NMR-LIGAND BINDING
B Y M I N G M I N G YA N G
KAIYU ZHANG

INTRODUCTION
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•
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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