Class 12: Anesthesia Flashcards
Structural biology
Looking to interpret the function of biology based on the structure.
Also looking for structural change during function.
What is the gold standard for structural biology?
X-ray crystallography
Cry-EM: both freeze proteins.
Static pictures of dynamic machines
NMR!!!-dynamic investigation.
How do we use NMR?
Direct structural analysis: seeing changes in NMR after adding ligand of some sort.
Changes in dynamic environment:
Rate of notional dynamics reflected in the __________
NMR line shape
Intermediate dynamic information
Coalesce the 2 individual chemical shifts.
Fast dynamic information:
One NMR spike seen for one structure.
Slow dynamic:
show both positions of AA
EPR
Electron paramagnetic resonance
NMR
Nucleic magnetic resonance
AA position changes can be relevant to:
The AP- ion channels
How anesthetic works:
Blocks ion channels-
Modulators
Agonist competes with __________ for binding site to change ….
Antagonist
Both are transmembrane:
ELIC- (and small intracellular domain): pentameric - prokaryotic protein
NaChBac: tetrameric. Sodium channel for bacteria
ELIC and NaChBac
Cation channels
This is what is inhibited w/ propofol.
TET was used to mutate the pore lining AAs.
What is the mechanism?
Sight-directed labeling
Steps of sight-directed spin labeling:
8
- Identify all native CYS residues
- Create a CYS-null mutant
- mutate novel non-native CYS
- Express and nullify
- label w/ CYS-specific spin-label
- remove free label (SEC,desalting, disfiltration)
- perform experiment: asses for absence of free label
- Profit?
Looking at mutated protein for:
Functional?
Native structure?
Similar biophysical properties?
HOw can you prove that a protein is still functional?
Electrophysiology
[show that it can be inhibited or excited again]
And Two-electrode voltage clamp (TEVC)
Photo-affinity labeling:
- Incubation of drug (Propofol) w/ receptor -noncolvanet interaction
- photolysis
- photactivated complex (covalent interaction)
- digestion
- Peptides analyzed w/ MS
Does binding site change based on functional state protein is in?
Yes:
M265
F308-top and bottom binding site for propofol
Saturation transfer difference NMR:
- Incorporate selectively excitable nucleus (19-F)
- saturate both on and off protein resonance
- Allow transfer to ligand w/ variable saturation time
- no ligand-protein=>ligand signal canceled in the difference spectra
Isoflourene binds to:
T 189
STD NMR shows:
Where the Rx is binding
Isoflourene may bind at different sites d/t:
orientation
F-19 NMR
provides a unique perspective of conformation, topology, and dynamics
Anesthetic binding sites:
ELIC and NaChBac
