2H95
Structure of the Amantadine-Blocked Influenza A M2 Proton Channel Trans-membrane Domain by Solid-state NMR spectroscopy
SOLID-STATE NMR
| NMR Experiment | ||||||||
|---|---|---|---|---|---|---|---|---|
| Experiment | Type | Sample Contents | Solvent | Ionic Strength | pH | Pressure | Temperature (K) | Spectrometer |
| 1 | solid-State NMR PISEMA | M2-TMD (~120 mg) and DMPC (~75 mg) were first co-dissolved in 10 ml TFE, followed by the removal of the solvent under vacuum. The peptide/lipid mixture was rehydrated and sonicated to make liposomes in a citrate-borate-phosphate (CBP) buffer (pH 8.8) with 1 mM EDTA and 10 mM amantadine at 310 K. The liposomes were pelleted by ultracentrifugation . Then the pellet was spread on glass slides and dehydrated in a 75% humidity chamber. The dehydrated slides were rehydrated with 1.5 microl liter CBP buffer per slide followed by being stacked into a glass tube and incubated at 316 K for 24 hours in 96% relative humidity. Finally, the glass tube was sealed at both ends with epoxy and two glasscaps. | oriented peptide/lipid bilayer of M2_TMD and DMPC | 8.8 | AMBIENT | 308 | ||
| NMR Spectrometer Information | |||
|---|---|---|---|
| Spectrometer | Manufacturer | Model | Field Strength |
| 1 | Bruker | AVANCE | 400 |
| NMR Refinement | ||
|---|---|---|
| Method | Details | Software |
| ENERGY MINIMIZATION WITH ORIENTATIONAL CONSTRAINTS | REFINEMENT WAS CARRIED OUT IN VACUO ON INITIAL MONOMER COORDINATES CONSISTING OF TWO ALPHA-HELICAL FRAGMENTS (3.6 RESIDUES PER TURN) HAVING TILT AND ROTATIONAL ORIENTATIONS WITH RESPECT TO THE BILAYER DERIVED FROM PISEMA DIPOLAR WAVE ANALYSIS. ENERGY MINIMIZATION USED A GLOBAL PENALTY FUNCTION INCORPORATING ORIENTATIONAL RESTRAINTS, HYDROGEN BONDING AND THE CHARMM EMPIRICAL FUNCTION. THE ORIENTATIONAL RESTRAINTS IMPOSED ON THE STRUCTURE DURING REFINEMENT ARE 16 15N CHEMICAL SHIFTS AND 16 15N-1H DIPOLAR COUPLINGS FROM PISEMA EXPERIMENTS. A SYMMETRIC, TETRAMERIC BUNDLE MODEL OF M2-TMD WAS CONSTRUCTED BY A SERIES OF RIGID-BODY TRANSFORMATIONS OF THE REFINED M2-TMD MONOMER. THE RESULTING HOMO-TETRAMER IS THE LOWEST FREE ENERGY CONFORMER BASED ON ROTATIONAL CONFORMATIONAL SEARCH. NOTE THAT THE HIS37 AND TRP41 SIDECHAIN POSITIONS ARE CONSISTENT WITH MEASURED ORIENTATIONAL CONSTRAINTS. THE ROTAMERIC STATES OF OTHER RESIDUES ARE TAKEN FROM A BACKBONE DEPENDENT SIDECHAIN ROTAMER LIBRARY (SCRWL). | XPLOR-NIH |
| NMR Ensemble Information | |
|---|---|
| Conformer Selection Criteria | structures with the lowest energy |
| Conformers Calculated Total Number | 72 |
| Conformers Submitted Total Number | 1 |
| Representative Model | 1 (lowest energy) |
| Computation: NMR Software | ||||
|---|---|---|---|---|
| # | Classification | Version | Software Name | Author |
| 1 | refinement | XPLOR-NIH | 2.9.9 | Schwieters, Kuszewski, Tjandra, Clore |
