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 [superscript 15]N–[superscript 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 [superscript 15]N– [superscript 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.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-002926)National Institutes of Health. National Institute for Biomedical Imaging and Bioengineering (Grant EB-001035

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 [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

Action Spectroscopy on Dense Samples of Photosynthetic Reaction Centers of Rhodobacter sphaeroides WT Based on Nanosecond Laser-Flash 13C 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 13C 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

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) 13C 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

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.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

Dynamic Nuclear Polarization of Oxygen-17

Oxygen-17-detected DNP NMR of a water/glycerol glass enabled an 80-fold enhancement of signal intensity at 82 K, using the biradical TOTAPOL. The >6000-fold savings in acquisition time enable [superscript 17]O–[superscript 1]H distance measurements and heteronuclear correlation experiments. These experiments are the initial demonstration of the feasibility of DNP NMR on quadrupolar [superscript 17]O.National Institute for Biomedical Imaging and Bioengineering (U.S.) (Award EB-001960)National Institute for Biomedical Imaging and Bioengineering (U.S.) (Award EB-002804)National Institute for Biomedical Imaging and Bioengineering (U.S.) (Award EB-001035)National Institute for Biomedical Imaging and Bioengineering (U.S.) (Award EB-002026

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, PL and PM, 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, PL is strongly disturbed, carrying a slightly negative charge. In the radical cation state, the ratio of total electron spin densities between PL and PM 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 PL. 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

Dynamic Nuclear Polarization of Oxygen-17

Oxygen-17-detected DNP NMR of a water/glycerol glass enabled an 80-fold enhancement of signal intensity at 82 K, using the biradical TOTAPOL. The >6000-fold savings in acquisition time enable ¹⁷O–¹H distance measurements and heteronuclear correlation experiments. These experiments are the initial demonstration of the feasibility of DNP NMR on quadrupolar ¹⁷O

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 M_o 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