Skills and Experience

Kai Li PhD

1

Assay development and optimization

2

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

Competition assay

4

Exchanging assay

K. Li, et al, Biochemistry 2007, 46(16), 4840-48495

PAPS

2OST

Y94A

Epimerase assay

6

In vitro FATylation – time course

7

Pull down assay

-- 220 kD

-- 97 kD

-- 66 kD

-- 45 kD

-- 30 kD

2 X HeLa 1 X HeLa 8

Cell based assays

9

BaF3 proliferation assay

Concentration (µg/mL)

0.0 0.5 1.0 1.5 2.0

[3H

] T

hym

idin

e I

nco

rpo

ratio

n (

%)

0

20

40

60

80

100

Heparin

Synthesized

10

FACS analysis of splicing factors

11

Cell cycle and cell imaging

12

Other assays used

13

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 assayGLDH coupled enzyme assay for glutaminase activity

14

NADH � NAD+

Pyrophosphate assayCoupled 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

HPLC assayDNFB 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

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

Enzyme kinetics and simulation

18

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

appar

ent

KM

(mM

)L-asparagine (mM)

19

E + Gln

+

Asn

EAsn

E + GluEGlnk

1

k2

kcat

k3

k4

[Gln]

[Gln]max

+=

'

mK

vv )

]Asn[1(

I

m

'

m

KKK +=

mM 11.03

4 ==k

kK

I

Kinetic model - 1

20

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)

app

aren

t K

M(m

M)EE + Gln

+

Asn

EEAsn

AsnEEAsn

+

Asn

+Asn

AsnEEGln

EE + GluEEGlnk

1

k2

kcat

AsnEAsnEAsn

+Asn

KIs2

KIs3

KIiK

Is1

M

Ii

IsIsIsIsIsIs

MK

K

I

KKK

I

KK

I

K

I

K ×

+

+++

=][

1

][][][1

' 321

3

21

2

1

mM 33.0mM 09.0

mM 06.0

mM 7.0

mM) 116.0(

mM) 33.0(

mM) 117.0(

21

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

7.8 x 10-3 [14NH3] s-1E + 14Gln TE

E + Glu

E + 15Gln

7.8 x 10-3 [15NH3] s-1

1.06 x 10-4 [H2O] s-15100 M-1s-1

Enzyme kinetics – 3

22

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

Simulation – 3

23

E.ATP.Aspk-1

k1[Gln]E.ATP.Asp.Gln

k2E + Glu + 14Asn

k13

E.ATP.Asp + Glu

k-3

k3[15NH3]E.ATP.Asp.15NH3

k4E + 15Asn

k12[15Asn] k-12

E.ATP.Asp.15NH3.15Asn

k8[15Asn] k-8

E.ATP.Asp.15Asnk-9

k9[15NH3]E.15Asn + 15Asn

k10

k11[14Asn] k-11 k5[14Asn] k-5

E.ATP.Asp.15NH3.14Asn E.ATP.Asp.14Asnk-6

k6[15NH3]E.15Asn + 14Asn

k7

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

Kinetic model – 4

24

Simulation – 4

25

Simulation at physiological conditions

0.1

0.6

1.1

1.6

0

0.2

0.4

0.6

0.81

1.2

1.4

1.6

1.82

00.250.50.7511.251.51.7522.252.52.7533.253.53.754

ve

locit

y (

s-1

)

[Gln] m

M

[Asn] mM

0.1

0.4

0.7

1

1.3

1.6

1.9

0

0.2

0.4

0.6

0.81

1.2

1.4

1.6

1.82

00.250.50.7511.251.51.7522.252.52.7533.253.53.754

ve

locit

y (

s-1)

[Gln

] mM

[Asn] mM

26

Instrumental analysis

27

RPIP-HPLC analysis of carbohydrate

Time (min)

0 10 20 30 40 50 60 70 80 90 100 110 120

Radio

activity (cpm

)

0

500

1000

1500

2000

2500

3000

3500

28

SEC/HPLC

29

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

gHMQC 1H-NMR

30

LC-MS analysis of disaccharide

31

Protein expression, purification and characterization

32

Plasmid Construction

33

In vitro translation using reticulocyte cell lysate

34

In vitro translation using wheat germ extract

35

Prediction of secondary structure

36

SEC analysis of quaternary structure changes

upon inhibitor binding

0 - 5 mM Asn

ASB

0 and 5 mM Asn

hASNS

37

SEC analysis of quaternary structure changes

with increasing concentrations

38

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

Probing the key residue of epimerase

Kai Li et al, JBC40

Bioinformatics and protein engineering

41

Multivariate analysis

42

Cluster analysis

43

Network analysis

44

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. 235-24245

Computer aided protein engineering - 2

46

Computer aided protein engineering - 3

47