Removal of H-decoupling sidebands in CHD C-CEST profiles.

A unique aspect of NMR is its capacity to provide integrated insight into both the structure and intrinsic dynamics of biomolecules. Chemical exchange phenomena that often serve as probes of dynamic processes in biological macromolecules can be quantitatively investigated with chemical exchange saturation transfer (CEST) experiments. H-decoupling sidebands, however, always occur in the profiles of CHD C-CEST experiments when using the simple CW (continuous wave) method, which may obscure the detection of minor dips of excited states.

The activation loop of PIP5K functions as a membrane sensor essential for lipid substrate processing.

Phosphatidylinositol 4-phosphate 5-kinase (PIP5K), a representative member of the phosphatidylinositol phosphate kinase (PIPK) family, is a major enzyme that biosynthesizes the signaling molecule PI(4,5)P (phosphatidylinositol 4,5-bisphosphate) in eukaryotic cells. The stringent specificity toward lipid substrates and the high sensitivity to the membrane environment strongly suggest a membrane-sensing mechanism, but the underlying structural basis is still largely unknown. We present a nuclear magnetic resonance (NMR) study on a peptide commensurate with a PIP5K's activation loop, which has been reported to be a determinant of lipid substrate specificity and subcellular localization of PIP5K.

Structural determinants of host specificity of complement Factor H recruitment by Streptococcus pneumoniae.

Many human pathogens have strict host specificity, which affects not only their epidemiology but also the development of animal models and vaccines. Complement Factor H (FH) is recruited to pneumococcal cell surface in a human-specific manner via the N-terminal domain of the pneumococcal protein virulence factor choline-binding protein A (CbpAN). FH recruitment enables Streptococcus pneumoniae to evade surveillance by human complement system and contributes to pneumococcal host specificity.

Structure of bacterial transcription factor SpoIIID and evidence for a novel mode of DNA binding.

SpoIIID is evolutionarily conserved in endospore-forming bacteria, and it activates or represses many genes during sporulation of Bacillus subtilis. An SpoIIID monomer binds DNA with high affinity and moderate sequence specificity. In addition to a predicted helix-turn-helix motif, SpoIIID has a C-terminal basic region that contributes to DNA binding.

Role of glutamate 64 in the activation of the prodrug 5-fluorocytosine by yeast cytosine deaminase.

Yeast cytosine deaminase (yCD) catalyzes the hydrolytic deamination of cytosine to uracil as well as the deamination of the prodrug 5-fluorocytosine (5FC) to the anticancer drug 5-fluorouracil. In this study, the role of Glu64 in the activation of the prodrug 5FC was investigated by site-directed mutagenesis, biochemical, nuclear magnetic resonance (NMR), and computational studies. Steady-state kinetics studies showed that the mutation of Glu64 causes a dramatic decrease in k(cat) and a dramatic increase in K(m), indicating Glu64 is important for both binding and catalysis in the activation of 5FC.

Hydrogen-bond detection, configuration assignment and rotamer correction of side-chain amides in large proteins by NMR spectroscopy through protium/deuterium isotope effects.

The configuration and hydrogen-bonding network of side-chain amides in a 35 kDa protein were determined by measuring differential and trans-hydrogen-bond H/D isotope effects by using the isotopomer-selective (IS)-TROSY technique, which leads to a reliable recognition and correction of erroneous rotamers that are frequently found in protein structures. First, the differential two-bond isotope effects on carbonyl (13)C' shifts, which are defined as Delta(2)Delta(13)C'(ND) = (2)Delta(13)C'(ND(E))-(2)Delta(13)C'(ND(Z)), provide a reliable means for the configuration assignment for side-chain amides, because environmental effects (hydrogen bonds and charges, etc.) are greatly attenuated over the two bonds that separate the carbon and hydrogen atoms, and the isotope effects fall into a narrow range of positive values.

Using structural analysis to generate parasite-selective monoclonal antibodies.

Diagnosis of eukaryotic parasitic infection using antibody-based tests such as ELISAs (enzyme-linked immunosorbent assays) is often problematic because of the need to differentiate between homologous host and pathogen proteins and to ensure that antibodies raised against a peptide will also bind to the peptide in the context of its three-dimensional protein structure. Filariasis caused by the nematode, Brugia malayi, is an important worldwide tropical disease in which parasites disappear from the bloodstream during daylight hours, thus hampering standard microscopic diagnostic methods. To address this problem, a structural approach was used to develop monoclonal antibodies (mAbs) that detect asparaginyl-tRNA synthetase (AsnRS) secreted from B.

TROSY of side-chain amides in large proteins.

By using the mixed solvent of 50% H2O/50% D2O and employing deuterium decoupling, TROSY experiments exclusively detect NMR signals from semideuterated isotopomers of carboxamide groups with high sensitivities for proteins with molecular weights up to 80 kDa. This isotopomer-selective strategy extends TROSY experiments from exclusively detecting backbone to both backbone and side-chain amides, particularly in large proteins. Because of differences in both TROSY effect and dynamics between 15N-H(E){D(Z)} and 15N-H(Z){D(E)} isotopomers of the same carboxamide, the 15N transverse magnetization of the latter relaxes significantly faster than that of the former, which provides a direct and reliable stereospecific distinction between the two configurations.

Simultaneous NMR assignment of backbone and side chain amides in large proteins with IS-TROSY.

A new strategy for the simultaneous NMR assignment of both backbone and side chain amides in large proteins with isotopomer-selective transverse-relaxation-optimized spectroscopy (IS-TROSY) is reported. The method considers aspects of both the NMR sample preparation and the experimental design. First, the protein is dissolved in a buffer with 50%H2O/50%D2O in order to promote the population of semideuterated NHD isotopomers in side chain amides of Asn/Gln residues.

Product release is rate-limiting in the activation of the prodrug 5-fluorocytosine by yeast cytosine deaminase.

Yeast cytosine deaminase (yCD), a zinc metalloenzyme, catalyzes the hydrolytic deamination of cytosine to uracil. The enzyme is of great biomedical interest because it also catalyzes the deamination of the prodrug 5-fluorocytosine (5FC) to form the anticancer drug 5-fluorouracil (5FU). yCD/5FC is one of the most widely used enzyme/prodrug combinations for gene-directed enzyme prodrug therapy for the treatment of cancers.

Three Dimensional Solution Structure of Neurotoxin CM-11 from the Ophiophagus hannah.

The Ophiophagus hannah (King Cobra) neurotoxin CM-11 is a small protein with 72 amino acid residues. Based on complete assignments of (1)H-NMR resonances and determination of secondary structures of CM-11, 349 distance and 27 dihedral angle constraints including 19 psi's and 8 Xi (1)'s were collected from NOESY and DQF-COSY, and the chemical stereospecific assignment of Beta(1)H was partially achieved. Twelve structures with lower energy were obtained via metric matrix distance geometry and refinement with simulated annealing.

Determination of the Secondary Structure of the king Cobra Neurotoxin CM-11.

The king cobra neurotoxin CM-11 is a small protein with 72 amino acid residues. After its complete assignments of (1)H-NMR resonance's were obtained using various 2D-NMR technologies, including DQF-COSY, clean-TOCSY and NOESY, the secondary structure was analysed by studying the various NOEs extracted from the NOESY spectra and the distribution of chemical shifts. The secondary structure was finally determined by MCD as follows: a triple-strand antiparallel beta sheet with I20-W26, R37-A43 and V53-S59 as its beta strands, a short alpha helix formed by W30-G35 and four turns formed by P7-K1O, C14-G17, K50-V53 and D61-N64.

Complete Assignment of (1)H-NMR Resonances of the King Cobra Neurotoxin CM-11.

The king cobra (Ophiophagus Hannah) neurotoxin CM-Il is long-chain peptide with 72 amino acid residues. Its complete assignment of (1)H-NMR resonances was obtained using various 2D-NMR technologies, including DQF-COSY, clean-TOCSY and NOESY.