13C CPMAS spectroscopy of deuterated proteins: CP dynamics, line shapes, and T1 relaxation

(13)C CPMAS NMR has been investigated in application to protein samples with a variety of deuteration patterns. Samples were prepared with protons in either all hydrogen positions, only in the exchangeable sites, or in the exchangeable sites plus select methyl groups. CP dynamics, T(1) relaxation times, and (13)C line widths have been compared. Using ubiquitin as a model system, reasonable (1)H-(13)C CP transfer is observed for the extensively deuterated samples. In the absence of deuterium decoupling, the (13)C line widths observed for the deuterated samples are identical to those observed for the perprotio samples with a MAS rate of 20 kHz. Extensive deuteration has little effect on the T(1) of the exchangeable protons. On the basis of these observations, it is clear that there are no substantive compromises accompanying the use of extensive deuteration in the design of (1)H, (15)N, or (13)C solid-state NMR methods.     

Assignments of carbon NMR resonances for microcrystalline ubiquitin

Solid-state NMR 2D spectroscopy was used to correlate carbon backbone and side-chain chemical shifts for uniformly (13)C,(15)N-enriched microcrystalline ubiquitin. High applied field strengths, 800 MHz for protons, moderate proton decoupling fields, 80-100 kHz, and high magic angle sample spinning frequencies, 20 kHz, were used to narrow the most of the carbon line widths to 0.5-0.8 ppm. Homonuclear magnetization transfer was effected by matching the proton RF field to the spinning frequency, the so-called dipolar-assisted rotational resonance (DARR) (Takegoshi, K.; Nakamura, S.; Terao, T. Chem. Phys. Lett. 2001, 344, 631-637), and a mixing time of 20 ms was used to maximize the intensity of one-bond transfers between carbon atoms. This polarization transfer sequence resulted in roughly 14% transfer efficiencies for directly bonded carbon pairs and 4% transfer efficiencies for carbons separated by a third carbon. With this simple procedure, the majority of the one-bond correlations was observed with moderate transfer efficiencies, and many two-bond correlations were also observed with weaker intensities. Spin systems could be identified for more than half of the amino acid side chains, and site-specific assignments were readily possible via comparison with 400 MHz (15)N-(13)C-(13)C correlation spectroscopy (described separately).     

Sensitive high resolution inverse detection NMR spectroscopy of proteins in the solid state

A new indirect detection scheme for obtaining (15)N/(1)H shift correlation spectra in crystalline proteins is described. Excellent water suppression is achieved without the need for pulsed field gradients, and using only a 2-step phase cycle. Careful attention to overall NMR instrument stability was found critical for obtaining the best resolution and sensitivity. Magnetic dilution by deuteration of the protein in combination with high-speed magic angle spinning produces (1)H resonances averaging only 0.22 ppm in width, and in some cases lines as narrow as 0.17 ppm are obtained. In application to two different polymorphs of ubiquitin, structure dependent differences in both (15)N and (1)H amide chemical shifts are observed. In one case, distinct shifts for different molecules in the asymmetric unit are seen, and all differ substantially from solution NMR shifts. A gain of 7 in sensitivity makes the method competitive with solution NMR as long as nanocrystalline samples are available.     

Variable temperature system using vortex tube cooling and fiber optic temperature measurement for low temperature magic angle spinning NMR

We describe the construction and operation of a variable temperature (VT) system for a high field fast magic angle spinning (MAS) probe. The probe is used in NMR investigations of biological macromolecules, where stable setting and continuous measurement of the temperature over periods of several days are required in order to prevent sample overheating and degradation. The VT system described is used at and below room temperature. A vortex tube is used to provide cooling in the temperature range of -20 to 20 degrees C, while a liquid nitrogen-cooled heat exchanger is used below -20 degrees C. Using this arrangement, the lowest temperature that is practically achievable is -140 degrees C. Measurement of the air temperature near the spinning rotor is accomplished using a fiber optic thermometer that utilizes the temperature dependence of the absorption edge of GaAs. The absorption edge of GaAs also has a magnetic field dependence that we have measured and corrected for. This dependence was calibrated at several field strengths using the well-known temperature dependence of the (1)H chemical shift difference of the protons in methanol.     

19F NMR chemical shifts. 1. Aliphatic fluorides

The (19)F NMR shielding for the alkyl fluorides from methyl fluoride to tert-butyl fluoride has been calculated using IGAIM and has been separated into the contribution from each of the molecular orbitals. The relatively large change in fluorine shielding, in contrast to the adjacent carbon, was found to be due to the tensor components normal to the C-F bond axis. As the number of adjacent p-orbitals increases, the lone-pair p orbitals at fluorine become involved with MOs using these orbitals. The increase in the number of occupied orbitals associated with the fluorine leads to increased opportunities for mixing with virtual orbitals and to the increase in paramagnetic deshielding. The same pattern is seen on going from acetylene to 2-tert-butylacetylene and is also seen in the methyl (13)C shielding in the series ethane, propane, isobutane, and neopentane. The fluorine shielding in the series of fluoromethanes also decreases with increasing fluorine substitution. With carbon tetrafluoride, the decreased shielding arises from the highest occupied MO, which is a nonbonding linear combination of pure p functions at the fluorines.     

Improvement of spectral editing in solids: A sequence for obtaining (13)CH + (13)CH(2)-only (13)C spectra

An improved spectral editing method for solids is described which allows one to obtain a set of subspectra in roughly two-thirds the amount of time as our original CPPI editing method for the same signal to noise. This improvement is afforded by a new pulse sequence that is used to acquire a (13)CH + (13)CH(2) spectrum which has very little (13)CH(3) or nonprotonated carbon contamination. By using this new sequence the (13)CH-only subspectrum is obtained much more efficiently. Criteria for optimizing the signal to noise in the edited subspectra are discussed. Copyright 2000 Academic Press.     

Further conventions for NMR shielding and chemical shifts (IUPAC Recommendations 2008)

IUPAC has published a number of recommendations regarding the reporting of nuclear magnetic resonance (NMR) data, especially chemical shifts. The most recent publication [Pure Appl. Chem. 73, 1795 (2001)] recommended that tetramethylsilane (TMS) serve as a universal reference for reporting the shifts of all nuclides, but it deferred recommendations for several aspects of this subject. This document first examines the extent to which the (1)H shielding in TMS itself is subject to change by variation in temperature, concentration, and solvent. On the basis of recently published results, it has been established that the shielding of TMS in solution [along with that of sodium-3-(trimethylsilyl)propanesulfonate, DSS, often used as a reference for aqueous solutions] varies only slightly with temperature but is subject to solvent perturbations of a few tenths of a part per million (ppm). Recommendations are given for reporting chemical shifts under most routine experimental conditions and for quantifying effects of temperature and solvent variation, including the use of magnetic susceptibility corrections and of magic-angle spinning (MAS). This document provides the first IUPAC recommendations for referencing and reporting chemical shifts in solids, based on high-resolution MAS studies. Procedures are given for relating (13)C NMR chemical shifts in solids to the scales used for high-resolution studies in the liquid phase. The notation and terminology used for describing chemical shift and shielding tensors in solids are reviewed in some detail, and recommendations are given for best practice.     

High-sensitivity observation of dipolar exchange and NOEs between exchangeable protons in proteins by 3D solid-state NMR spectroscopy

A highly sensitive new 1H-detected 3D solid-state NMR method is described for characterizing 1H-1H spin exchange in nanocrystalline samples of 15N- and 2H-enriched protein. Long-range contacts are observed in human ubiquitin. The method is also used to show that numerous NOEs between backbone amides and crystal water protons can be observed.     

Diluting abundant spins by isotope edited radio frequency field assisted diffusion

A new solid-state NMR method is described for obtaining long-range distance constraints in nanocrystalline samples of 13C-, 15N-, and 2H-enriched protein. The method selects only those 13C or 15N nuclei close to 1Hs for dipolar recoupling. When used with extensive deuteration, the bath of abundant 13C spins is made to appear dilute. Contacts over 4.5 A are readily observed in human ubiquitin.     

Preparation of protein nanocrystals and their characterization by solid state NMR

Preparation of proteins in their crystalline state has been found to be important in producing stable therapeutic protein formulations, cross-linked enzyme crystals for application in industrial processes, generating novel porous media for separations, and of course in structure elucidation. Of these applications only X-ray crystallography requires large crystals, defined here as being crystals 100s of microns or greater in size. Smaller crystals have attractive attributes in many instances, and are just as useful in structure determination by solid state NMR (ssNMR) as are large crystals. In this paper we outline a simple set of procedures for preparing nanocrystalline protein samples for ssNMR or other applications and describe the characterization of their crystallinity by ssNMR and X-ray powder diffraction. The approach is demonstrated in application to five different proteins: ubiquitin, lysozyme, ribonuclease A, streptavidin, and cytochrome c. In all instances the nanocrystals produced are found to be highly crystalline as judged by natural abundance 13C ssNMR and optical and electron microscopy. We show for ubiquitin that nanocrystals prepared by rapid batch crystallization yield equivalent 13C ssNMR spectra to those of larger X-ray diffraction quality crystals. Single crystal and powder X-ray diffraction measurements are made to compare the degree of order present in polycrystalline, nanocrystalline, and lyophilized ubiquitin. Solid state 13C NMR is also used to show that ubiquitin nanocrystals are thermally robust, giving no indication of loss of local order after repeated temperature cycling between liquid nitrogen and room temperature. The methods developed are rapid and should scale well from the tenths of milligram to multi-gram scales, and as such should find wide utility in the preparation of protein nanocrystals for applications in catalysis, separations, and especially in sample preparation for structural studies using ssNMR.