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The structure of two protected amino acids, FMOC-l-leucine and FMOC-l-valine, and a dipeptide, N-acetyl-l-valyl-l-leucine (N-Ac-VL), were studied via one- and two-dimensional solid-state nuclear magnetic resonance (NMR) spectroscopy. Utilizing (17)O magic-angle spinning (MAS) NMR at multiple magnetic fields (17.6–35.2 T/750–1500 MHz for (1)H) the (17)O quadrupolar and chemical shift parameters were determined for the two oxygen sites of each FMOC-protected amino acids and the three distinct oxygen environments of the dipeptide. The one- and two-dimensional, (17)O, (15)N─(17)O, (13)C─(17)O, and (1)H─(17)O double-resonance correlation experiments performed on the uniformly (13)C,(15)N and 70% (17)O-labeled dipeptide prove the attainability of (17)O as a probe for structure studies of biological systems. (15)N─(17)O and (13)C─(17)O distances were measured via one-dimensional REAPDOR and ZF-TEDOR experimental buildup curves and determined to be within 15% of previously reported distances, thus demonstrating the use of (17)O NMR to quantitate interatomic distances in a fully labeled dipeptide. Through-space hydrogen bonding of N-Ac-VL was investigated by a two-dimensional (1)H-detected (17)O R(3)-R-INEPT experiment, furthering the importance of (17)O for studies of structure in biomolecular solids.

Dynamic nuclear polarization (DNP) is theoretically able to enhance the signal in nuclear magnetic resonance (NMR) experiments by a factor γe/γn, where γ’s are the gyromagnetic ratios of an electron and a nuclear spin. However, DNP enhancements currently achieved in high-field, high-resolution biomolecular magic-angle spinning NMR are well below this limit because the continuous-wave DNP mechanisms employed in these experiments scale as ω−n0 where n ~ 1–2. In pulsed DNP methods, such as nuclear orientation via electron spin-locking (NOVEL), the DNP efficiency is independent of the strength of the main magnetic field. Hence, these methods represent a viable alternative approach for enhancing nuclear signals. At 0.35 T, the NOVEL scheme was demonstrated to be efficient in samples doped with stable radicals, generating 1H NMR enhancements of ~430. However, an impediment in the implementation of NOVEL at high fields is the requirement of sufficient microwave power to fulfill the on-resonance matching condition, ω0I = ω1S, where ω0I and ω1S are the nuclear Larmor and electron Rabi frequencies, respectively. Here, we exploit a generalized matching condition, which states that the effective Rabi frequency, ωeff1S, matches ω0I. By using this generalized off-resonance matching condition, we generate 1H NMR signal enhancement factors of 266 (~70% of the on-resonance NOVEL enhancement) with ω1S/2π = 5 MHz. We investigate experimentally the conditions for optimal transfer of polarization from electrons to 1H both for the NOVEL mechanism and the solid-effect mechanism and provide a unified theoretical description for these two historically distinct forms of DNP. published

The N-terminus of the voltage-dependent anion channel (VDAC) has been proposed to contain the mechanistically important gating helices that modulate channel opening and closing. In this study, we utilize magic angle spinning nuclear magnetic resonance (MAS NMR) to determine the location and structure of the N-terminus for functional channels in lipid bilayers by measuring long-range (13)C-(13)C distances between residues in the N-terminus and other domains of VDAC reconstituted into DMPC lipid bilayers. Our structural studies show that the distance between A14 Cβ in the N-terminal helix and S193 Cβ is ∼4-6 Å. Furthermore, VDAC phosphorylation by a mitochondrial kinase at residue S193 has been claimed to delay mitochondrial cell death by causing a conformational change that closes the channel, and a VDAC-Ser193Glu mutant has been reported to show properties very similar to those of phosphorylated VDAC in a cellular context. We expressed VDAC-S193E and reconstituted it into DMPC lipid bilayers. Two-dimensional (13)C-(13)C correlation experiments showed chemical shift perturbations for residues located in the N-terminus, indicating possible structural perturbations to that region. However, electrophysiological data recorded on VDAC-S193E showed that channel characteristics were identical to those of wild type samples, indicating that phosphorylation of S193 does not directly affect channel gating. The combination of NMR and electrophysiological results allows us to discuss the validity of proposed gating models.

The study of inorganic crystalline materials by solid-state NMR spectroscopy is often complicated by the low sensitivity of heavy nuclei. However, these materials often contain or can be prepared with paramagnetic dopants without significantly affecting the structure of the crystalline host. Dynamic nuclear polarization (DNP) is generally capable of enhancing NMR signals by transferring the magnetization of unpaired electrons to the nuclei. Therefore, the NMR sensitivity in these paramagnetically doped crystals might be increased by DNP. In this paper we demonstrate the possibility of efficient DNP transfer in polycrystalline samples of [Co(en)3Cl3]2·NaCl·6H2O (en = ethylenediamine, C2H8N2) doped with Cr(III) in varying concentrations between 0.1 and 3 mol %. We demonstrate that (1)H, (13)C, and (59)Co can be polarized by irradiation of Cr(III) with 140 GHz microwaves at a magnetic field of 5 T. We further explain our findings on the basis of electron paramagnetic resonance spectroscopy of the Cr(III) site and analysis of its temperature-dependent zero-field splitting, as well as the dependence of the DNP enhancement factor on the external magnetic field and microwave power. This first demonstration of DNP transfer from one paramagnetic metal ion to its diamagnetic host metal ion will pave the way for future applications of DNP in paramagnetically doped materials or metalloproteins.

The catalytic activity of tin-containing zeolites, such as Sn-Beta, is critically dependent on the successful incorporation of the tin metal center into the zeolite framework. However, synchrotron-based techniques or solid-state nuclear magnetic resonance (ssNMR) of samples enriched with (119)Sn isotopes are the only reliable methods to verify framework incorporation. This work demonstrates, for the first time, the use of dynamic nuclear polarization (DNP) NMR for characterizing zeolites containing ~2 wt % of natural abundance Sn without the need for (119)Sn isotopic enrichment. The biradicals TOTAPOL, bTbK, bCTbK, and SPIROPOL functioned effectively as polarizing sources, and the solvent enabled proper transfer of spin polarization from the radical's unpaired electrons to the target nuclei. Using bCTbK led to an enhancement (ε) of 75, allowing the characterization of natural-abundance (119)Sn-Beta with excellent signal-to-noise ratios in24 h. Without DNP, no (119)Sn resonances were detected after 10 days of continuous analysis.

National Institutes of Health (U.S.) (EB-002804), National Institutes of Health (U.S.) (EB-002026)

We illustrate the ability to place a water-insoluble biradical, bTbk, into a glycerol/water matrix with the assistance of a surfactant, sodium octyl sulfate (SOS). This surfactant approach enables a previously water insoluble biradical, bTbk, with favorable electron-electron dipolar coupling to be used for dynamic nuclear polarization (DNP) nuclear magnetic resonance (NMR) experiments in frozen, glassy, aqueous media. Nuclear Overhauser enhancement (NOE) and paramagnetic relaxation enhancement (PRE) experiments are conducted to determine the distribution of urea and several biradicals within the SOS macromolecular assembly. We also demonstrate that SOS assemblies are an effective approach by which mixed biradicals are created through an assembly process.

Protein sedimentation sans cryoprotection is a new approach to magic angle spinning (MAS) and dynamic nuclear polarization (DNP) nuclear magnetic resonance (NMR) spectroscopy of proteins. It increases the sensitivity of the experiments by a factor of ∼4.5 in comparison to the conventional DNP sample preparation and circumvents intense background signals from the cryoprotectant. In this paper, we investigate sedimented samples and concentrated frozen solutions of natural abundance bovine serum albumin (BSA) in the absence of a glycerol-based cryoprotectant. We observe DNP signal enhancements of ε ∼ 66 at 140 GHz in a BSA pellet sedimented from an aqueous solution containing the biradical polarizing agent TOTAPOL and compare this with samples prepared using the conventional protocol (i.e., dissolution of BSA in a glycerol/water cryoprotecting mixture). The dependence of DNP parameters on the radical concentration points to the presence of an interaction between TOTAPOL and BSA, so much so that a frozen solution sans cryoprotectant still gives ε ∼ 50. We have studied the interaction of BSA with another biradical, SPIROPOL, that is more rigid than TOTAPOL and has been reported to give higher enhancements. SPIROPOL was also found to interact with BSA, and to give ε ∼ 26 close to its maximum achievable concentration. Under the same conditions, TOTAPOL gives ε ∼ 31, suggesting a lesser affinity of BSA for SPIROPOL with respect to TOTAPOL. Altogether, these results demonstrate that DNP is feasible in self-cryoprotecting samples.

Despite tremendous advances in recent years, solution NMR remains fundamentally restricted due to its inherent insensitivity. Dynamic nuclear polarization (DNP) potentially offers significant improvements in this respect. The basic DNP strategy is to irradiate the EPR transitions of a stable radical and transfer this nonequilibrium polarization to the hydrogen spins of water, which will in turn transfer polarization to the hydrogens of the macromolecule. Unfortunately, these EPR transitions lie in the microwave range of the electromagnetic spectrum where bulk water absorbs strongly, often resulting in catastrophic heating. Furthermore, the residence times of water on the surface of the protein in bulk solution are generally too short for efficient transfer of polarization. Here we take advantage of the properties of solutions of encapsulated proteins dissolved in low viscosity solvents to implement DNP in liquids. Such samples are largely transparent to the microwave frequencies required and thereby avoid significant heating. Nitroxide radicals are introduced into the reverse micelle system in three ways: attached to the protein, embedded in the reverse micelle shell, and free in the aqueous core. Significant enhancements of the water resonance ranging up to ∼−93 at 0.35 T were observed. We also find that the hydration properties of encapsulated proteins allow for efficient polarization transfer from water to the protein. These and other observations suggest that merging reverse micelle encapsulation technology with DNP offers a route to a significant increase in the sensitivity of solution NMR spectroscopy of proteins and other biomolecules.

Protein magic angle spinning (MAS) NMR spectroscopy has generated structural models of several amyloid fibril systems, thus providing valuable information regarding the forces and interactions that confer the extraordinary stability of the amyloid architecture. Despite these advances, however, obtaining atomic resolution information describing the higher levels of structural organization within the fibrils remains a significant challenge. Here, we detail MAS NMR experiments and sample labeling schemes designed specifically to probe such higher order amyloid structure and we have applied them to the fibrils formed by an eleven-residue segment of the amyloidogenic protein transthyretin (TTR(105-115)). These experiments have allowed us to define unambiguously not only the arrangement of the peptide β-strands into β-sheets but also the β-sheet interfaces within each protofilament, and in addition to identify the nature of the protofilament-to-protofilament contacts that lead to the formation of the complete fibril. Our efforts have resulted in 111 quantitative distance and torsion angle restraints (10 per residue) that describe the various levels of structure organization. The experiments benefited extensively from the use of dynamic nuclear polarization (DNP), which in some cases allowed us to shorten the data acquisition time from days to hours and to improve significantly the signal-to-noise ratios of the spectra. The β-sheet interface and protofilament interactions identified here revealed local variations in the structure that result in multiple peaks for the exposed N- and C-termini of the peptide and in inhomogeneous line-broadening for the side-chains buried within the interior of the fibrils.

We demonstrate the use of dynamic nuclear polarization (DNP) to elucidate ligand binding to a membrane protein using dipolar recoupling magic angle spinning (MAS) NMR. In particular, we detect drug binding in the proton transporter M2(18-60) from influenza A using recoupling experiments at room temperature and with cryogenic DNP. The results indicate that the pore binding site of rimantadine is correlated with previously reported widespread chemical shift changes, suggesting functional binding in the pore. Futhermore, the N-15-labeled ammonium of rimantadine was observed near A30 C-13 beta and G34 C-13 alpha, suggesting a possible hydrogen bond to A30 carbonyl. Cryogenic DNP was required to observe the weaker external binding site(s) in a ZF-TEDOR spectrum. This approach is generally applicable, particularly for weakly bound ligands, in which case the application of MAS NMR dipolar recoupling requires the low temperatures to quench dynamic exchange processes. For the fully protonated samples investigated, we observed DNP signal enhancements of similar to 10 at 400 MHz using only 4-6 mM of the polarizing agent TOTAPOL. At 600 MHz and with DNP, we measured a distance between the drug and the protein to a precision of 0.2 angstrom.

Dynamic nuclear polarization (DNP) of amorphous and crystalline ortho-terphenyl (OTP) in the absence of glass forming agents is presented in order to gauge the feasibility of applying DNP to pharmaceutical solid-state nuclear magnetic resonance experiments and to study the effect of intermolecular structure, or lack thereof, on the DNP enhancement. By way of (1)H-(13)C cross-polarization, we obtained a DNP enhancement (ε) of 58 for 95% deuterated OTP in the amorphous state using the biradical bis-TEMPO terephthalate (bTtereph) and ε of 36 in the crystalline state. Measurements of the (1)H T1 and electron paramagnetic resonance experiments showed the crystallization process led to phase separation of the polarization agent, creating an inhomogeneous distribution of radicals within the sample. Consequently, the effective radical concentration was decreased in the bulk OTP phase, and long-range (1)H-(1)H spin diffusion was the main polarization propagation mechanism. Preliminary DNP experiments with the glass-forming anti-inflammation drug, indomethacin, showed promising results, and further studies are underway to prepare DNP samples using pharmaceutical techniques.

We investigate complexes of two paramagnetic metal ions Gd3+ and Mn2+ to serve as polarizing agents for solid-state dynamic nuclear polarization (DNP) of 1H, 13C, and 15N at magnetic fields of 5, 9.4, and 14.1 T. Both ions are half-integer high-spin systems with a zero-field splitting and therefore exhibit a broadening of the mS = −1/2 ↔ +1/2 central transition which scales inversely with the external field strength. We investigate experimentally the influence of the chelator molecule, strong hyperfine coupling to the metal nucleus, and deuteration of the bulk matrix on DNP properties. At small Gd-DOTA concentrations the narrow central transition allows us to polarize nuclei with small gyromagnetic ratio such as 13C and even 15N via the solid effect. We demonstrate that enhancements observed are limited by the available microwave power and that large enhancement factors of >100 (for 1H) and on the order of 1000 (for 13C) can be achieved in the saturation limit even at 80 K. At larger Gd(III) concentrations (≥10 mM) where dipolar couplings between two neighboring Gd3+ complexes become substantial a transition towards cross effect as dominating DNP mechanism is observed. Furthermore, the slow spin-diffusion between 13C and 15N, respectively, allows for temporally resolved observation of enhanced polarization spreading from nuclei close to the paramagnetic ion towards nuclei further removed. Subsequently, we present preliminary DNP experiments on ubiquitin by site-directed spin-labeling with Gd3+ chelator tags. The results hold promise towards applications of such paramagnetically labeled proteins for DNP applications in biophysical chemistry and/or structural biology., Physical Chemistry Chemical Physics, 18 (39), ISSN:1463-9084, ISSN:1463-9076

The structure and dynamics of the bound water in barium chlorate monohydrate were studied with (17)O nuclear magnetic resonance (NMR) spectroscopy in samples that are stationary and spinning at the magic-angle in magnetic fields ranging from 14.1 to 21.1 T. (17)O NMR parameters of the water were determined, and the effects of torsional oscillations of the water molecule on the (17)O quadrupolar coupling constant (CQ) were delineated with variable temperature MAS NMR. With decreasing temperature and reduction of the librational motion, we observe an increase in the experimentally measured CQ explaining the discrepancy between experiments and predictions from density functional theory. In addition, at low temperatures and in the absence of (1)H decoupling, we observe a well-resolved (1)H-(17)O dipole splitting in the spectra, which provides information on the structure of the H2O molecule. The splitting arises because of the homogeneous nature of the coupling between the two (1)H-(17)O dipoles and the (1)H-(1)H dipole.

We described a versatile spectrometer designed for the study of dynamic nuclear polarization (DNP) at low temperatures and high fields. The instrument functions both as an NMR spectrometer operating at 212 MHz ([superscript 1]H frequency) with DNP capabilities, and as a pulsed-EPR operating at 140 GHz. A coiled TE[subscript 011] resonator acts as both an NMR coil and microwave resonator, and a double balanced ([superscript 1]H, [superscript 13]C) radio frequency circuit greatly stabilizes the NMR performance. A new 140 GHz microwave bridge has also been developed, which utilizes a four-phase network and ELDOR channel at 8.75 GHz, that is then multiplied and mixed to obtain 140 GHz microwave pulses with an output power of 120 mW. Nutation frequencies obtained are as follows: 6 MHz on S = 1/2 electron spins, 100 kHz on [superscript 1]H, and 50 kHz on [superscript 13]C. We demonstrate basic EPR, ELDOR, ENDOR, and DNP experiments here. Our solid effect DNP results demonstrate an enhancement of 144 and sensitivity gain of 310 using OX063 trityl at 80 K and an enhancement of 157 and maximum sensitivity gain of 234 using Gd-DOTA at 20 K, which is significantly better performance than previously reported at high fields (⩾3 T)., National Institutes of Health (U.S.) (EB002804), National Institutes of Health (U.S.) (EB002026), National Institutes of Health (U.S.) (EB001965), National Institutes of Health (U.S.) (EB004866), Deutsche Forschungsgemeinschaft (Postdoctoral Fellowship)

Molecules that exhibit emission in the solid state, especially those known as aggregation-induced emission (AIE) chromophores, have found applications in areas as varied as light-emitting diodes and biological sensors. Despite numerous studies, the mechanism of fluorescence quenching in AIE chromophores is still not completely understood. To this end, much interest has focused on understanding the low-frequency vibrational dynamics of prototypical systems, such as tetraphenylethylene (TPE), in the hope that such studies would provide more general principles toward the design of new sensors and electronic materials. We hereby show that a perdeuterated TPE-based metal-organic framework (MOF) serves as an excellent platform for studying the low-energy vibrational modes of AIE-type chromophores. In particular, we use solid-state (2)H and (13)C NMR experiments to investigate the phenyl ring dynamics of TPE cores that are coordinatively trapped inside a MOF and find a phenyl ring flipping energy barrier of 43(6) kJ/mol. DFT calculations are then used to deconvolute the electronic and steric contributions to this flipping barrier. Finally, we couple the NMR and DFT studies with variable-temperature X-ray diffraction experiments to propose that both the ethylenic C═C bond twist and the torsion of the phenyl rings are important for quenching emission in TPE, but that the former may gate the latter. To conclude, we use these findings to propose a set of design criteria for the development of tunable turn-on porous sensors constructed from AIE-type molecules, particularly as applied to the design of new multifunctional MOFs.

The synthesis of air-stable, highly water-soluble organic radicals containing a 1,3-bis(diphenylene)-2-phenylallyl (BDPA) core is reported. A sulfonated derivative, SA-BDPA, retains the narrow electron paramagnetic resonance linewidth (

International audience; The synthesis and characterization of oxidized bis-thioketal-trispiro dinitroxide biradicals that orient the nitroxides in a rigid, approximately orthogonal geometry are reported. The biradicals show better performance as polarizing agents in dynamic nuclear polarization (DNP) NMR experiments as compared to biradicals lacking the constrained geometry. In addition, the biradicals display improved solubility in aqueous media due to the presence of polar sulfoxides. The results suggest that the orientation of the radicals is not dramatically affected by the oxidation state of the sulfur atoms in the biradical, and we conclude that a biradical polarizing agent containing a mixture of oxidation states can be used for improved solubility without a loss in performance.

The paper describes two-dimensional solid state NMR experiments that use powdered dephased anti-phase coherence (γ preparation) to encode chemical shifts in the indirect dimension. Both components of this chemical shift encoded gamma-prepared states can be refocused into inphase coherence by a recoupling element. This helps to achieve sensitivity enhancement in 2D NMR experiments by quadrature detection. The powder dependence of the gamma-prepared states allows for manipulating them by suitable insertion of delays in the recoupling periods. This helps to design experiments that suppress diagonal peaks in 2D spectra, leading to improved resolution. We describe some new phase modulated heteronuclear and homonuclear recoupling pulse sequences that simplify the implementation of the described experiments based on γ prepared states. Recoupling in the heteronuclear spin system is achieved by matching the difference in the amplitude of the sine/cosine modulated phase on the two rf-channels to the spinning frequency while maintaining the same power on the two rf-channels.

Solid-state magic angle spinning nuclear magnetic resonance (MAS NMR) is maturing rapidly. Progress is highlighted by recent examples that demonstrate its capability to yield site-specific assignments and atomic resolution structural information on fibrillar,[1–3] membrane-associated,[4–6] and non-crystalline proteins.[7–9] Furthermore, applications to systems of ever-increasing molecular size are limited only by the signal-to-noise ratio (S/N) in the multidimensional spectra required for adequate resolution,, rather than by more fundamental limitations from spin relaxation. Therefore, the most pressing need in MAS NMR is arguably for more efficient data acquisition methods.

Gas vesicles are gas-filled buoyancy organelles with walls that consist almost exclusively of gas vesicle protein A (GvpA). Intact, collapsed gas vesicles from the cyanobacterium Anabaena flos-aquae were studied by solid-state NMR spectroscopy, and most of the GvpA sequence was assigned. Chemical shift analysis indicates a coil-α-β-β-α-coil peptide backbone, consistent with secondary-structure-prediction algorithms, and complementary information about mobility and solvent exposure yields a picture of the overall topology of the vesicle subunit that is consistent with its role in stabilizing an air-water interface.

The use of pseudocontact shifts arising from paramagnetic metal ions in a microcrystalline protein sample is proposed as a strategy to obtain unambiguous signal assignments in solid-state NMR spectra enabling distance extraction for protein structure calculation. With this strategy, 777 unambiguous (281 sequential, 217 medium-range, and 279 long-range) distance restraints could be obtained from PDSD, DARR, CHHC, and the recently introduced PAR and PAIN-CP solid-state experiments for the cobalt(II)-substituted catalytic domain of matrix metalloproteinase 12 (159 amino acids, 17.6 kDa). The obtained structure is a high resolution one, with backbone rmsd of 1.0 +/- 0.2 A, and is in good agreement with the X-ray structure (rmsd to X-ray 1.3 A). The proposed strategy, which may be generalized for nonmetalloproteins with the use of paramagnetic tags, represents a significant step ahead in protein structure determination using solid-state NMR.

A new polarizing agent with superior performance in dynamic nuclear polarization experiments is introduced, and utilizes two TEMPO (2,2,6,6-tetramethylpiperidine-1-oxyl) moieties connected through a rigid spiro tether (see structure). The observed NMR signal intensities were enhanced by a factor of 1.4 compared to those of TOTAPOL, a previously described TEMPO-based biradical with a flexible tether., National Institutes of Health (U.S.) (EB002804), National Institutes of Health (U.S.) (EB002026), National Institutes of Health (U.S.) (EB009866), National Institutes of Health (U.S.) (EB001965), National Institutes of Health (U.S.) (EB001035), Sixth Framework Programme (European Commission) (EU-Design Study Bio-DNP), Deutsche Forschungsgemeinschaft (postdoctoral fellowship), Naito Foundation

By exploiting dynamic nuclear polarization (DNP) at 90 K, we observe the first NMR spectrum of the K intermediate in the ion-motive photocycle of bacteriorhodopsin. The intermediate is identified by its reversion to the resting state of the protein in red light and by its thermal decay to the L intermediate. The 15 N chemical shift of the Schiff base in K indicates that contact has been lost with its counterion. Under these circumstances, the visible absorption of K is expected to be more red-shifted than is observed and this suggests torsion around single bonds of the retinylidene chromophore. This is in contrast to the development of a strong counterion interaction and double bond torsion in L. Thus, photon energy is stored in electrostatic modes in K and is transferred to torsional modes in L. This transfer is facilitated by the reduction in bond alternation that occurs with the initial loss of the counterion interaction, and is driven by the attraction of the Schiff base to a new counterion. Nevertheless, the process appears to be difficult, as judged by the multiple L substates, with weaker counterion interactions, that are trapped at lower temperatures. The double-bond torsion ultimately developed in the first half of the photocycle is probably responsible for enforcing vectoriality in the pump by causing a decisive switch in the connectivity of the active site once the Schiff base and its counterion are neutralized by proton transfer.

© 2015 WILEY VCH Verlag GmbH Co. KGaA Weinheim. In the Spring of 2013 NMR spectroscopists convened at the Weizmann Institute in Israel to brainstorm on approaches to improve the sensitivity of NMR experiments particularly when applied in biomolecular settings. This multi author interdisciplinary Review presents a state of the art description of the primary approaches that were considered. Topics discussed included the future of ultrahigh field NMR systems emerging NMR detection technologies new approaches to nuclear hyperpolarization and progress in sample preparation. All of these are orthogonal efforts whose gains could multiply and thereby enhance the sensitivity of solid and liquid state experiments. While substantial advances have been made in all these areas numerous challenges remain in the quest of endowing NMR spectroscopy with the sensitivity that has characterized forms of spectroscopies based on electrical or optical measurements. These challenges and the ways by which scientists and engineers are striving to solve them are also addressed. A new spin on bio NMR: This Review presents a state of the art description of the leading approaches being considered today to improve the sensitivity of NMR spectroscopy particularly as applied in biomolecular settings. The focus is on the future of ultrahigh field NMR systems emerging NMR detection technologies new approaches to nuclear hyperpolarization and progress in sample preparation.

Enzymes are used as environmentally friendly catalysts in many industrial applications, and are frequently immobilized in a matrix to improve their chemical stability for long-term storage and reusability. Recently, it was shown that an atomic-level description of proteins immobilized in a biosilica matrix can be attained by examining their magic-angle spinning (MAS) NMR spectra. However, even though MAS NMR is an excellent tool for determining structure, it is severely hampered by sensitivity. In this work we provide the proof of principle that NMR characterization of biosilica-entrapped enzymes could be assisted by high-field dynamic nuclear polariza tion (DNP). A closer look at entrapped enzymes: Enzymes entrapped in bioinspired materials are gaining traction in green applications, and solid-state NMR promises to be the technique to study them at atomic detail. However, sensitivity is usually limited. Dynamic nuclear polarization can be applied to increase sensitivity and assess the preservation of the enzyme fold., National Institute of Biomedical Imaging and Bioengineering (U.S.) (Grant EB-003151), National Institute of Biomedical Imaging and Bioengineering (U.S.) (Grant EB-002026), National Institute of Biomedical Imaging and Bioengineering (U.S.) (Grant EB-002804)

Magic angle spinning (MAS) NMR studies of amyloid and membrane proteins and large macromolecular complexes are an important new approach to structural biology. However, the applicability of these experiments, which are based on 13C- and 15N-detected spectra, would be enhanced if the sensitivity were improved. Here we discuss two advances that address this problem: high-frequency dynamic nuclear polarization (DNP) and 1H-detected MAS techniques. DNP is a sensitivity enhancement technique that transfers the high polarization of exogenous unpaired electrons to nuclear spins via microwave irradiation of electron–nuclear transitions. DNP boosts NMR signal intensities by factors of 102 to 103, thereby overcoming NMR's inherent low sensitivity. Alternatively, it permits structural investigations at the nanomolar scale. In addition, 1H detection is feasible primarily because of the development of MAS rotors that spin at frequencies of 40 to 60 kHz or higher and the preparation of extensively 2H-labeled proteins.

Computer-optimized selective pulses are routinely used in solution-state NMR spectroscopy. At the same time, their utility and importance for solid-state applications has yet to be fully realized. We suggest a new computational approach that makes the design of soft selective pulses with desired properties relatively straightforward. By applying this technique to the generic selective excitation problem, we have arrived at a family of high performance selective excitation pulses, dubbed E-Family, that allows more flexibility and better performance than analogous pulses previously reported in the literature. The new pulses have been successfully tested in both solid- and solution-state NMR experiments. A theoretical treatment of the effects of chemical shift anisotropy (CSA) on the selective excitation in magic-angle spinning (MAS) experiments in solids is presented. The set of heuristics that comprise our new strategy were incorporated into a general NMR simulation program SPINEVOLUTION.

We show how high-resolution magic angle-spinning NMR spectroscopy can be used to characterize 13C-labeled saccharides that have been prepared using solid-phase synthesis techniques while they are still bound to a solid-support resin. With the use of 13C-labeled glucose as the starting material, we have successfully synthesized mono-, di- and trisaccharides with uniform 13C labeling of the saccharide rings. Using these materials, we have been able to assign the 13C and 1H spectra and to characterize various impurities on the resin beads.

Amyloid fibrils are self-assembled filamentous structures associated with protein deposition conditions including Alzheimer's disease and the transmissible spongiform encephalopathies. Despite the immense medical importance of amyloid fibrils, no atomic-resolution structures are available for these materials, because the intact fibrils are insoluble and do not form diffraction-quality 3D crystals. Here we report the high-resolution structure of a peptide fragment of the amyloidogenic protein transthyretin, TTR(105–115), in its fibrillar form, determined by magic angle spinning NMR spectroscopy. The structure resolves not only the backbone fold but also the precise conformation of the side chains. Nearly complete 13 C and 15 N resonance assignments for TTR(105–115) formed the basis for the extraction of a set of distance and dihedral angle restraints. A total of 76 self-consistent experimental measurements, including 41 restraints on 19 backbone dihedral angles and 35 13 C– 15 N distances between 3 and 6 Å were obtained from 2D and 3D NMR spectra recorded on three fibril samples uniformly 13 C, 15 N-labeled in consecutive stretches of four amino acids and used to calculate an ensemble of peptide structures. Our results indicate that TTR(105–115) adopts an extended β-strand conformation in the amyloid fibrils such that both the main- and side-chain torsion angles are close to their optimal values. Moreover, the structure of this peptide in the fibrillar form has a degree of long-range order that is generally associated only with crystalline materials. These findings provide an explanation of the unusual stability and characteristic properties of this form of polypeptide assembly.

We describe three-dimensional magic-angle-spinning NMR experiments for the simultaneous measurement of multiple carbon-nitrogen distances in uniformly (13)C,(15)N-labeled solids. The approaches employ transferred echo double resonance (TEDOR) for (13)C-(15)N coherence transfer and (15)N and (13)C frequency labeling for site-specific resolution, and build on several previous 3D TEDOR techniques. The novel feature of the 3D TEDOR pulse sequences presented here is that they are specifically designed to circumvent the detrimental effects of homonuclear (13)C-(13)C J-couplings on the measurement of weak (13)C-(15)N dipolar couplings. In particular, homonuclear J-couplings lead to two undesirable effects: (i) they generate anti-phase and multiple-quantum (MQ) spin coherences, which lead to spurious cross-peaks and phase-twisted lines in the 2D (15)N-(13)C correlation spectra, and thus degrade the spectral resolution and prohibit the extraction of reliable cross-peak intensities, and (ii) they significantly reduce cross-peak intensities for strongly J-coupled (13)C sites (e.g., CO and C(alpha)). The first experiment employs z-filter periods to suppress the anti-phase and MQ coherences and generates 2D spectra with purely absorptive peaks for all TEDOR mixing times. The second approach uses band-selective (13)C pulses to refocus J-couplings between (13)C spins within the selective pulse bandwidth and (13)C spins outside the bandwidth. The internuclear distances are extracted by using a simple analytical model, which accounts explicitly for multiple spin-spin couplings contributing to cross-peak buildup. The experiments are demonstrated in two U-(13)C,(15)N-labeled peptides, N-acetyl-L-Val-L-Leu (N-ac-VL) and N-formyl-L-Met-L-Leu-L-Phe (N-f-MLF), where 20 and 26 (13)C-(15)N distances up to approximately 5-6 A were measured, respectively. Of the measured distances, 10 in N-ac-VL and 13 in N-f-MLF are greater than 3 A and provide valuable structural constraints.

In recent years, structural information about bacteriorhodopsin has grown substantially with the publication of several crystal structures. However, precise measurements of the chromophore conformation in the various photocycle states are still lacking. This information is critical because twists about the chromophore backbone chain can influence the Schiff base nitrogen position, orientation, and proton affinity. Here, we focus on the C14-C15 bond, using solid-state nuclear magnetic resonance spectroscopy to measure the H-C14-C15-H dihedral angle. In the resting state (bR(568)), we obtain an angle of 164 +/- 4 degrees, indicating a 16 degrees distortion from a planar all-trans chromophore. The dihedral angle is found to decrease to 147 +/- 10 degrees in the early M intermediate (M(o)) and to 150 +/- 4 degrees in the late M intermediate (M(n)). These results demonstrate changes in the chromophore conformation undetected by recent X-ray diffraction studies.

Solid-state NMR is a powerful technique for the investigation of complex biological systems such as membrane proteins and amyloid fibrils. In magic-angle spinning (MAS) NMR, structural information is obtained via the reintroduction of anisotropic interactions.[1] In particular, a large number of homonuclear dipolar recoupling schemes have been developed and applied to record correlation spectra and measure internuclear distances in peptides and proteins.[1] While many recoupling techniques efficiently transfer polarization between directly bonded 13C nuclei, their effectiveness can be reduced significantly when distant 13C spins with weak dipolar couplings are involved. This limitation is generally imposed both by the experimental constraints that must be maintained during long mixing periods and by the inherent complexities of multiple-spin systems such as dipolar truncation,[2,3] that is the attenuation of weak dipolar couplings by stronger dipolar couplings in the recoupled dipolar Hamiltonian. As a consequence, spin diffusion techniques,[4–8] which circumvent some of these limitations,[9] have been widely utilized to estimate long-range homonuclear structural constraints in protein solid-state NMR studies.[10,11] In this communication we present an experimental approach that provides highly sensitive long-range correlations between aliphatic 13C nuclei through the combination of isotope dilution and efficient polarization transfer via a band selective radio frequency dipolar recoupling (BASE RFDR) scheme. We demonstrate this method with a sample of PI3-SH3 (the SH3 domain of the p85α subunit of phosphatidylinositol 3 kinase) in amyloid fibril form.

Constraints on the proximity of the carboxyl carbons of the Asp-85 and Asp-212 side chains to the 14-carbon of the retinal chromophore have been established for the bR(555), bR(568), and M(412) states of bacteriorhodopsin (bR) using solid-state NMR spectroscopy. These distances were examined via (13)C-(13)C magnetization exchange, which was observed in two-dimensional RF-driven recoupling (RFDR) and spin diffusion experiments. A comparison of relative RFDR cross-peak intensities with simulations of the NMR experiments yields distance measurements of 4.4 +/- 0.6 and 4.8 +/- 1.0 A for the [4-(13)C]Asp-212 to [14-(13)C]retinal distances in bR(568) and M(412), respectively. The spin diffusion data are consistent with these results and indicate that the Asp-212 to 14-C-retinal distance increases by 16 +/- 10% upon conversion to the M-state. The absence of cross-peaks from [14-(13)C]retinal to [4-(13)C]Asp-85 in all states and between any [4-(13)C]Asp residue and [14-(13)C]retinal in bR(555) indicates that these distances exceed 6.0 A. For bR(568), the NMR distance constraints are in agreement with the results from recent diffraction studies on intact membranes, while for the M state the NMR results agree with theoretical simulations employing two bound waters in the region of the Asp-85 and Asp-212 residues. The structural information provided by NMR should prove useful for refining the current understanding of the role of aspartic acid residues in the proton-pumping mechanism of bR.

To enforce vectorial proton transport in bacteriorhodopsin (bR), it is necessary that there be a change in molecular structure between deprotonation and reprotonation of the chromophore-i.e., there must be at least two different M intermediates in the functional photocycle. We present here the first detection of multiple M intermediates in native wild-type bacteriorhodopsin by solid-state NMR. Illumination of light-adapted [zeta-15N-Lys]-bR at low temperatures shifts the 15N signal of the retinal Schiff base (SB) downfield by about 150 ppm, indicating a deprotonated chromophore. In 0.3 M Gdn-HCl at pH 10.0, two different M states are obtained, depending on the temperature during illumination. The M state routinely prepared at the lower temperature, Mo, decays to the newly observed M state, Mn, and the N intermediate, as the temperature is increased. Both relax to bR568 at 0 degreesC. A unique reaction sequence is derived: bR568-->Mo-->(Mn+N)-->bR568. Mo and Mn have similar chemical shifts at [12-13C]ret, [14-13C]ret, and [epsilon-13C]Lys216, indicating that Mn, like Mo, has a 13-cis and C=N anti chromophore. However, a small splitting in the [14-13C]ret signal of Mo reveals that it has at least two substates. The 7 ppm greater shielding of the SB nitrogen in Mn compared to Mo suggests an increase in basicity and/or hydrogen bonding. Probing the peptide backbone of the protein, via [1-13C]Val labeling, reveals a substantial structural change between Mo and Mn including the relaxation of perturbations at some sites and the development of new perturbations at other sites. The combination of the change in the protein structure and the increase in the pKa of the SB suggests that the demonstrated Mo-->Mn transition may function as the "reprotonation switch" required for vectorial proton transport.

High-resolution solid state 17 O NMR spectra of 17 O-enriched compounds were observed with multiple-quantum magic-angle-spinning (MQMAS) experiments. The resolution of the 17 O MQMAS spectra is approximately 30- to 150-fold higher than that found in conventional 17 O MAS spectra, making it possible to detect crystallographically distinct oxygen sites. It is shown that 17 O MQMAS studies are feasible for systems with 17 O quadrupole coupling constants up to 7 MHz, provided that sufficiently high radio-frequency field strengths (> 120 kHz and spinning speeds (∼ 20 kHz) can be achieved.

The L550 intermediate in the bacteriorhodopsin (bR) photocycle has drawn much attention with respect to the mechanism of light-driven proton transport because it selectively releases the Schiff base (SB) proton to the extracellular proton channel in the L--M transition. Here we extend our solid-state NMR studies of bR photocycle intermediates to the L state. Under conditions that stabilize L550, a new SB signal is detected in the 15N NMR spectrum which disappears upon thermal relaxation. This signal is in the range for a protonated SB, but downfield from the SB signals of bR568 and N520. Since steric interactions would have the opposite effect on shielding, the data argue against a 13,14-dicis chromophore in L550. Comparison with the 15N chemical shifts of halide salts of protonated Schiff bases (pSB's) of retinal suggests that the interaction of the SB with its counterion is significantly stronger in L550 than in N520 (which in turn is stronger than in bR568). This is consistent with models of the early photocycle in which the electrostatic interaction between the SB and its counterion constitutes an important constraint. Although the L counterion interaction is comparable to that of a 6-s-trans,13-cis chloride salt, the visible spectrum is strongly red-shifted from the lambdamax = 491 nm of the chloride. This suggests some double bond strain in L550, particularly about the C=N bond. This strain is apparently gone in the N intermediate, which has a normal relationship between the 15N chemical shift and lambdamax. Such a relaxed chromophore is consistent with orientation of the SB proton toward the cytoplasmic surface in the N intermediate.

We present electron paramagnetic resonance experiments for which solid effect dynamic nuclear polarization transitions were observed indirectly via polarization loss on the electron. This use of indirect observation allows characterization of the dynamic nuclear polarization (DNP) process close to the electron. Frequency profiles of the electron-detected solid effect obtained using trityl radical showed intense saturation of the electron at the usual solid effect condition, which involves a single electron and nucleus. However, higher order solid effect transitions involving two, three, or four nuclei were also observed with surprising intensity, although these transitions did not lead to bulk nuclear polarization--suggesting that higher order transitions are important primarily in the transfer of polarization to nuclei nearby the electron. Similar results were obtained for the SA-BDPA radical where strong electron-nuclear couplings produced splittings in the spectrum of the indirectly observed solid effect conditions. Observation of high order solid effect transitions supports recent studies of the solid effect, and suggests that a multi-spin solid effect mechanism may play a major role in polarization transfer via DNP.

Dynamic nuclear polarization of 17O was studied using four different polarizing agents – the biradical TOTAPOL, and the monoradicals trityl and SA-BDPA, as well as a mixture of the latter two. Field profiles, DNP mechanisms and enhancements were measured to better understand and optimize directly polarizing this low-gamma quadrupolar nucleus using both mono- and bi-radical polarizing agents. Enhancements were recorded < 88 K and were > 100 using the trityl (OX063) radical and < 10 with the other polarizing agents. The > 10,000 fold savings in acquisition time enabled a series of biologically relevant small molecules to be studied with small sample sizes and the measurement of various quadrupolar parameters. The results are discussed with comparison to room temperature studies and GIPAW quantum chemical calculations. These experimental results illustrate the strength of high field DNP and the importance of radical selection for studying low-gamma nuclei.

Yeast prions are self-templating protein-based mechanisms of inheritance whose conformational changes lead to the acquisition of diverse new phenotypes. The best studied of these is the prion domain (NM) of Sup35, which forms an amyloid that can adopt several distinct conformations (strains) that produce distinct phenotypes. Using magic-angle spinning nuclear magnetic resonance spectroscopy, we provide a detailed look at the dynamic properties of these forms over a broad range of timescales. We establish that different prion strains have distinct amyloid structures, with many side chains in different chemical environments. Surprisingly, the prion strain with a larger fraction of rigid residues also has a larger fraction of highly mobile residues. Differences in mobility correlate with differences in interaction with the prion-partitioning factor Hsp104 in vivo, perhaps explaining strain-specific differences in inheritance., Howard Hughes Medical Institute (Investigator), Howard Hughes Medical Institute (HHMI fellow of the Life Science Research Foundation), National Institutes of Health (U.S.) (NIH grant GM025874), National Institutes of Health (U.S.) (NIH grant EB003151), National Institutes of Health (U.S.) (NIH grant EB002026)

The power of nuclear magnetic resonance spectroscopy derives from its site-specific access to chemical, structural and dynamic information. However, the corresponding multiplicity of interactions can be difficult to tease apart. Complimentary approaches involve spectral editing on the one hand and selective isotope substitution on the other. Here we present a new “redox” approach to the latter: acetate is chosen as the sole carbon source for the extreme oxidation numbers of its two carbons. Consistent with conventional anabolic pathways for the amino acids, [1-[superscript 13]C] acetate does not label α carbons, labels other aliphatic carbons and the aromatic carbons very selectively, and labels the carboxyl carbons heavily. The benefits of this labeling scheme are exemplified by magic angle spinning spectra of microcrystalline immunoglobulin binding protein G (GB1): the elimination of most J-couplings and one- and two-bond dipolar couplings provides narrow signals and long-range, intra- and inter-residue, recoupling essential for distance constraints. Inverse redox labeling, from [2-[superscript 13]C] acetate, is also expected to be useful: although it retains one-bond couplings in the sidechains, the removal of CA–CO coupling in the backbone should improve the resolution of NCACX spectra., National Institutes of Health. National Institute for Biomedical Imaging and Bioengineering (Grant EB-001035), National Institutes of Health. National Institute for Biomedical Imaging and Bioengineering (Grant EB-001960), National Institutes of Health. National Institute for Biomedical Imaging and Bioengineering (Grant EB-002026)

During the three decades 1980-2010, magic angle spinning (MAS) NMR developed into the method of choice to examine many chemical, physical, and biological problems. In particular, a variety of dipolar recoupling methods to measure distances and torsion angles can now constrain molecular structures to high resolution. However, applications are often limited by the low sensitivity of the experiments, due in large part to the necessity of observing spectra of low-γ nuclei such as the I = 1/2 species (13)C or (15)N. The difficulty is still greater when quadrupolar nuclei, such as (17)O or (27)Al, are involved. This problem has stimulated efforts to increase the sensitivity of MAS experiments. A particularly powerful approach is dynamic nuclear polarization (DNP) which takes advantage of the higher equilibrium polarization of electrons (which conventionally manifests in the great sensitivity advantage of EPR over NMR). In DNP, the sample is doped with a stable paramagnetic polarizing agent and irradiated with microwaves to transfer the high polarization in the electron spin reservoir to the nuclei of interest. The idea was first explored by Overhauser and Slichter in 1953. However, these experiments were carried out on static samples, at magnetic fields that are low by current standards. To be implemented in contemporary MAS NMR experiments, DNP requires microwave sources operating in the subterahertz regime, roughly 150-660 GHz, and cryogenic MAS probes. In addition, improvements were required in the polarizing agents, because the high concentrations of conventional radicals that are required to produce significant enhancements compromise spectral resolution. In the last two decades, scientific and technical advances have addressed these problems and brought DNP to the point where it is achieving wide applicability. These advances include the development of high frequency gyrotron microwave sources operating in the subterahertz frequency range. In addition, low temperature MAS probes were developed that permit in situ microwave irradiation of the samples. And, finally, biradical polarizing agents were developed that increased the efficiency of DNP experiments by factors of ∼4 at considerably lower paramagnet concentrations. Collectively, these developments have made it possible to apply DNP on a routine basis to a number of different scientific endeavors, most prominently in the biological and material sciences. This Account reviews these developments, including the primary mechanisms used to transfer polarization in high frequency DNP, and the current choice of microwave sources and biradical polarizing agents. In addition, we illustrate the utility of the technique with a description of applications to membrane and amyloid proteins that emphasizes the unique structural information that is available in these two cases.

The cross-β amyloid form of peptides and proteins represents an archetypal and widely accessible structure consisting of ordered arrays of β-sheet filaments. These complex aggregates have remarkable chemical and physical properties, and the conversion of normally soluble functional forms of proteins into amyloid structures is linked to many debilitating human diseases, including several common forms of age-related dementia. Despite their importance, however, cross-β amyloid fibrils have proved to be recalcitrant to detailed structural analysis. By combining structural constraints from a series of experimental techniques spanning five orders of magnitude in length scale—including magic angle spinning nuclear magnetic resonance spectroscopy, X-ray fiber diffraction, cryoelectron microscopy, scanning transmission electron microscopy, and atomic force microscopy—we report the atomic-resolution (0.5 Å) structures of three amyloid polymorphs formed by an 11-residue peptide. These structures reveal the details of the packing interactions by which the constituent β-strands are assembled hierarchically into protofilaments, filaments, and mature fibrils.

We demonstrate the utility of a two-pulse sequence in obtaining high-resolution solid state NMR spectra of half-integer quadrupolar nuclei with magic-angle-spinning (MAS). The experiment, which utilizes multiple/single-quantum correlation, was first described in a different form by Frydman and Harwood [J. Am. Chem. Soc. 117 (1995) 5367] and yields high-resolution isotropic NMR spectra where shifts are determined by the sum of resonance offset (chemical shift) and second-order quadrupolar effects. The two-pulse sequence described here is shown to provide a higher and more uniform excitation of multiple-quantum coherence than the three-pulse sequence used previously.

We describe a new approach for observation of multiple-quantum (MQ) NMR spectra of S = 3/2 nuclei with magic-angle spinning (MAS). The new method employs the Rotation-Induced Adiabatic Coherence Transfer (RIACT) that occurs between triple-quantum (3Q) and central-transition (1Q) coherences in S = 3/2 systems. In contrast to currently available coherence-transfer techniques, RIACT is relatively insensitive to the magnitude of the quadrupole interaction for e2qQ/h ≤ 4 MHz for both 3Q excitation and 3Q-to-1Q conversion. Thus, RIACT provides a means of extracting quantitative information about site populations from isotropic MQ NMR spectra. We illustrate the utility of the approach with 23Na (S = 3/2) MQ NMR spectra of a series of sodium salts exhibiting crystallographically distinct sites. The spectra provide quantitative measurements of quadrupolar parameters, chemical shifts, and relative site populations for each of the crystallographically distinct sodium sites.

Amyloid fibrils can be generated from proteins with diverse sequences and folds. Although amyloid fibrils assembled in vitro commonly involve a single protein precursor, fibrils formed in vivo can contain more than one protein sequence. How fibril structure and stability differ in fibrils composed of single proteins (homopolymeric fibrils) from those generated by co-polymerization of more than one protein sequence (heteropolymeric fibrils) is poorly understood. Here we compare the structure and stability of homo and heteropolymeric fibrils formed from human β2-microglobulin and its truncated variant ΔN6. We use an array of approaches (limited proteolysis, magic angle spinning NMR, Fourier transform infrared spectroscopy, and fluorescence) combined with measurements of thermodynamic stability to characterize the different fibril types. The results reveal fibrils with different structural properties, different side-chain packing, and strikingly different stabilities. These findings demonstrate how co-polymerization of related precursor sequences can expand the repertoire of structural and thermodynamic polymorphism in amyloid fibrils to an extent that is greater than that obtained by polymerization of a single precursor alone.

In this paper, we describe the design and experimental results from the rebuild of a 250 GHz gyrotron used for Dynamic Nuclear Polarization enhanced Nuclear Magnetic Resonance spectroscopy on a 380 MHz spectrometer. Tuning bandwidth of approximately 2 GHz is easily achieved at a fixed magnetic field of 9.24 T and a beam current of 95 mA producing an average output power of >10 W over the entire tuning band. This tube incorporates a double disk output sapphire window in order to maximize the transmission at 250.58 GHz. DNP Signal enhancement of >125 is achieved on a [superscript 13]C-Urea sample using this gyrotron., National Institutes of Health (U.S.) (Grant EB002804), National Institutes of Health (U.S.) (Grant EB003151), National Institutes of Health (U.S.) (Grant EB002026), National Institutes of Health (U.S.) (Grant EB001960), National Institutes of Health (U.S.) (Grant EB001035), National Institutes of Health (U.S.) (Grant EB001965), National Institutes of Health (U.S.) (Grant EB004866)

We employ a combination of (13)C/(15)N magic angle spinning (MAS) NMR and (2)H NMR to study the structural and functional consequences of different membrane environments on VDAC1 and, conversely, the effect of VDAC1 on the structure of the lipid bilayer. MAS spectra reveal a well-structured VDAC1 in 2D crystals of dimyristoylphosphatidylcholine (DMPC) and diphytanoylphosphatidylcholine (DPhPC), and their temperature dependence suggests that the VDAC structure does not change conformation above and below the lipid phase transition temperature. The same data show that the N-terminus remains structured at both low and high temperatures. Importantly, functional studies based on electrophysiological measurements on these same samples show fully functional channels, even without the presence of Triton X-100 that has been found necessary for in vitro-refolded channels. (2)H solid-state NMR and differential scanning calorimetry were used to investigate the dynamics and phase behavior of the lipids within the VDAC1 2D crystals. (2)H NMR spectra indicate that the presence of protein in DMPC results in a broad lipid phase transition that is shifted from 19 to ~27 °C and show the existence of different lipid populations, consistent with the presence of both annular and bulk lipids in the functionally and structurally homogeneous samples.