG and DMFPD suggests that the hydroxyl proton of DMDAG is involved in hydrogen bonding interactions, which appear to be largely responsible for maintaining the orientational inequivalence of its two acyl chains. Bilayer-associated DMFPD also appears to exhibit a higher degree of whole-molecule disorder than DMDAG, again suggestive of an important structural role for the hydroxyl proton of DMDAG. Finally, comparison of 13C-NMR data for oriented DMPC in the presence and absence of low levels of DMDAG and DMFPD indicated that neither compound induces a significant change in the averaged conformational state of DMPC comprising the bilayer matrix of the oriented samples.

Simulation of NMR data from oriented membrane proteins: practical information for experimental design

Several hundred solid state NMR dipolar couplings and chemical shift anisotropies were simulated for the polytopic membrane protein, bacteriorhodopsin, and for an idealized transmembrane peptide conforming to several different secondary structures (alpha- and 3(10)-helices and parallel and antiparallel beta-sheets), each at several tilt angles with respect to the bilayer normal. The use of macroscopically oriented samples was assumed. The results of these simulations suggest: (i) Because of the r-3 dependence of dipolar coupling, it is likely to prove difficult to successfully execute uniform isotopic enrichment strategies to generate large numbers of quantitatively interpretable structural measurements in oriented sample NMR studies of membrane proteins. (ii) There are a number of readily implementable specific isotopic labeling schemes which can yield data patterns sufficient to identify local secondary structure for transmembrane segments of idealized proteins which are tilted by < 10 degrees with respect to the bilayer normal. (iii) The measurement of dipolar coupling constants between 13C-, 19F-, and/or 3H-labeled side chains of proximal residues may prove effective as routes to long range tertiary structural data constraints.

Orientation and dynamics of .beta.-dodecyl glucopyranoside in phospholipid bilayers by oriented sample NMR and order matrix analysis

ADVERTISEMENT RETURN TO ISSUEPREVArticleNEXTOrientation and dynamics of .beta.-dodecyl glucopyranoside in phospholipid bilayers by oriented sample NMR and order matrix analysisCharles R. Sanders II and James H. PrestegardCite this: J. Am. Chem. Soc. 1991, 113, 6, 1987–1996Publication Date (Print):March 1, 1991Publication History Published online1 May 2002Published inissue 1 March 1991https://doi.org/10.1021/ja00006a019RIGHTS & PERMISSIONSArticle Views161Altmetric-Citations40LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit PDF (1 MB) Get e-Alerts Get e-Alerts

Direct determination of the oriented sample nmr spectrum from the powder spectrum for systems with local axial symmetry

A procedure is described for extracting the nuclear magnetic resonance spectrum of an oriented system from the spectrum of a superposition of randomly oriented domains. The procedure is applicable to dipolar, quadrupolar and anisotropic chemical shift interactions in systems having local axial symmetry and is illustrated using simulated and experimental spectra.