Alex Nevzorov Group
NC State University
Department of Chemistry
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Alex Nevzorov GroupNC State UniversityDepartment of Chemistry |
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Membrane proteins comprise about 30% of the human genome. They play major role in signal transduction, ion transport, cell recognition, and energy activation. Some membrane proteins, such as g-protein coupled receptors (GPCRs) are now recognized as the most important drug targets. GPCRs represent about 60% of drug targets aimed at treating a broad spectrum of diseases, including AIDS, cancer, asthma, allergies, stomach ulcers, and pain. However, d espite the current interest in membrane proteins in the pharmaceutical industry and the vital role they play, merely 1% of their structures have been determined by X-ray crystallography and solution NMR methods. This is largely due to the difficulties in crystallization of membrane proteins and the presence of the lipid molecules to maintain their native folds. By contrast, solid-state NMR is now emerging as a powerful tool for the structure determination of fully hydrated membrane and membrane-associated proteins in their native lipid environment.
Channel blockers of AchR. A.) QX-314 and QX-222 blockers; B.) The orientation of the QX-314 blocker within the AchR transmembrane domain from molecular dynamics simulations J. M. Pascual, A. Karlin, J. Gen. Physiol. 112 (1998) 611-621. 15N or 13C spin labeling of the blocker will allow one to determine its orientation experimentally by using solid-state NMR.
Flower, BBA (1999)
Bruker Avance II 500 MHz solid-state NMR spectrometer equipped with an UltraShield Magnet and probes for static and rotating samples (exclusive use).
Bruker Ultrashield (TM) 500 MHz Magnet with Avance II (TM) Solids console.
Bruker Biosolids E-free™ triple-resonance probe for static samples.
I. Development of new solid-state NMR methodology for the structural studies of membrane proteins. Unlike solution NMR, in solid-state it is essential to deal with multiple spin-bearing nuclei which are strongly coupled to each other. Many-spin dynamics is used to simulate and create pulse sequences for the homonuclear decoupling and selective polarization transfer in multiple NMR dimensions. The spectrum below shows the application of a new pulse sequence, SAMPI4 (Nevzorov and Opella, 2006 [760 KB]) to a sample containing three n-acetyl leucine single crystals. Each peak corresponds to the distinct orientation of an N-H bond from which structural information can be deduced. SAMPI4 provides uniform decoupling and yields sharp resonances for the entire range of orientations present in peptide planes.
In solid-state NMR of oriented samples each peak corresponds to unique orientation of the NH bond. Here the sample containing three n-acetyl Leucine single crystals yields a total of 12 peaks; the resulting linewidths are indicated.
II. Development of Membrane Mimetics for NMR Structural Studies and Detection of Ligand Binding. Bicelles represent flat bilayer-forming structures made by the mixing of long-chain lipids (e.g. DMPC) with short-chain lipids (e.g. DHPC) at appropriate concentrations in solution. When placed in a strong magnetic field, the bicelles orient themselves so that their normals are on average perpendicular to the magnetic field. Since each bicelle is fully surrounded by water it guarantees 100% hydration. Other factors such as pH, ionic concentration, etc. can be easily controlled. In addition, soluble ligands or small-molecule drugs can be introduced into the sample. In solution NMR, the bicelle-receptor-ligand complex yields no detectable signal; by contrast, solid-state NMR methods are directly applicable since the ligand is oriented together with the bicelle-receptor complex, which allows for the cross-polarization (CP) transfer from 1H to 15N nuclei of the ligand. Moreover magnetically oriented bicelles preserve angular information, which can be used to determine the orientation of the ligand relative to the bicelle normal.
Detection of ligand binding in bicelles. A.) Unbound soluble ligand or drug molecule; B.) In the bound form, the ligand becomes oriented relative to the magnetic field; thus, solid-state NMR methods can be used to determine the ligand orientation and structural changes in the protein.
III. Structural fitting algorithms using angular constraints. A combination of bioinformatics, global minimization, and torsion dynamics is used to obtain protein backbone conformations using angular restraints derived from solid-state NMR (Nevzorov and Opella, 2006 [3,105KB]). These include chemical shift anisotropy and heteronuclear (e.g. N-H and C-H) dipolar couplings. The example of a structure calculation using solid-state NMR data of a mercury transporter protein, MerF is given below (DeAngelis et al, 2006 [335 KB]). The search for the torsion angular solutions for the specific aminoacid residue is restricted by the differential Ramachandran plots.
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