Class 12: Anesthesia

Question 1

Structural biology

Answer 1

Looking to interpret the function of biology based on the structure.

Also looking for structural change during function.

Question 2

What is the gold standard for structural biology?

Answer 2

X-ray crystallography
Cry-EM: both freeze proteins.

Static pictures of dynamic machines

NMR!!!-dynamic investigation.

Question 3

How do we use NMR?

Answer 3

Direct structural analysis: seeing changes in NMR after adding ligand of some sort.

Changes in dynamic environment:

Question 4

Rate of notional dynamics reflected in the __________

Answer 4

NMR line shape

Question 5

Intermediate dynamic information

Answer 5

Coalesce the 2 individual chemical shifts.

Question 6

Fast dynamic information:

Answer 6

One NMR spike seen for one structure.

Question 7

Slow dynamic:

Answer 7

show both positions of AA

Question 8

EPR

Answer 8

Electron paramagnetic resonance

Question 9

NMR

Answer 9

Nucleic magnetic resonance

Question 10

AA position changes can be relevant to:

Answer 10

The AP- ion channels

Question 11

How anesthetic works:

Answer 11

Blocks ion channels-

Modulators

Question 12

Agonist competes with __________ for binding site to change ….

Answer 12

Antagonist

Question 13

Both are transmembrane:

Answer 13

ELIC- (and small intracellular domain): pentameric - prokaryotic protein

NaChBac: tetrameric. Sodium channel for bacteria

Question 14

ELIC and NaChBac

Answer 14

Cation channels

This is what is inhibited w/ propofol.

Question 15

TET was used to mutate the pore lining AAs.

What is the mechanism?

Answer 15

Sight-directed labeling

Question 16

Steps of sight-directed spin labeling:

8

Answer 16

1. Identify all native CYS residues
2. Create a CYS-null mutant
3. mutate novel non-native CYS
4. Express and nullify
5. label w/ CYS-specific spin-label
6. remove free label (SEC,desalting, disfiltration)
7. perform experiment: asses for absence of free label
8. Profit?

Question 17

Looking at mutated protein for:

Answer 17

Functional?

Native structure?

Similar biophysical properties?

Question 18

HOw can you prove that a protein is still functional?

Answer 18

Electrophysiology
[show that it can be inhibited or excited again]

And Two-electrode voltage clamp (TEVC)

Question 19

Photo-affinity labeling:

Answer 19

1. Incubation of drug (Propofol) w/ receptor -noncolvanet interaction
2. photolysis
3. photactivated complex (covalent interaction)
4. digestion
5. Peptides analyzed w/ MS

Question 20

Does binding site change based on functional state protein is in?

Answer 20

Yes:

M265
F308-top and bottom binding site for propofol

Question 21

Saturation transfer difference NMR:

Answer 21

1. Incorporate selectively excitable nucleus (19-F)
2. saturate both on and off protein resonance
3. Allow transfer to ligand w/ variable saturation time
4. no ligand-protein=>ligand signal canceled in the difference spectra

Question 22

Isoflourene binds to:

Answer 22

T 189

Question 23

STD NMR shows:

Answer 23

Where the Rx is binding

Question 24

Isoflourene may bind at different sites d/t:

Answer 24

orientation

Question 25

F-19 NMR

Answer 25

provides a unique perspective of conformation, topology, and dynamics

Question 26

Anesthetic binding sites:

Answer 26

ELIC and NaChBac