Biomedical subjects

Sorin Luca

Publications and source records attributed to Sorin Luca.

9 recordsLinked to original sources

PubMed2005

Probing conformational disorder in neurotensin by two-dimensional solid-state NMR and comparison to molecular dynamics simulations.

An approach is introduced to characterize conformational ensembles of intrinsically unstructured peptides on the atomic level using two-dimensional solid-state NMR data and their combination with molecular dynamics simulations. For neurotensin, a peptide that binds with high affinity to a G-protein coupled receptor, this method permits the investigation of the changes in conformational preferences of a neurotransmitter transferred from a frozen aqueous solution via a lipid model phase to the receptor-bound form. The results speak against a conformational pre-organization of the ligand in detergents in which the receptor has been shown to be functional. Further extensions to the study of protein folding are possible.

Detergents↗

PubMed2005

Investigation of ligand-receptor systems by high-resolution solid-state NMR: recent progress and perspectives.

Solid-state Nuclear Magnetic Resonance (NMR) provides a general method to study molecular structure and dynamics in a non-crystalline and insoluble environment. We discuss the latest methodological progress to construct 3D molecular structures from solid-state NMR data obtained under magic-angle-spinning conditions. As shown for the neurotensin/NTS-1 system, these methods can be readily applied to the investigation of ligand-binding to G-protein coupled receptors.

Chemistry, Pharmaceutical↗

PubMed2004

Multiple-spin analysis of chemical-shift-selective (13C, 13C) transfer in uniformly labeled biomolecules.

Chemical-shift-selective (13C, 13C) polarization transfer is analyzed in uniformly labeled biomolecules. It is shown that the spin system dynamics remain sensitive to the distance of interest and can be well reproduced within a quantum-mechanical multiple-spin analysis. These results lead to a general approach on how to describe chemical-shift-selective transfer in uniformly labeled systems. As demonstrated in the case of ubiquitin, this methodology can be used to detect long-range distance constraints in uniformly labeled proteins.

Biopolymers↗

PubMed2003

Analysis of proton-proton transfer dynamics in rotating solids and their use for 3D structure determination.

A detailed analysis of proton-proton-transfer dynamics under magic angle spinning NMR is presented. Results obtained on model compounds are evaluated under different experimental conditions and NMR mixing schemes. It is shown that the resulting buildup rates can be interpreted in terms of internuclear proton-proton distances provided that an appropriate theoretical description is chosen. As demonstrated in two test applications, these dependencies can be used in the context of a three-dimensional structure determination in the solid state.

Alanine↗

PubMed2003

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 beta-strand conformation. Our results provide a viable structural template for further pharmacological investigations.

GTP-Binding Proteins↗

PubMed2003

High-resolution solid-state NMR applied to polypeptides and membrane proteins.

Solid-state NMR provides unique possibilities to study insoluble or noncrystalline molecules at the atomic level. High-resolution conditions can be established by employing magic-angle spinning at ultrahigh magnetic fields. We discuss NMR methods that make use of these experimental improvements and allow for the study of multiply or uniformly [(13)C,(15)N]-labeled polypeptides and proteins. Recent biophysical applications are reviewed.

Membrane Proteins↗

PubMed2003

Solid state NMR sequential resonance assignments and conformational analysis of the 2x10.4 kDa dimeric form of the Bacillus subtilis protein Crh.

Solid state NMR sample preparation and resonance assignments of the U-[13C,15N] 2x10.4 kDa dimeric form of the regulatory protein Crh in microcrystalline, PEG precipitated form are presented. Intra- and interresidue correlations using dipolar polarization transfer methods led to nearly complete sequential assignments of the protein, and to 88% of all 15N, 13C chemical shifts. For several residues, the resonance assignments differ significantly from those reported for the monomeric form analyzed by solution state NMR. Dihedral angles obtained from a TALOS-based statistical analysis suggest that the microcrystalline arrangement of Crh must be similar to the domain-swapped dimeric structure of a single crystal form recently solved using X-ray crystallography. For a limited number of protein residues, a remarkable doubling of the observed NMR resonances is observed indicative of local static or dynamic conformational disorder. Our study reports resonance assignments for the largest protein investigated by solid state NMR so far and describes the conformational dimeric variant of Crh with previously unknown chemical shifts.

Amino Acid Sequence↗

PubMed2002

Structural constraints from proton-mediated rare-spin correlation spectroscopy in rotating solids.

A novel solid-state NMR concept is introduced that allows for the detection of through-space proton-proton contacts in high spectral resolution. The proposed method not only can be utilized to infer sequential assignments and backbone or side chain conformation in solid-phase polypeptides, but it also provides information about the three-dimensional arrangement of the molecule of interest. As a result, the molecular structure can be studied without additional restrictions regarding sample labeling or magic angle spinning rates.

Carbon Isotopes↗

PubMed2002

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.

Nuclear Magnetic Resonance, Biomolecular↗