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Examining the role of quaternary structure for the catalysis and regulation of DAH7PS from Neisseria meningitidis (Nme) Vicky Zhang Supervisor: Prof. Emily Parker 1

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Page 1: BSc Hon Presentation

Examining the role of quaternary structure for the catalysis and

regulation of DAH7PS from Neisseria meningitidis (Nme)

Vicky Zhang Supervisor: Prof. Emily Parker

1

Page 2: BSc Hon Presentation

IntroductionThe shikimate pathway

Neisseria meningitidis

(Trp) (Phe) (Tyr)

2DAH7PS: 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase

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IntroductionNmeDAH7PS Tight dimer interface

Dimer-dimer interface

3PDB Entry: 4HSN; Cross, et al. Protein science, 2013, 22, 1087-1099

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Project Aims

1. To disrupt the tetrameric structure of NmeDAH7PS by removing key interactions.

2. Characterise the interface variant and compare to the wild type NmeDAH7PS.

4

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Dimer-dimer interface

5Glu27Arg126

Arginine (Arg)

Glutamate (Glu)

Serine (Ser)

Arg126Ser Mutation

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Arg126Ser NmeDAH7PS

(MW~38 kDa)

kDa

260

40

30

6

1 2 3 4 5 6 7 8

The Arg126Ser variant protein

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1. Analytical Size Exclusion Chromatography

1.1 1.2 1.3 1.4 1.5 1.6 1.7 1.8 1.9 2 2.10

0.5

1

1.5

2

2.5

3

Ve / Vo

Log

(mol

ecul

ar w

eigh

t)Dimer vs Tetramer

7

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0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.20

0.5

1

1.5

2

2.5

3

Ve / Vo

Log(

mol

ecul

ar w

eigh

t)

Wild-type protein MW~142kDa

Dimer vs Tetramer

8

8 9 10 11 12 13 14 15 16 17 18 0

100

200

300

400

500

WT

Protein elution volume (mL)

Abs

orba

nce

280

nm (

mA

u)

1. Analytical Size Exclusion Chromatography

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0.6 0.8 1 1.2 1.4 1.6 1.8 2 2.20

0.5

1

1.5

2

2.5

3

Ve / Vo

Log

(mol

ecul

ar w

eigh

t)

Wild-type protein MW~142kDa

Arg126Ser variant MW~ 66kDa

Dimer vs Tetramer

9

8 9 10 11 12 13 14 15 16 17 18 0

100

200

300

400

500

WT

Arg126Ser

Protein elution volume (mL)

Abs

orba

nce

280

nm (

mA

u)

1. Analytical Size Exclusion Chromatography

WT Arg126Ser

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Dimer vs Tetramer

10

2. Small Angle X-ray Scattering (SAXS)Wild-type protein (Tetrameric)

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Dimer vs Tetramer

11

Arg126Ser variant (Dimeric)

2. Small Angle X-ray Scattering (SAXS)

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12

Characterisation1. Metal Ion Dependency

Mn Cd Co Zn Fe Cu Mg0%

20%

40%

60%

80%

100%

120%

Arg126SerWT protein

Divalent metal ions

Spec

ific

activ

ity

(%)

1. Cross, et al. Protein science, 2013, 22, 1087-1099

1

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DAH7PS KmPEP (μM) Km

E4P (μM) kcat (S-1)

NmeDAH7PS-WT1

11 ± 1 43 ± 4 25.5 ± 0.5

NmeDAH7PS-Arg126Ser

100 ± 7 22 ± 3 26.3 ± 0.4

2. Michaelis-Menten Kinetics

Characterisation

131. Cross, et al. Protein science, 2013, 22, 1087-1099

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0 100 200 300 400 500 600 700 800 900 10000%

20%

40%

60%

80%

100% PheTyrTrp

Inhibitor concentration (µM)

Spec

ific

act

ivit

y re

mai

nin

g (%

)

14

Characterisation3. Regulatory Properties

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Characterisation3. Regulatory Properties

DAH7PS KmPEP(μM) Km

E4P(μM) kcat (S-1)

NmeDAH7PS-WT1 (With 300 μM Phe)

21 ± 1 92 ± 10 6.9 ± 0.4

NmeDAH7PS-Arg126Ser(With 300 μM Phe)

25 ± 2 121 ± 12 15.2 ± 1

1. Cross, et al. Protein science, 2013, 22, 1087-1099

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PEP

Glu27

Arg126

Ser126

Mutation site

Active site

(rmsd=0.6 Å)

Crystal Structure

Yellow=WT structureGreen=Variant structure

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17

Arg126SerMutation

Conclusions

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Conclusions

18

The dimeric form of

NmeDAH7PS is a

FUNCTIONAL UNIT

Similar metal ion

dependency

Similar catalytic

efficiency

Similar regulatory properties

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Conclusions

19

The dimeric form of

NmeDAH7PS is a

FUNCTIONAL UNIT

Similar metal ion

dependency

Similar catalytic

efficiency

Similar regulatory properties

Crystal structure with Phe binding

Protein flexibility

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Acknowledgements

20

Prof. Emily Parker

Dr. Penelope Cross

Dr. Ali Nazmi

Dr. Marie Squire

Gerd Mittelstaedt

Dmitri Joseph

Logan Heyes

Nicky Blackmore

Sarah Wilson-Coutts

Tammie Cookson

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21

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Site-directed mutagenesis----Arg126Ser

5’ 3’

5’ 3’

3’ 5’

3’ 5’

5’ 3’3’ 5’

5’ 3’3’ 5’

Mutated plasmidParental plasmid

Transformation

Site-directed mutagenesis

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Protein purification

(http://chromacademy.com/Introduction_to_Ion_Chromatography_Essential_Guide.asp)

• Anion-exchange chromatography

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• Size-exclusion chromatographyProtein purification

Arg126Ser NmeDAH7PS(~38 kDa)

kDa 260

40

30

(http://en.wikipedia.org/wiki/Size-exclusion_chromatography)

Arg126Ser NmeDAH7PS

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Trp Tyr Phe45

46

47

48

49

50

51

52

53

48.3 48.1

52.3

Mel

ting

Tem

pera

ture

(⁰C

)

0

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X-ray Crystallography

Crystal growth: Hanging-drop diffusion at 20 °C for 6-8 days