Primary Transfer Step in the Light-Driven Ion Pump Bacteriorhodopsin: An Irreversible U-Turn Revealed by Dynamic Nuclear Polarization-Enhanced Magic Angle Spinning NMR.

Despite much attention, the path of the highly consequential primary proton transfer in the light-driven ion pump bacteriorhodopsin (bR) remains mysterious. Here we use DNP-enhanced magic angle spinning (MAS) NMR to study critical elements of the active site just before the Schiff base (SB) deprotonates (in the L intermediate), immediately after the SB has deprotonated and Asp85 has become protonated (in the Mo intermediate), and just after the SB has reprotonated and Asp96 has deprotonated (in the N intermediate). An essential feature that made these experiments possible is the 75-fold signal enhancement through DNP. 15N(SB)-1H correlations reveal that the newly deprotonated SB is accepting a hydrogen bond from an alcohol and 13C-13C correlations show that Asp85 draws close to Thr89 before the primary proton transfer. Concurrently, 15N-13C correlations between the SB and Asp85 show that helices C and G draw closer together just prior to the proton transfer and relax thereafter. Together, these results indicate that Thr89 serves to relay the SB proton to Asp85 and that creating this pathway involves rapprochement between the C and G helices as well as chromophore torsion.

High frequency dynamic nuclear polarization.

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.

An amyloid organelle, solid-state NMR evidence for cross-ß assembly of gas vesicles.

Functional amyloids have been identified in a wide range of organisms, taking on a variety of biological roles and being controlled by remarkable mechanisms of directed assembly. Here, we report that amyloid fibrils constitute the ribs of the buoyancy organelles of Anabaena flos-aquae. The walls of these gas-filled vesicles are known to comprise a single protein, GvpA, arranged in a low pitch helix. However, the tertiary and quaternary structures have been elusive. Using solid-state NMR correlation spectroscopy we find detailed evidence for an extended cross-ß structure. This amyloid assembly helps to account for the strength and amphiphilic properties of the vesicle wall. Buoyancy organelles thus dramatically extend the scope of known functional amyloids.

Efficient resonance assignment of proteins in MAS NMR by simultaneous intra- and inter-residue 3D correlation spectroscopy.

Resonance assignment is the first step in NMR structure determination. For magic angle spinning NMR, this is typically achieved with a set of heteronuclear correlation experiments (NCaCX, NCOCX, CONCa) that utilize SPECIFIC-CP (15)N-(13)C transfers. However, the SPECIFIC-CP transfer efficiency is often compromised by molecular dynamics and probe performance. Here we show that one-bond ZF-TEDOR (15)N-(13)C transfers provide simultaneous NCO and NCa correlations with at least as much sensitivity as SPECIFIC-CP for some non-crystalline samples. Furthermore, a 3D ZF-TEDOR-CC experiment provides heteronuclear sidechain correlations and robustness with respect to proton decoupling and radiofrequency power instabilities. We demonstrate transfer efficiencies and connectivities by application of 3D ZF-TEDOR-DARR to a model microcrystalline protein, GB1, and a less ideal system, GvpA in intact gas vesicles.

A High-Throughput Workflow for FFPE Tissue Proteomics.

Laser capture microdissection (LCM) has become an indispensable tool for mass spectrometry-based proteomic analysis of specific regions obtained from formalin-fixed paraffin-embedded (FFPE) tissue samples in both clinical and research settings. Low protein yields from LCM samples along with laborious sample processing steps present challenges for proteomic analysis without sacrificing protein and peptide recovery. Automation of sample preparation workflows is still under development, especially for samples such as laser-capture microdissected tissues. Here, we present a simplified and rapid workflow using adaptive focused acoustics (AFA) technology for sample processing for high-throughput FFPE-based proteomics. We evaluated three different workflows: standard extraction method followed by overnight trypsin digestion, AFA-assisted extraction and overnight trypsin digestion, and AFA-assisted extraction simultaneously performed with trypsin digestion. The use of AFA-based ultrasonication enables automated sample processing for high-throughput proteomic analysis of LCM-FFPE tissues in 96-well and 384-well formats. Further, accelerated trypsin digestion combined with AFA dramatically reduced the overall processing times. LC-MS/MS analysis revealed a slightly higher number of protein and peptide identifications in AFA accelerated workflows compared to standard and AFA overnight workflows. Further, we did not observe any difference in the proportion of peptides identified with missed cleavages or deamidated peptides across the three different workflows. Overall, our results demonstrate that the workflow described in this study enables rapid and high-throughput sample processing with greatly reduced sample handling, which is amenable to automation.

Whole cell nuclear magnetic resonance characterization of two photochemically active states of the photosynthetic reaction center in heliobacteria.

Applying photo-CIDNP (photochemically induced dynamic nuclear polarization) MAS (magic-angle spinning) nuclear magnetic resonance to whole cells of Heliobacillus (Hb.) mobilis, we demonstrate that heliobacterial reaction centers are operational in two different states as indicated by the occurrence of a light-induced spin-correlated radical pair. A culture maintained anaerobically is called "Braunstoff" (German for "brown substance"). After exposure to oxygen, Braunstoff is converted to "Grünstoff" ("green substance") as indicated by a color change due to the conversion of BChl g to Chl a(F). It is shown that electron transfer occurs symmetrically via both branches of cofactors in both forms. The donor and acceptor cofactors remain identical and unchanged upon conversion, while the intermediate accessory cofactors are transformed from BChl g to Chl a(F). The donor triplet state in Braunstoff is localized on the special pair donor and lives for 100 µs, demonstrating the absence of nearby carotenoids. In Grünstoff, the donor triplet becomes mobile and appears to be formed on an accessory cofactor.

The electronic structure of the primary electron donor of reaction centers of purple bacteria at atomic resolution as observed by photo-CIDNP 13C NMR.

Composed of the two bacteriochlorophyll cofactors, P(L) and P(M), the special pair functions as the primary electron donor in bacterial reaction centers of purple bacteria of Rhodobacter sphaeroides. Under light absorption, an electron is transferred to a bacteriopheophytin and a radical pair is produced. The occurrence of the radical pair is linked to the production of enhanced nuclear polarization called photochemically induced dynamic nuclear polarization (photo-CIDNP). This effect can be used to study the electronic structure of the special pair at atomic resolution by detection of the strongly enhanced nuclear polarization with laser-flash photo-CIDNP magic-angle spinning NMR on the carotenoid-less mutant R26. In the electronic ground state, P(L) is strongly disturbed, carrying a slightly negative charge. In the radical cation state, the ratio of total electron spin densities between P(L) and P(M) is 2:1, although it is 2.5:1 for the pyrrole carbons, 2.2:1 for all porphyrinic carbons, and 4:1 for the pyrrole nitrogen. It is shown that the symmetry break between the electronic structures in the electronic ground state and in the radical cation state is an intrinsic property of the special pair supermolecule, which is particularly attributable to a modification of the structure of P(L). The significant difference in electron density distribution between the ground and radical cation states is explained by an electric polarization effect of the nearby histidine.

Biradical Polarizing Agents at High Fields.

The sensitivity enhancements available from dynamic nuclear polarization (DNP) are rapidly reshaping the research landscape and expanding the field of nuclear magnetic resonance (NMR) spectroscopy as a tool for solving complex chemical and structural problems. The past decade has seen considerable advances in this burgeoning method, while efforts to further improve its capabilities continue along many avenues. In this report, we examine the influence of static magnetic field strength and temperature on the reported 1H DNP enhancements from three conventional organic biradicals: TOTAPOL, AMUPol, and SPIROPOL. In contrast to the conventional wisdom, our findings show that at liquid nitrogen temperatures and 700 MHz/460.5 GHz, these three bisnitroxides all provide similar 1H DNP enhancements, ε ≈ 60. Furthermore, we investigate the influence of temperature, microwave power, magnetic field strength, and protein sample deuteration on the NMR experimental results.

A Novel Dry-Stabilized Whole Blood Microsampling and Protein Extraction Method for Testing of SARS-CoV-2 Antibody Titers.

The COVID-19 pandemic has revealed a crucial need for rapid, straightforward collection and testing of biological samples. Serological antibody assays can analyze patient blood samples to confirm immune response following mRNA vaccine administration or to verify past exposure to the SARS-CoV-2 virus. While blood tests provide vital information for clinical analysis and epidemiology, sample collection is not trivial; this process requires a visit to the doctor's office, a professionally trained phlebotomist to draw several milliliters of blood, processing to yield plasma or serum, and necessitates appropriate cold chain storage to preserve the specimen. A novel whole blood collection kit (truCOLLECT) allows for a lancet-based, decentralized capillary blood collection of metered low volumes and eliminates the need for refrigerated transport and storage through the process of active desiccation. Anti-SARS-CoV-2 spike (total and neutralizing) and nucleocapsid protein antibody titers in plasma samples obtained via venipuncture were compared to antibodies extracted from desiccated whole blood using Adaptive Focused Acoustics (AFA). Paired plasma versus desiccated blood extracts yields Pearson correlation coefficients of 0.98; 95% CI [0.96, 0.99] for anti-SARS-CoV-2 spike protein antibodies, 0.97; 95% CI [0.95, 0.99] for neutralizing antibodies, and 0.97; 95% CI [0.94, 0.99] for anti-SARS-CoV-2 nucleocapsid protein antibodies. These data suggest that serology testing using desiccated and stabilized whole blood samples can be a convenient and cost-effective alternative to phlebotomy.

A 10,000-fold nuclear hyperpolarization of a membrane protein in the liquid phase via a solid-state mechanism.

Several techniques rely on electron-nuclear interactions to boost the polarization of nuclear spins in the solid phase. Averaging out of anisotropic interactions as a result of molecular tumbling strongly reduces the applicability of such hyperpolarization approaches in liquids. Here we show for the first time that anisotropic electron-nuclear interactions in solution can survive sufficiently long to generate nuclear spin polarization by the solid-state photo-CIDNP mechanism. A 10,000-fold NMR signal increase in solution was observed for a giant biomolecular complex of a photosynthetic membrane protein with a tumbling correlation time in the submicrosecond regime, corresponding to a molecular weight close to 1 MDa.

Observation of the solid-state photo-CIDNP effect in entire cells of cyanobacteria Synechocystis.

Cyanobacteria are widely used as model organism of oxygenic photosynthesis due to being the simplest photosynthetic organisms containing both photosystem I and II (PSI and PSII). Photochemically induced dynamic nuclear polarization (photo-CIDNP) (13)C magic-angle spinning (MAS) NMR is a powerful tool in understanding the photosynthesis machinery down to atomic level. Combined with selective isotope enrichment this technique has now opened the door to study primary charge separation in whole living cells. Here, we present the first photo-CIDNP observed in whole cells of the cyanobacterium Synechocystis.

Action Spectroscopy on Dense Samples of Photosynthetic Reaction Centers of Rhodobacter sphaeroides WT Based on Nanosecond Laser-Flash C Photo-CIDNP MAS NMR.

Photochemically induced dynamic nuclear polarization magic-angle spinning nuclear magnetic resonance (photo-CIDNP MAS NMR) allows for the investigation of the electronic structure of the photochemical machinery of photosynthetic reaction centers (RCs) at atomic resolution. For such experiments, either continuous radiation from white xenon lamps or green laser pulses are applied to optically dense samples. In order to explore their optical properties, optically thick samples of isolated and quinone-removed RCs of the purple bacteria of Rhodobacter sphaeroides wild type are studied by nanosecond laser-flash (13)C photo-CIDNP MAS NMR using excitation wavelengths between 720 and 940 nm. Action spectra of both the transient nuclear polarization as well as the nuclear hyperpolarization, remaining in the electronic ground state at the end of the photocycle, are obtained. It is shown that the signal intensity is limited by the amount of accessible RCs and that the different mechanisms of the photo-CIDNP production rely on the same photophysical origin, which is the photocycle induced by one single photon.

Photo-CIDNP MAS NMR beyond the T1 limit by fast cycles of polarization extinction and polarization generation.

In nanosecond-laser flash photo-CIDNP MAS NMR, polarization generation (PG) proceeds much faster than longitudinal spin relaxation. With a nanosecond-laser setup linked to the NMR console the repetition time of the experiment is then limited by the minimum recycle delay of the NMR spectrometer and the maximum repetition rate of laser flashes. These limits can only be reached if polarization left after the NMR experiment is completely canceled before the next laser flash. We introduce a presaturation pulse sequence, based on three (pi/2) (13)C pulses and optimized timing and phase cycling that allows for such efficient polarization extinction (PE). The technique is demonstrated on selectively isotope labeled bacterial reaction centers (RCs) of Rhodobacter (Rb.) sphaeroides wildtype (WT). High-quality (13)C photo-CIDNP MAS NMR spectra are obtained using cycle rates up to 4 Hz. The PE-PG strategy proposed here provides a general experimental scheme for reduction of measurement time in magnetic resonance experiments based on fast PG.

Peptide and Protein Dynamics and Low-Temperature/DNP Magic Angle Spinning NMR.

In DNP MAS NMR experiments at ∼80-110 K, the structurally important -13CH3 and -15NH3+ signals in MAS spectra of biological samples disappear due to the interference of the molecular motions with the 1H decoupling. Here we investigate the effect of these dynamic processes on the NMR line shapes and signal intensities in several typical systems: (1) microcrystalline APG, (2) membrane protein bR, (3) amyloid fibrils PI3-SH3, (4) monomeric alanine-CD3, and (5) the protonated and deuterated dipeptide N-Ac-VL over 78-300 K. In APG, the three-site hopping of the Ala-Cß peak disappears completely at 112 K, concomitant with the attenuation of CP signals from other 13C's and 15N's. Similarly, the 15N signal from Ala-NH3+ disappears at ∼173 K, concurrent with the attenuation in CP experiments of other 15N's as well as 13C's. In bR and PI3-SH3, the methyl groups are attenuated at ∼95 K, while all other 13C's remain unaffected. However, both systems exhibit substantial losses of intensity at ∼243 K. Finally, with spectra of Ala and N-Ac-VL, we show that it is possible to extract site specific dynamic data from the temperature dependence of the intensity losses. Furthermore, 2H labeling can assist with recovering the spectral intensity. Thus, our study provides insight into the dynamic behavior of biological systems over a wide range of temperatures, and serves as a guide to optimizing the sensitivity and resolution of structural data in low temperature DNP MAS NMR spectra.

Spectral editing through laser-flash excitation in two-dimensional photo-CIDNP MAS NMR experiments.

In solid-state photochemically induced dynamic nuclear polarization (photo-CIDNP) MAS NMR experiments, strong signal enhancement is observed from molecules forming a spin-correlated radical pair in a rigid matrix. Two-dimensional (13)C-(13)C dipolar-assisted rotational resonance (DARR) photo-CIDNP MAS NMR experiments have been applied to obtain exact chemical shift assignments from those cofactors. Under continuous illumination, the signals are enhanced via three-spin mixing (TSM) and differential decay (DD) and their intensity corresponds to the electron spin density in pz orbitals. In multiple-(13)C labelled samples, spin diffusion leads to propagation of signal enhancement to all (13)C spins. Under steady-state conditions, direct signal assignment is possible due to the uniform signal intensity. The original intensities, however, are inaccessible and the information of the local electron spin density is lost. Upon laser-flash illumination, the signal is enhanced via the classical radical pair mechanism (RPM). The obtained intensities are related to isotropic hyperfine interactions aiso and both enhanced absorptive and emissive lines can be observed due to differences in the sign of the local isotropic hyperfine interaction. Exploiting the mechanism of the polarization, selectivity can be increased by the novel time-resolved two-dimensional dipolar-assisted rotational resonance (DARR) MAS NMR experiment which simplifies the signal assignment compared to complex spectra of the same RCs obtained by continuous illumination. Here we present two-dimensional time-resolved photo-CIDNP MAS NMR experiments providing both directly: signal assignment and spectral editing by sign and strength of aiso. Hence, this experiment provides a direct key to the electronic structure of the correlated radical pair.

Gas vesicles across kingdoms: a comparative solid-state nuclear magnetic resonance study.

The buoyancy organelles of aquatic microorganisms have to meet stringent specifications: allowing gases to equilibrate freely across the proteinaceous shell, preventing the condensation of water vapor inside the hollow cavity and resisting collapse under hydrostatic pressures that vary with column depth. These properties are provided by the 7- to 8-kDa gas vesicle protein A (GvpA), repeats of which form all but small, specialized portions of the shell. Magic angle spinning nuclear magnetic resonance is uniquely capable of providing high-resolution information on the fold and assembly of GvpA. Here we compare results for the gas vesicles of the haloarchaea Halobacterium salinarum with those obtained previously for the cyanobacterium Anabaena flos-aquae. The data suggest that the two organisms follow similar strategies for avoiding water condensation. On the other hand, in its relatively shallow habitat, H. salinarum is able to avoid collapse with a less costly GvpA fold than is adopted by A. flos-aquae.

Continuously Tunable 250 GHz Gyrotron with a Double Disk Window for DNP-NMR Spectroscopy.

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 13C-Urea sample using this gyrotron.

Dynamic Nuclear Polarization of Oxygen-17.

Oxygen-17 detected DNP NMR of a water/glycerol glass enabled an 80-fold enhancement of signal intensities at 82 K, using the biradical TOTAPOL. The >6,000-fold savings in acquisition time enables (17)O-(1)H distance measurements and heteronuclear correlation experiments. These experiments are the initial demonstration of the feasibility of DNP NMR on quadrupolar (17)O.

Dynamic nuclear polarization at 700 MHz/460 GHz.

We describe the design and implementation of the instrumentation required to perform DNP-NMR at higher field strengths than previously demonstrated, and report the first magic-angle spinning (MAS) DNP-NMR experiments performed at (1)H/e(-) frequencies of 700 MHz/460 GHz. The extension of DNP-NMR to 16.4 T has required the development of probe technology, cryogenics, gyrotrons, and microwave transmission lines. The probe contains a 460 GHz microwave channel, with corrugated waveguide, tapers, and miter-bends that couple microwave power to the sample. Experimental efficiency is increased by a cryogenic exchange system for 3.2 mm rotors within the 89 mm bore. Sample temperatures ≤85 K, resulting in improved DNP enhancements, are achieved by a novel heat exchanger design, stainless steel and brass vacuum jacketed transfer lines, and a bronze probe dewar. In addition, the heat exchanger is preceded with a nitrogen drying and generation system in series with a pre-cooling refrigerator. This reduces liquid nitrogen usage from >700 l per day to <200 l per day and allows for continuous (>7 days) cryogenic spinning without detrimental frost or ice formation. Initial enhancements, ε=-40, and a strong microwave power dependence suggests the possibility for considerable improvement. Finally, two-dimensional spectra of a model system demonstrate that the higher field provides excellent resolution, even in a glassy, cryoprotecting matrix.

Molecular structure of humin and melanoidin via solid state NMR.

Sugar-derived humins and melanoidins figure significantly in food chemistry, agricultural chemistry, biochemistry, and prebiotic chemistry. Despite wide interest and significant experimental attention, the amorphous and insoluble nature of the polymers has made them resistant to conventional structural characterization. Here we make use of solid-state NMR methods, including selective (13)C substitution, (1)H-dephasing, and double quantum filtration. The spectra, and their interpretation, are simplified by relying exclusively on hydronium for catalysis. The results for polymers derived from ribose, deoxyribose, and fructose indicate diverse pathways to furans, suggest a simple route to pyrroles in the presence of amines, and reveal a heterogeneous network-type polymer in which sugar molecules cross-link the heterocycles.