Using a set of six 1H-detected triple-resonance NMR experiments, we establish a method for sequence-specific backbone resonance assignment of magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectra of 5–30 kDa proteins. The approach relies on perdeuteration, amide 2H/1H exchange, high magnetic fields, and high-spinning frequencies (ωr/2π ≥ 60 kHz) and yields high-quality NMR data, enabling the use of automated analysis. The method is validated with five examples of proteins in different condensed states, including two microcrystalline proteins, a sedimented virus capsid, and two membrane-embedded systems. In comparison to contemporary 13C/15N-based methods, this approach facilitates and accelerates the MAS NMR assignment process, shortening the spectral acquisition times and enabling the use of unsupervised state-of-the-art computational data analysis protocols originally developed for solution NMR.

We introduce a new approach to improve structural and dynamical determination of large metalloproteins using solid-state nuclear magnetic resonance (NMR) with 1 H detection under ultrafast magic angle spinning (MAS). The approach is based on the rapid and sensitive acquisition of an extensive set of 15 N and 13 C nuclear relaxation rates. The system on which we demonstrate these methods is the enzyme Cu, Zn superoxide dismutase (SOD), which coordinates a Cu ion available either in Cu þ (diamagnetic) or Cu 2þ (paramagnetic) form. Paramagnetic relaxation enhancements are obtained from the difference in rates measured in the two forms and are employed as structural constraints for the determination of the protein structure. When added to 1 H-1 H distance restraints, they are shown to yield a twofold improvement of the precision of the structure. Site-specific order parameters and timescales of motion are obtained by a Gaussian axial fluctuation (GAF) analysis of the relaxation rates of the diamagnetic molecule, and interpreted in relation to backbone structure and metal binding. Timescales for motion are found to be in the range of the overall correlation time in solution, where internal motions characterized here would not be observable.paramagnetism | nuclear relaxation rates | copper | microcrystal S tructure determination of proteins plays a central role in understanding key events in biology. Although the structure of many proteins can be obtained from single-crystal X-ray diffraction, or by solution-state nuclear magnetic resonance (NMR) spectroscopy, there is nevertheless a range of important substrates for which structures cannot be determined today. These include immobile systems lacking long-range order such as protein aggregates, large complexes and membrane-bound systems.Solid-state NMR has the unique potential to study, with atomic resolution, systems of this nature, and spectacular progress has been made in this area over the last decade (1). There are today a small handful of structures obtained by solid-state NMR, from microcrystalline samples to fibrils and membrane-associated systems (2). Additionally, solid-state NMR is uniquely sensitive to site-specific protein dynamics over a broad range of timescales, and a number of demonstration studies have recently appeared for model proteins (3).The use of perdeuterated proteins has very recently opened the way to highly sensitive proton-detected solid-state experiments (4). Despite early proof-of-principle papers (5), this approach only became popular with the realization that amide sites must be only partially reprotonated (typically 10-30% back exchange) (6-8) to yield well-resolved 1 H spectra. This represented a significant compromise in sensitivity to gain resolution, and effectively made the determination of internuclear distances impractical, with few exceptions (9-11). We have recently shown how this problem can be completely overcome by using 100% reprotonation of exchangeable sites, without loss of resolution, if perdeuteration is combined wit...

Re‐protonation is key: A combination of a high magnetic field (1 GHz) and ultra‐fast magic‐angle spinning (60 kHz) allows easy detection of NMR spectra revealing details of secondary and tertiary structures of medium‐sized proteins. The technique was applied to the 153‐residue microcrystalline ZnII‐loaded superoxide dismutase (ZnII‐SOD) fully [2H,13C,15N]‐labeled and 100 % re‐protonated at the exchangeable sites.

Isolated pancreatic islets were transplanted isogenoieally into the subcutaneus tissue, peritoneum and portal vein of diabetic rats. implantation of islets subcutaneously did not modify the diabetic state. Intraperitoneal islets ameliorated the effects of diabetes but normal urine vohmaes, blood glucose and urine glucose Ievels were not achieved. Direct injection of islets into the portal vein resulted in normal urine volumes, normo-glycemia and abolition of glyeosuria in the rats studied. These effects have been maintained 2 months later.-It is suggested that the portal environment may be the most effective site for transplanted pancreatic islets.

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Narrow 1H NMR linewidths can be obtained for fully protonated protein samples in the solid state by using ultrafast magic‐angle spinning (60 kHz). Medium‐size microcrystalline and noncrystalline proteins can be analyzed without any need for deuteration of the protein sample. This approach provides assignments of the backbone 1H, 15N, 13Cα, and 13CO resonances and yields information about 1H–1H proximities.

Metal ions are ubiquitous in biochemical and cellular processes. Since many metal ions are paramagnetic due to the presence of unpaired electrons, paramagnetic molecules are an important class of targets for research in structural biology and related fields. Today, NMR spectroscopy plays a central role in the investigation of the structure and chemical properties of paramagnetic metalloproteins, linking the observed paramagnetic phenomena directly to electronic and molecular structure. A major step forward in the study of proteins by solid-state NMR came with the advent of ultrafast magic angle spinning (MAS) and the ability to use (1)H detection. Combined, these techniques have allowed investigators to observe nuclei that previously were invisible in highly paramagnetic metalloproteins. In addition, these techniques have enabled quantitative site-specific measurement of a variety of long-range paramagnetic effects. Instead of limiting solid-state NMR studies of biological systems, paramagnetism provides an information-rich phenomenon that can be exploited in these studies. This Account emphasizes state-of-the-art methods and applications of solid-state NMR in paramagnetic systems in biological chemistry. In particular, we discuss the use of ultrafast MAS and (1)H-detection in perdeuterated paramagnetic metalloproteins. Current methodology allows us to determine the structure and dynamics of metalloenzymes, and, as an example, we describe solid-state NMR studies of microcrystalline superoxide dismutase, a 32 kDa dimer. Data were acquired with remarkably short times, and these experiments required only a few milligrams of sample.

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