Hyperpolarized 133Cs is a sensitive probe for real-time monitoring of biophysical environments.

133Cs NMR is a valuable tool for non-invasive analysis of biological systems, where chemical shift and relaxation properties report on changes in the physical environment. Hyperpolarization can increase the liquid-state 133Cs NMR signal by several orders of magnitude and allow real-time monitoring of physical changes in cell based systems.

In vivo and in vitro liver cancer metabolism observed with hyperpolarized [5-(13)C]glutamine.

Glutamine metabolism is, with its many links to oncogene expression, considered a crucial step in cancer metabolism and it is thereby a key target for alteration in cancer development. In particular, strong correlations have been reported between oncogene expression and expression and activity of the enzyme glutaminase. This mitochondrial enzyme, which is responsible for the deamidation of glutamine to form glutamate, is overexpressed in many tumour tissues. In animal models, glutaminase expression is correlated with tumour growth rate and it is readily possible to limit tumour growth by suppression of glutaminase activity. In principle, hyperpolarized (13)C MR spectroscopy can provide insight to glutamine metabolism and should hence be a valuable tool to study changes in glutaminase activity as tumours progress. However, no such successful in vivo studies have been reported, even though several good biological models have been tested. This may, at least partly, be due to problems in preparing glutamine for hyperpolarization. This paper reports a new and improved preparation of hyperpolarized [5-(13)C]glutamine, which provides a highly sensitive (13)C MR marker. With this preparation of hyperpolarized [5-(13)C]glutamine, glutaminase activity in vivo in a rat liver tumour was investigated. Moreover, this marker was also used to measure response to drug treatment in vitro in cancer cells. These examples of [5-(13)C]glutamine used in tumour models warrant the new preparation to allow metabolic studies with this conditionally essential amino acid.

Barley lipid transfer protein, LTP1, contains a new type of lipid-like post-translational modification.

In plants a group of proteins termed nonspecific lipid transfer proteins are found. These proteins bind and catalyze transfer of lipids in vitro, but their in vivo function is unknown. They have been suggested to be involved in different aspects of plant physiology and cell biology, including the formation of cutin and involvement in stress and pathogen responses, but there is yet no direct demonstration of an in vivo function. We have found and characterized a novel post-translational modification of the barley nonspecific lipid transfer protein, LTP1. The protein-modification bond is of a new type in which an aspartic acid in LTP1 is bound to the modification through what most likely is an ester bond. The chemical structure of the modification has been characterized by means of two-dimensional homo- and heteronuclear nuclear magnetic resonance spectroscopy as well as mass spectrometry and is found to be lipid-like in nature. The modification does not resemble any standard lipid post-translational modification but is similar to a compound with known antimicrobial activity.

Identification of a neuritogenic ligand of the neural cell adhesion molecule using a combinatorial library of synthetic peptides.

The neural cell adhesion molecule (NCAM) plays a key role in neural development, regeneration, and learning. In this study, we identified a synthetic peptide-ligand of the NCAM Ig1 module by combinatorial chemistry and showed it could modulate NCAM-mediated cell adhesion and signal transduction with high potency. In cultures of dissociated neurons, this peptide, termed C3, stimulated neurite outgrowth by activating a signaling pathway identical to that activated by homophilic NCAM binding. A similar effect was shown for the NCAM Ig2 module, the endogenous ligand of NCAM Ig1. By nuclear magnetic resonance spectroscopy, the C3 binding site in the NCAM Ig1 module was mapped and shown to be different from the binding site of the NCAM Ig2 module. The C3 peptide may prove useful as a lead in development of therapies for neurodegenerative disorders, and the C3 binding site of NCAM Ig1 may represent a target for discovery of nonpeptide drugs.

Pulse sequences for measurement of one-bond (15)N-(1)H coupling constants in the protein backbone.

A set of three improved two-dimensional (2D) NMR methods for measuring one-bond (15)N-(1)H coupling constants in the protein backbone is presented. They are tailored to suit the size of the TROSY effect, i.e., the degree of interference between dipolar and chemical shift anisotropy relaxation mechanisms. The methods edit 2D spectra into two separate subspectra corresponding to the two possible spin states of the coupling partner. Cross talk between the two subspectra is a second order effect in the difference between the actual coupling constants and the one used in setting the pertinent delays of the pulse sequences. This relatively high degree of editing accuracy makes the methods useful for applications to molecules subjected to weak alignment where the one-bond coupling constants are linear combinations of a scalar J and a residual dipolar contribution containing important structural information. A demonstration of the new methods is shown for the (15)N-labeled protein chymotrypsin inhibitor 2 in a lipid bicelle mixture.

Solution structure of barley lipid transfer protein complexed with palmitate. Two different binding modes of palmitate in the homologous maize and barley nonspecific lipid transfer proteins.

The structure of a nonspecific lipid transfer protein from barley (ns-LTPbarley) in complex with palmitate has been determined by NMR spectroscopy. The structure has been compared to the structure of ns-LTPbarley in the absence of palmitate, to the structure of ns-LTPbarley in complex with palmitoyl coenzyme A, to the structure of ns-LTPmaize in its free form, and to the maize protein complexed with palmitate. Binding of palmitate only affects the structure of ns-LTPbarley moderately in contrast to the binding of palmitoyl coenzyme A, which leads to a considerable expansion of the protein. The modes of binding palmitate to the maize and barley protein are different. Although in neither case there are major conformational changes in the protein, the orientation of the palmitate in the two proteins is exactly opposite.

Barley lipid-transfer protein complexed with palmitoyl CoA: the structure reveals a hydrophobic binding site that can expand to fit both large and small lipid-like ligands.

BACKGROUND: . Plant nonspecific lipid-transfer proteins (nsLTPs) bind a variety of very different lipids in vitro, including phospholipids, glycolipids, fatty acids and acyl coenzyme As. In this study we have determined the structure of a nsLTP complexed with palmitoyl coenzyme A (PCoA) in order to further our understanding of the structural mechanism of the broad specificity of these proteins and its relation to the function of nsLTPs in vivo. RESULTS: . 1H and 13C nuclear magnetic resonance spectroscopy (NMR) have been used to study the complex between a nsLTP isolated from barley seeds (bLTP) and the ligand PCoA. The resonances of 97% of the 1H atoms were assigned for the complexed bLTP and nearly all of the resonances were assigned in the bound PCoA ligand. The palmitoyl chain of the ligand was uniformly 13C-labelled allowing the two ends of the hydrocarbon chain to be assigned. The comparison of a subset of 20 calculated structures to an average structure showed root mean square deviations of 1.89 +/- 0.19 for all C, N, O, P and S atoms of the entire complex and of 0.57 +/- 0.09 for the peptide backbone atoms of the four alpha helices of the complexed bLTP. The four-helix topology of the uncomplexed bLTP is maintained in the complexed form of the protein. The bLTP only binds the hydrophobic parts of PCoA with the rest of the ligand remaining exposed to the solvent. The palmitoyl chain moiety of the ligand is placed in the interior of the protein and bent in a U-shape. This part of the ligand is completely buried within a hydrophobic pocket of the protein. CONCLUSIONS: . A comparison of the structures of bLTP in the free and bound forms suggests that bLTP can accommodate long olefinic ligands by expansion of the hydrophobic binding site. This expansion is achieved by a bend of one helix, HA, and by conformational changes in both the C terminus and helix HC. This mode of binding is different from that seen in the structure of maize nsLTP in complex with palmitic acid, where binding of the ligand is not associated with structural changes.