kinetic simulation/modeling; Assay development; Enzymology; Instrumental analysis; protein engineering; Bioinformatics/pathway analysis

1. Skills and Experience
Kai Li PhD
1

2. Assay development and
optimization
2

3. NMR assay
quench, centrifugation,
aliquot pH 9 buffer + DMSO
+ DNFB in EtOH
45 mins at 50 °C
quench, neutralize, spin,
analyze using RP-HPLC
15N-Asn + 14N-Asn
T. M. Larsen, et al, Biochemistry 1999, 38, 16146-16157
K. Li, et al, Biochemistry 2007, 46(16), 4840-4849
pH 8 buffer + 15NH4Cl + 14N-Gln
(+ substrate) + enzyme
10 mins at 37 °C
Ajust pH to 5.0 by adding 10M NaOH
1H-NMR
15N-Asn
3

4. Competition assay
4

5. Exchanging assay
K. Li, et al, Biochemistry 2007, 46(16), 4840-4849
5

6. PAPS
2OST
Y94A
Epimerase assay
6

7. In vitro FATylation – time course
7

8. Pull down assay
-- 220 kD
-- 97 kD
-- 66 kD
-- 45 kD
-- 30 kD
2 X HeLa 1 X HeLa
8

9. Cell based assays
9

10. BaF3 proliferation assay
Concentration (µg/mL)
0.0 0.5 1.0 1.5 2.0
[
3
H]ThymidineIncorporation(%)
0
20
40
60
80
100
Heparin
Synthesized
10

11. FACS analysis of splicing factors
11

12. Cell cycle and cell imaging
12

13. Other assays used
13

14. quenchpH 8 buffer + substrates
+ enzyme
10 mins at 37 °C
pH 9 buffer + GLDH
30 mins at RT
Abs340 at t=0 and t=30 mins
Standard curve
Glu
NAD+ NADH
Boehlein, S., et al, J. Biol. Chem., 1994, 269(10), 7450-7
Glutaminase assay
GLDH coupled enzyme assay for glutaminase activity
14

15. NADH NAD+
Pyrophosphate assay
Coupled enzyme assay for synthetase activity
pH 8 buffer + substrates +
PPi reagent (Sigma) + enzyme
at 37 °C
monitor Abs340 for 10 mins
PPiAsn
Boehlein, S., et al, J. Biol. Chem., 1994, 269(43), 26789-9515

16. HPLC assay
DNFB derivation
pH 8 buffer + substrate
+ enzyme
10 mins at 37 °C
quench, centrifugation,
aliquot pH 9 buffer + DMSO
+ DNFB in EtOH
45 mins at 50 °C
quench, neutralize, spin,
analyze using RP-HPLC
Asn, Gln, Asp, or Glu
T. M. Larsen, et al, Biochemistry 1999, 38, 16146-1615716

17. Ubiquitination assay
UbcH10
Degradation mix
[35S] -securin
Cell extract + + + + + + + + + + + +
+ + + + + + + + + + + +
+ + + + + + + + + + + +
- + + + + + - - - + + +
Time (min) 0 30 60 0 30 60 0 30 60 0 30 60
Second First
Room temperature
17

18. Enzyme kinetics and
simulation
18

19. Enzyme kinetics - 1
0
10
20
30
40
50
60
70
80
0 0.5 1 1.5 2
1/v
1/[Gln] (mM-1)
0
5
10
15
20
0 0.2 0.4 0.6 0.8 1
apparentKM(mM)
L-asparagine (mM)
19

20. E + Gln
+
Asn
EAsn
E + GluEGln
k1
k2
kcat
k3k4
[Gln]
[Gln]max
+
= '
mK
v
v )
]Asn[
1(
I
m
'
m
K
KK +=
mM11.0
3
4
==
k
k
KI
Kinetic model - 1
20

21. Enzyme kinetics and simulation - 2
0
5
10
15
20
25
30
0 0.1 0.2 0.3 0.4 0.5
L-asparagine (mM)
apparentKM(mM)
EE + Gln
+
Asn
EEAsn
AsnEEAsn
+
Asn
+
Asn
AsnEEGln
EE + GluEEGln
k1
k2
kcat
AsnEAsnEAsn
+
Asn
KIs2
KIs3
KIiKIs1
M
Ii
IsIsIsIsIsIs
M K
K
I
KKK
I
KK
I
K
I
K ×
+
+++
=
][
1
][][][
1
' 321
3
21
2
1
mM33.0mM09.0
mM06.0
mM7.0
mM)116.0(
mM)33.0(
mM)117.0(
21

22. K. Li, et al, Biochemistry 2007, 46(16), 4840-4849
7.8 x 10-3
[14
NH3] s-1
E + 14
Gln TE
E + Glu
E + 15
Gln
7.8 x 10-3
[15
NH3] s-1
1.06 x 10-4
[H2O] s-1
5100 M-1
s-1
Enzyme kinetics – 3
22

23. K. Li, et al, Biochemistry 2007, 46(16), 4840-4849
Simulation – 3
23

24. E.ATP.Asp
k-1
k1[Gln]
E.ATP.Asp.Gln
k2
E + Glu + 14Asn
k13
E.ATP.Asp + Glu
k-3
k3[15
NH3]
E.ATP.Asp.15NH3
k4
E + 15Asn
k12[15
Asn] k-12
E.ATP.Asp.15NH3.15Asn
k8[15
Asn] k-8
E.ATP.Asp.15Asn
k-9
k9[15NH3]
E.15Asn + 15Asn
k10
k11[14Asn] k-11 k5[14Asn] k-5
E.ATP.Asp.15NH3.14Asn E.ATP.Asp.14Asn
k-6
k6[15
NH3]
E.15Asn + 14Asn
k7
K. Li, et al, Biochemistry 2007, 46(16), 4840-4849
Kinetic model – 4
24

25. Simulation – 4
25

26. Simulation at physiological conditions
0.1
0.6
1.1
1.6
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0
0.25
0.5
0.75
1
1.25
1.5
1.75
2
2.25
2.5
2.75
3
3.25
3.5
3.75
4
velocity(s-1
)
[G
ln]m
M
[Asn] mM
0.1
0.4
0.7
1
1.3
1.6
1.9
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
2
0
0.25
0.5
0.75
1
1.25
1.5
1.75
2
2.25
2.5
2.75
3
3.25
3.5
3.75
4
velocity(s-1
)
[Gln]mM
[Asn] mM
26

27. Instrumental analysis
27

28. RPIP-HPLC analysis of carbohydrate
Time (min)
0 10 20 30 40 50 60 70 80 90 100 110 120
Radioactivity(cpm)
0
500
1000
1500
2000
2500
3000
3500
28

29. SEC/HPLC
29

30. K. Li, et al, Biochemistry 2007, 46(16), 4840-4849
gHMQC 1H-NMR
30

31. LC-MS analysis of disaccharide
31

32. Protein expression,
purification and
characterization
32

33. Plasmid Construction
33

34. In vitro translation using reticulocyte cell lysate
34

35. In vitro translation using wheat germ extract
35

36. Prediction of secondary structure
36

37. SEC analysis of quaternary structure changes
upon inhibitor binding
0 - 5 mM Asn
ASB
0 and 5 mM Asn
hASNS
37

38. SEC analysis of quaternary structure changes
with increasing concentrations
38

39. Enzymatic activity of truncated mutants
Cut 1
Cut 4
Cut 2
Cut 3
Cut 5
Cut 6
TM
Conserved
region 1
Conserved
region 2
39

40. Probing the key residue of epimerase
Kai Li et al, JBC 40

41. Bioinformatics and
protein engineering
41

42. Multivariate analysis
42

43. Cluster analysis
43

44. Network analysis
44

45. Computer aided protein engineering - 1
T. M. Larsen, et al, Biochemistry 1999, 38, 16146-16157
Berman, H.M., et al., Nucleic Acids Research, 2000. 28(1): p. 235-242
45

46. Computer aided protein engineering - 2
46

47. Computer aided protein engineering - 3
47