B Y M I N G M I N G YA N G
KAIYU ZHANG
INTRODUCTION
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NMR
STD NMR
Experiment
Analysis
Conclusion
NUCLEAR MAGNETIC RESONANCE
SPECTROSCOPY
• Nuclear magnetic resonance spectroscopy(NMR)
• Determine structure of organic compounds
• Nuclear Spin
• Lower energy state
• Higher energy state
NUCLEAR MAGNETIC RESONANCE
SPECTROSCOPY
SATURATION TRANSFER DIFFERENCE
SPECTROSCOPY
• Saturation Transfer Difference Spectroscopy (STD
NMR)
• Interactions between protein and ligand
• Intermolecular transfer of magnetization
• NMR signals—magnetization transfer
SATURATION TRANSFER DIFFERENCE
SPECTROSCOPY
• Exchange between bound and the free ligand state • For bound ligand, only hydrogen in close contact(
• < 5 A) with protein would receive magnetization transfer • Signal Intensity proportional to the distance from protein COMPETITIVE STD NMR
• Competition between high affinity and low affinity ligands • Measure the loss of low affinity ligand STD signal
• Significant reduction/disappearance of low-affinity ligand EXPERIMENT-SACCHARIN
• 5 uM mutant hCAII
• 5mM Saccharin
• 0.5mM TSP
• 10% D2O
• 4% d6-DMSO
EXPERIMENT-COMPETITIVE
• 50uM hCAII
• 5mM Saccharin
• 57.2/497 mM Furosemide for 50/90% reduction
• 0.5mM TSP
• 10%D2O
• 4% d6-DMSO
SACCHARIN
Sulfonamide in 5-membered ring
Lactam carbonyl group
SACCHARIN
Hillerbrecht, etc, 2007
SACCHARIN
Hillerbrecht, et, al, 2007
SACCHARIN
• Lower binding affinity
-sulfonamide group within a ring
- stabilized by hydrogen bonding
FUROSEMIDE
Exocyclic sulfonamide group
FUROSEMIDE
Honndorf, V.S. (not published yet)
FUROSEMIDE
• Stabilized by 4 hydrogen bonds and one NH bond
• Bind through nitrogen on sulfonamide Honndorf, V.S. (not published yet)
ithout Furosemide
RESULTS
(hCAII Wild type)
57.2 uM Furosemide
497 uM Furosemide
RESULTS
STD
Signal
Grou p1 Grou p2 Grou p3 Grou p4 Grou p5 Grou p6 WT
Without
Furosemid
e
73.0
9
83.1
4
18.5
0
40.8
7
61.4
8
66.2
0
59.9
2
57.2 uM
Furosemid
e
56.0
2
70.4
7
18.0
0
37.3
6
59.5
1
47.9
8
49.0
4
497 uM
Furosemid
e
54.1
6
67.6
0
12.8
4
31.8
1
51.2
3
41.9
3
38.3
3
(average)
RESULTS
Ki
(uM)
Grou p1 Grou Grou Grou Grou Grou WT p2 p3 p4 p5 p6 50%STD reduction 0.2188 3.2849 2.4373 0.7178 2.0396 0.1748 0.3017
90%STD reduction 1.6869 2.5683 1.3394 2.0683 2.9482 1.0172 1.0452
Average
0.9529 2.9266 1.8882 1.3930 2.4939 0.5959 0.6735
Range of Ki is approximately 10-3-10-8 M
(T . Haselhorst et al, 2009)
RESULTS
Ki from 50% reduction STD VS. Literature value
Group 2
Group 3
Group 4
Group 5
Group 6
WT
Literature value for Ki: 0.065 uM ( Temperini et al, 2009)
RESULTS
Binding affinity
External factor that affect Ki:
• Enzyme
• Inpurity
• Solution condition:
Buffer: HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid tris (hydroxymethyl)aminomethane
DMSO percentage
Temperature: kinetic and solubility
H2O
• Experimental error
RESULTS
Ki from 90% reduction STD VS. Literature value
Group
Group
Group
Group
Group
Group
WT
Data Sets
Literature value for Ki: 0.065 uM ( Temperini et al, 2009)
1
2
3
4
5
6
RESULTS
STD signal reduction of indicator
• Saturation ( 50%: 57.2 uM, 90%: 497 uM)
• Need more inhibitors
• Suggestion
10-4
104
10-3
105
10-2 10-1
1
101
102
103
Concentration of inhibitor (uM)(Wang Y-S et al, 2004)
RESULT
Internal factor that affect Ki
• Group 2-N67A-Saccharin
Red: O
Yellow: S
Blue; N
RESULT
• Group 2-N67A-Furosemide
RESULT
• Group 3-E106A-Saccharin
RESULT
• Group 3-E106A-Furosemide
RESULT
• Group 4-L197E -Saccharin
RESULT
• Group 4-L197E-Furosemide
RESULT
• Group 5-Y7H-Saccharin
RESULT