A novel strategy for NMR resonance assignment and protein structure determination.

The quality of protein structures determined by nuclear magnetic resonance (NMR) spectroscopy is contingent on the number and quality of experimentally-derived resonance assignments, distance and angular restraints. Two key features of protein NMR data have posed challenges for the routine and automated structure determination of small to medium sized proteins; (1) spectral resolution - especially of crowded nuclear Overhauser effect spectroscopy (NOESY) spectra, and (2) the reliance on a continuous network of weak scalar couplings as part of most common assignment protocols. In order to facilitate NMR structure determination, we developed a semi-automated strategy that utilizes non-uniform sampling (NUS) and multidimensional decomposition (MDD) for optimal data collection and processing of selected, high resolution multidimensional NMR experiments, combined it with an ABACUS protocol for sequential and side chain resonance assignments, and streamlined this procedure to execute structure and refinement calculations in CYANA and CNS, respectively. Two graphical user interfaces (GUIs) were developed to facilitate efficient analysis and compilation of the data and to guide automated structure determination. This integrated method was implemented and refined on over 30 high quality structures of proteins ranging from 5.5 to 16.5 kDa in size.

Membrane protein preparation for TROSY NMR screening.

The first steps toward undertaking an NMR structural study of a new protein is very often to purify the protein and then to acquire an HSQC or TROSY NMR spectrum, the quality of which is used to assess the feasibility of an NMR-based structural determination. Relatively few integral membrane proteins (IMPs) have been subjected even to this very preliminary stage of NMR analysis. Here, NMR feasibility testing methods are outlined that are tailored for hexahistidine-tagged IMPs that have been expressed in Escherichia coli. Generally applicable protocols are presented for expression testing, purification, and NMR sample preparation. A 2D TROSY pulse sequence that has been optimized for use with IMPs is also presented.

Solution nuclear magnetic resonance structure of membrane-integral diacylglycerol kinase.

Escherichia coli diacylglycerol kinase (DAGK) represents a family of integral membrane enzymes that is unrelated to all other phosphotransferases. We have determined the three-dimensional structure of the DAGK homotrimer with the use of solution nuclear magnetic resonance. The third transmembrane helix from each subunit is domain-swapped with the first and second transmembrane segments from an adjacent subunit. Each of DAGK's three active sites resembles a portico. The cornice of the portico appears to be the determinant of DAGK's lipid substrate specificity and overhangs the site of phosphoryl transfer near the water-membrane interface. Mutations to cysteine that caused severe misfolding were located in or near the active site, indicating a high degree of overlap between sites responsible for folding and for catalysis.

Molecular basis of Pirh2-mediated p53 ubiquitylation.

Pirh2 (p53-induced RING-H2 domain protein; also known as Rchy1) is an E3 ubiquitin ligase involved in a negative-feedback loop with p53. Using NMR spectroscopy, we show that Pirh2 is a unique cysteine-rich protein comprising three modular domains. The protein binds nine zinc ions using a variety of zinc coordination schemes, including a RING domain and a left-handed beta-spiral in which three zinc ions align three consecutive small beta-sheets in an interleaved fashion. We show that Pirh2-p53 interaction is dependent on the C-terminal zinc binding module of Pirh2, which binds to the tetramerization domain of p53. As a result, Pirh2 preferentially ubiquitylates the tetrameric form of p53 in vitro and in vivo, suggesting that Pirh2 regulates protein turnover of the transcriptionally active form of p53.

Solution NMR spectroscopy of the human vasopressin V2 receptor, a G protein-coupled receptor.

The seven-transmembrane-spanning G protein-coupled receptor (GPCR) superfamily plays many important roles in basic biology, human health, and human disease. Here, well-resolved solution NMR spectra are presented for a human GPCR, the vasopressin V2 receptor in detergent micelles. The quality of the NMR spectra indicates that backbone resonance assignments for a majority of resonances are feasible. The key to obtaining high quality spectra appears to be the coupling of methods for expressing the receptor into membranes rather than into inclusion bodies, with use of a biochemically mild lysolipid detergent for membrane extraction, protein purification, and NMR sample preparation.

NMR characterization of Bronsted acid sites in faujasitic zeolites with use of perdeuterated trimethylphosphine oxide.

The interactions of Bronsted acid sites of H-Y (FAU) with perdeuterated trimethylphosphine oxide (TMPO-d9) are studied with a set of high-resolution solid-state NMR experiments. Double- and triple-resonance MAS NMR techniques (such as CP, TRAPDOR, and REDOR) verify that the lines in the 31P MAS NMR spectrum are indeed from TMPO interacting with Bronsted acid sites. Replacement of acidic hydrogens in the sodalite cages with sodium cations results in the disappearance of one of the peaks, leading to final assignments of the resonances.