Disulfide Bond Formation in vivo Dr. Jim BardwellThe screen versions of these slides have full...

Disulfide Bond Formation in vivoDr. Jim Bardwell

1The screen versions of these slides have full details of copyright and acknowledgements

Disulfide Bond Formation in vivo

1

Dr. Jim BardwellUniversity of Michigan

HHMIGraphics by D.Fass

Disulfide bond formation is an oxidation reaction

2e-

2

2

Thiol/thiolate Disulfide

2e

Graphics by D.Fass

Disulfide bonds

Stabilize proteins by reducing the entropy of the unfolded state

3

Allow redox control of protein activity

Probe protein folding pathways

Graphics by D.Fass



Disulfide bonds are formed in the E. coli periplasm

Cytoplasm

Periplasm

Extracellular space

4

Chris Anfinsen and the thermodynamic hypothesis

5Graphics by D.Fass

De novo disulfide formation in the E. coli periplasm

SH

SH

S

S

Protein

Protein

02 ?

DsbAS

S

SH

SHDsbA

6

Periplasm

Cytoplasmic membrane

Cytosol

DsbB

MQ

e-

Aerobic conditions

Anaerobic conditions

e-

e-

Anaerobic acceptors

Bardwell et al. Cell 67: 581-589

Bardwell et al. PNAS 90: 1038-42

Bardwell et al. Cell 98: 217-227

UQCytochrome oxidases

e-

02

e- e-

?



The secretory pathway

7Graphics by D.Fass

Ero1

ER thiol oxidases catalyze disulfide bond formation

Erv2Oxidative protein folding rescued

8

Substrate protein

2e-2e-

PDIO2 H2O2

Graphics by D.Fass

A model for QSOX-PDI cooperation

9Thorpe, C. et al., J. Biol. Chem. 2007; 282: 13929-13933Graphics by C. Thorpe



O2

Eukaryotic Endoplasmic reticulum

ProkaryoticPeriplasm

OxidationOxidation Substrate proteins

Eukaryotic vs. prokaryoticdisulfide bond formation

10Anaerobic electron acceptors

CytoplasmCytochromeoxidases

O2

DsbB

O2

Membrane

Ero1pFAD

Goldberger et al. (1963) JBC 238: 628Pollard et al. (1998) Mol. Cell 1: 171Frand and Kaiser (1998) Mol. Cell 1: 161

Ubiquinone or Menaquinone

Engineering a new pathway for the formation of disulfide bonds

11

Jean-Francois Collet

George Georgiou - U-Texas

DsbA and thioredoxin are structurally related

12CPHC CGPC

Martin, Bardwell and Kuriyan, Nature 365: 464-8



ThioredoxinS

S

S

SThioredoxin

SH

SH

S

SH

SH

DsbAS

S

Can we replace the entire pathway with thioredoxin?

13

ThioredoxinSH

SH

ThioredoxinS

S

Periplasm Cytoplasm

DsbB

S

Se-

e-

Electron transport

chain

SH

SH

DsbASH

SH

e-

?

1 . Produce more oxidizing thioredoxin mutants

Thioredoxin

…CGPC…

2. Thioredoxin is constructed to have export tag

14

into periplasm

ThioredoxinExport tag

Select for mutants rescuing the motility of a dsb-

Motility requires disulfide bonds

FlgI: component of the flagellum

15

Outer membranePeptidoglycan

Inner membrane



dsb+ dsb-

2 mutants are able to partially rescue dsb- phenotype

16

WT thioredoxin: CGPCThioredoxinCACCCPCC

Mutant protein is brown

17

Absorbance spectrum CACC

18Characteristic of an iron-sulfur cluster



Iron-sulfur clusters• Found in numerous proteins (ferredoxin, cytochrome bc1 complex)

• Two major groups

SCys

192Fe-2S 4Fe-4S

Fe FeSFe

FeSS

Cys Cys

Cys

S

S

Cys

CysCys

CysFe Fe

• Gel filtration: dimer• EXAFS: 2Fe-2S,

ferredoxin-type• Catalytically active in vitro

Mutant thioredoxin’s properties

20SH

S

S

S

SS

SFe Fe Thioredoxin

HS

Mutant has two exposed cysteines

21

CXXC CXC



Normal pathway

Oxidation Substrate proteinsOxidation

New pathway

SFe

SFe

SThioredoxin Thioredoxin

22Cytoplasm

MembraneUbiquinone

DsbB

O2

Masip et al., Science 303: 1184-89

O2

S

Fe FeSS

Thioredoxin Thioredoxin

DsbB

UQ

O

UQ

OH

DsbASH

S+ DsbA

S

S +

DsbB’s quinone reductase activity is the source of disulfides in the periplasm

23

O OHSH S

CC

S S

Current model for the mechanism of DsbB

SC e-

DsbASH

SH e- DsbAS

S

e-

24NH3

+COOH-

Q

SC



Lamp

Syringe 1

Drive

Monochromater

Kinetic characterization of the purple intermediate by stopped flow absorbance

hv

hv

25

Syringe 2 Stoppingsyringe

PMT/DA

hv

Tim TapleyTimo EichnerDave Ballou

Single wavelength kinetic measurement (510 nm)

26

k1 depends on [DsbA]

27



k2 is fairly DsbA independent

28

Kinetic measurements at 275 nm

29

Kinetic scheme for DsbB reaction cycle

30J. Biol. Chem. 282: 10263-71



Disulfide bond formation in prokaryotes

SH

SHSH

SH

SS

S

SMisfolded protein

DsbA

SS

SHSH

31

SHSH

Newly translocated protein

SS

SS

Native protein

DsbCDsbG

Alchemists were obsessed with turning common items into GOLD!!

32The alchemists, 1757Attempting to make

gold from eggs

E. coli — the modern alchemist

Glucose + air + salts Pharmaceuticalproteins

G CSF = $806 663/gram 2

# disulfides

33

G-CSF = $806,663/gram

GH = $35,000/gram

IGF-1 ≈ $1,000/gram

Insulin ≈ $400/gram

Gold ≈ $18/gram

2

2

3

3

0



De novo disulfide formation and isomerization in the E. coli periplasm

DsbASH

SH

S

S

S

DsbASH SHSH

SS

S

S SS

SS

SHSHSHSH

34

DsbB

S

DsbASH

e-

e-e-

Periplasm

Cytoplasmic membrane

Cytosol

QO2

Thioredoxin

SHSH

DsbD

e-

e-

SHSH

DsbCDsbGDsbC

What distinguishes DsbC from DsbG?

Annie Hiniker, Begoña Heras, Jeanne Stuckey, Jenny Martin

113Å 113Å

35

DsbG

50Å

DsbC

50Å

36



dsbC- is copper sensitiveCopper mis-oxidizes, DsbC repairs

37

WT DsbG does not rescue dsbC-

copper sensitivity

pDsbCpDsbG

38

Library of mutants Selection

Directed evolution of DsbG so it functions as DsbC

39

for copper resistanceDsbGStructure: Heras et al., 2004, PNAS

DsbCStructure: McCarthy et al., 2000, Nat. Struct. Biol.



DsbG K113E mutant reverses electrostatic charge near active site

DsbC WTCGYCH

DsbG WTCPYCK

DsbG K113ECPYCE

40

DsbG V216M mutant changes αc loop position

DsbC vs. DsbG WT DsbC vs. DsbG V216M

41

And reverses charge!

DsbG WTCPYCK

DsbG V216MCPYCK

42



43PNAS (in press)

Normal pathway Isomerase conversion

New pathway

Oxidation Oxidation

44

Ub Cytoplasm

CytOx

Membrane DsbBO2

DsbC

DsbG

S

SFe

SFe

SS

S

Funding: HHMINIH

Thioredoxin

45

Disulfide Bond Formation in vivo Dr. Jim BardwellThe screen versions of these slides have full...

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