TY - JOUR

T1 - Measurement of multiple torsional angles from one-dimensional solid-state NMR spectra

T2 - application to the conformational analysis of a ligand in its biological receptor site

AU - Edwards, Rachel

AU - Madine, Jillian

AU - Fielding, Lee

AU - Middleton, David A

PY - 2010/11/14

Y1 - 2010/11/14

N2 - Knowledge of the three-dimensional structure of a ligand in the binding site of its biological receptor is a valuable asset that can assist disease research and guide drug discovery. Solid-state nuclear magnetic resonance (SSNMR) is a useful high-resolution technique for the structural analysis of small molecule or peptide ligands when bound to receptors. SSNMR-derived constraints on the molecular conformations of isotopically (e.g., (13)C and (15)N) enriched ligands usually take the form of through-space distances between atomic nuclei that are separated by three or more bonds. It is advantageous to supplement such distance measurements with independent geometric constraints to resolve structural ambiguities arising from molecular symmetry. Here it is demonstrated that multiple torsional angle constraints can be measured directly for a uniformly labelled biological ligand at a realistically low concentration (150 nmoles) in a practicable experiment time. A simple adaptation of a standard one-dimensional (13)C double-quantum filtered SSNMR experiment is used to measure the relative orientations of C-H bonds in CH(2)-CH and CH(2)-CH(2) groups, which influence (13)C double quantum signal amplitudes in a predictable way. The methodology is applied to uniformly (13)C and (15)N labelled glutamate ([U-(13)C,(15)N]Glu) bound to the ligand binding domain of the ionotropic glutamate receptor 2 (GluR2) in a microcrystalline preparation. Two torsional angle constraints are sufficient to eliminate the structural ambiguities associated with (13)C-(15)N interatomic distance measurements, and thus provide a reliable representation of the conformation of glutamate in its receptor-bound state.

AB - Knowledge of the three-dimensional structure of a ligand in the binding site of its biological receptor is a valuable asset that can assist disease research and guide drug discovery. Solid-state nuclear magnetic resonance (SSNMR) is a useful high-resolution technique for the structural analysis of small molecule or peptide ligands when bound to receptors. SSNMR-derived constraints on the molecular conformations of isotopically (e.g., (13)C and (15)N) enriched ligands usually take the form of through-space distances between atomic nuclei that are separated by three or more bonds. It is advantageous to supplement such distance measurements with independent geometric constraints to resolve structural ambiguities arising from molecular symmetry. Here it is demonstrated that multiple torsional angle constraints can be measured directly for a uniformly labelled biological ligand at a realistically low concentration (150 nmoles) in a practicable experiment time. A simple adaptation of a standard one-dimensional (13)C double-quantum filtered SSNMR experiment is used to measure the relative orientations of C-H bonds in CH(2)-CH and CH(2)-CH(2) groups, which influence (13)C double quantum signal amplitudes in a predictable way. The methodology is applied to uniformly (13)C and (15)N labelled glutamate ([U-(13)C,(15)N]Glu) bound to the ligand binding domain of the ionotropic glutamate receptor 2 (GluR2) in a microcrystalline preparation. Two torsional angle constraints are sufficient to eliminate the structural ambiguities associated with (13)C-(15)N interatomic distance measurements, and thus provide a reliable representation of the conformation of glutamate in its receptor-bound state.

KW - Binding Sites

KW - Glutamates

KW - Ligands

KW - Magnetic Resonance Spectroscopy

KW - Molecular Conformation

KW - Protein Binding

KW - Protein Structure, Tertiary

KW - Proteins

KW - Quantum Theory

KW - Receptors, Glutamate

KW - Reproducibility of Results

U2 - 10.1039/c0cp00326c

DO - 10.1039/c0cp00326c

M3 - Journal article

C2 - 20877838

VL - 12

SP - 13999

EP - 14008

JO - June 2002. ISSN 1460-4582

JF - June 2002. ISSN 1460-4582

SN - 0893-3200

IS - 42

ER -