bsc hon presentation
TRANSCRIPT
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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
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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.
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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
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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
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2. Small Angle X-ray Scattering (SAXS)Wild-type protein (Tetrameric)
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Dimer vs Tetramer
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Arg126Ser variant (Dimeric)
2. Small Angle X-ray Scattering (SAXS)
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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
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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 (%
)
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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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Arg126SerMutation
Conclusions
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Conclusions
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The dimeric form of
NmeDAH7PS is a
FUNCTIONAL UNIT
Similar metal ion
dependency
Similar catalytic
efficiency
Similar regulatory properties
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Conclusions
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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
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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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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