Sorin Luca

The conformation of neurotensin bound to its G protein-coupled receptor

G protein-coupled receptors (GPCRs) mediate the perception of smell, light, taste, and pain. They are involved in signal recognition and cell communication and are some of the most important targets for drug development. Because currently no direct structural information on high-affinity ligands bound to GPCRs is available, rational drug design is limited to computational prediction combined with mutagenesis experiments. Here, we present the conformation of a high-affinity peptide agonist (neurotensin, NT) bound to its GPCR NTS-1, determined by direct structural methods. Functional receptors were expressed in Escherichia coli, purified in milligram amounts by using optimized procedures, and subsequently reconstituted into lipid vesicles. Solid-state NMR experiments were tailored to allow for the unequivocal detection of microgram quantities of 13C,15N-labeled NT(8–13) in complex with functional NTS-1. The NMR data are consistent with a disordered state of the ligand in the absence of receptor. Upon receptor binding, the peptide undergoes a linear rearrangement, adopting a β-strand conformation. Our results provide a viable structural template for further pharmacological investigations.

Secondary chemical shifts in immobilized peptides and proteins: a qualitative basis for structure refinement under magic angle spinning.

Resonance assignments recently obtained on immobilized polypeptides and a membrane protein aggregate under Magic Angle Spinning are compared to random coil values in the liquid state. The resulting chemical shift differences (secondary chemical shifts) are evaluated in light of the backbone torsion angle ψ previously reported using X-ray crystallography. In all cases, a remarkable correlation is found suggesting that the concept of secondary chemical shifts, well established in the liquid state, can be of similar importance in the context of multiple-labelled polypeptides studied under MAS conditions.

Enhanced spectral resolution in immobilized peptides and proteins by combining chemical shift sum and difference spectroscopy

A two-dimensional correlation experiment is introduced that records the sum and difference chemical shift of two scalar or dipolar coupled nuclei. Statistical results indicate that the suggested pulse scheme can significantly increase the possibility of separating chemical shift contributions due to residue type and backbone conformation in immobilized peptides and proteins. Experimental applications demonstrate the theoretical concept and lead to the predicted resolution enhancement between different amino acid types and among protein residues of different secondary structure.

Multiple-quantum spectroscopy of fully labeled polypeptides under MAS: A statistical and experimental analysis.

Spectral resolution represents a prerequisite for NMR based structural studies in multior fully labeled polypeptides such as membrane proteins, peptide ligands or protein aggregates. Recent applications in immobilized proteins [1–4] and a membrane protein aggregate [5] revealed that characteristic chemical shift information obtained in the liquid-state improves and expedites spectral assignment under MAS [6] conditions. In many cases, the observed NMR line width may limit applications in polypeptides and proteins of larger size. In this context, correlation experiments are desirable that maximize the spectral resolution without compromising the structural information contained in the spectra.