Purification of c7 type cytochromes from

Geobactar sulfurreducens

Argonne National LaboratoriesBioscience Division

Summer 2012

Tom Hajek

Overview

What is Geobactar sulfurreducens? What processes do we use to get the desired

protein? Why are we so interested in this particular

protein? Results Long Term Goals

Our FriendGeobactar sulfurreducens

Found in soil and sediments.

“Eats” U and other heavy metals.

Currently being used in bio-remediation of ground water.

Produces MANY multi-heme proteins (cytochromes).

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• Periplasmic cytochrome A (PpcA)

• The most abundant cytochrome in the periplasm of G. sulfurreducens

• Involved in iron (III) reduction and can reduce other metals, e.g. U(VI), Tc(VII)

Cytochrome c7, PpcA

Londer et al., BBA, 1554 (2002), 202-211

What are Cytochromes?

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Cytochromes c: cytochromes with covalently bound heme(s)

Electron transfer proteins that carry heme as a prosthetic group Involved in photosynthesis & aerobic/anaerobic respiration Function is related to the valence change of heme iron, Fe2+ Fe3+

First we must clone....

The gene that codes for PpcA (c7) is cloned into E. coli and selected for by plating with antibiotics

Only the cells that are resistant and contain our gene of interest can grow.

The lac-operon with c7 gene(region of interest)

lac promoter OmpA leader c7 gene (mature)

Y. Londer et al 2002

XbaI HindIII

Role of IPTGRole of IPTG RNA polymerase is

“stuck” on promoter while the repressor is bound to operator.of the lac operon.

IPTG acts as an analogous substrate of allolactase and transcription can begin.

Expression of PpcA in Periplasmic Space

Isolation of Periplasmic Fraction Centrifugation Centrifugation

15min at 4000 rpm 15min at 4000 rpm @ 4@ 4°°C.C.

Re-suspend pellet in Re-suspend pellet in TES buffer (100mM TES buffer (100mM Tris-HCL, pH 7.5, Tris-HCL, pH 7.5, 0.5mM EDTA, 20% 0.5mM EDTA, 20% sucrose)sucrose)

Add protease Add protease inhibitor.inhibitor.

Add lysozyme and Add lysozyme and incubate @ 4incubate @ 4°°C.C.

Cold HOH shock.Cold HOH shock.Centrifuge 15min at Centrifuge 15min at

20K rpm @ 420K rpm @ 4°°C.C.Supernatant = Supernatant =

Periplasmic fraction Periplasmic fraction (this has PpcA in it)(this has PpcA in it)

Cation exchange column

Ionoshpere resin

Protein Purification

CysHis

Typical cytochrome c7

Heme cofactor is covalently attached to the polypeptide through thioether bonds

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10 20 30 C7-1 A D D . I V L K A K N G D V K F P H K A H Q K A V P D C K K C H E . K G P G K I C7-2 A D T . M T F T A K N G N V T F D H K K H Q T I V P D C A V C H G . K T P G K I C7-3 I D K . I T Y P T R I G A V V F P H K K H Q D A L G E C R G C H E . K G P G R I C7-4 A D . V I L F P S K N G A V T F T H K R H S E F V R E C R S C H E . K T P G K I C7-5 H D K V V V L E A K N G N V T F D H K K H A G V K G E C K A C H E T E A G G K I 40 50 60 70 C7-1 E G F G K E M A H G K G C K G C H E E M K K G P T K C G E C H K K - - - - PpcA C7-2 E G F G K E M A H G K S C K G C H E E M K K G P T K C G E C H K K - - - - PpcB C7-3 D G F D K V M A H G K G C K G C H E E M K I G P V R C G D C H K G G S T H PpcC C7-4 R N F G K D Y A H . K T C K G C H E V R G A G P T K C K L C H T G - - - - PpcE C7-5 A G M G K D W A H . K T C T G C H K E M G K G P T K C G E C H K K - - - - PpcD

Aligned sequences of the homologs illustrating the different distribution of charged residues

(acidic residues Asp and Glu shown in red and basic residues Lys and Arg shown in blue).

Insertions and deletions of residues also result in different arrangements in space of the side

chains causing variation in surface electrostatic potential.

PpcA c7-1 amino acid sequence

K = Lysine E = Glutamic Acid

CxxCH at residues 27-31, 51-55, 65-69

E39C, K70C, K29C

71 amino acids. Contains 3 heme

groups. “Pocket” in protein

may accept a photosensitizer.

Cysteine(Cys)

K = Lysine E = Glutamic Acid

18

Heme I

Heme III

Heme IV

K70

K29

E39

Cysteine Mutation sites in PpcA studied (E39C, K29C, K70C)

K = Lysine E = Glutamic Acid

Resultsλ 408 210  

No. A1 A2 A1/A221 0.763 1.504 0.507522 1.135 1.857 0.611223 1.625 2.154 0.754324 1.918 2.266 0.846625 1.947 2.308 0.843926 1.715 2.24 0.765427 1.436 2.169 0.661928 1.315 2.167 0.60729 1.328 2.223 0.5971

E39Cλ 408 210  

No. A1 A2 A1/A29 0.144 0.404 0.356

10 0.189 0.452 0.417611 0.225 0.474 0.474812 0.26 0.522 0.497913 0.254 0.509 0.499414 0.218 0.484 0.450115 0.175 0.448 0.3917

K29Cλ 408 210  

No. A1 A2 A1/A255 0.375 0.795 0.471756 0.593 0.926 0.640557 0.578 0.903 0.640358 0.362 0.709 0.510759 0.188 0.56 0.3363

K70C

Heme:Peptide; 1:1Determined by mass

spectroscopy.A = ε c l

ε = molar extinction coefficientc = concentrationl = path length of cuvette

λ=408nm=heme absorbanceλ=210nm= peptide absorbance

Peak shifts are indicative of heme reduction.

Addition of reducing agent to samples

α

αβ

β

γ

γ

Long-term goalLong-term goal

Combine Chemical and Biological Electron Transfer Function in Metalloprotein Hybrid.

Replace Chemical Solar Catalysts with Bio-hybrid Solar Catalyst.

Create an unlimited supply of clean fuel with minimal input of mechanical energy.

Challenge: Combine Chemical and Biological Electron Transfer Function in Metalloprotein Hybrid

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hv

Catalyst

Photo-sensitizerLinker:

Photosen

sitizer

Catalyst

2H+ H2

2e-

Chemical Solar Catalyst Bio-hybrid Solar Catalyst

hv

hv

e-

fastfast

Problem: Efficiency limited by inability to “re-charge” PS

Solution: Use multi-heme protein frameworks as source for multiple e-

Artero et. al 2008 Ang. Chem. Int. Ed. 47: 564

X

PI: D. Tiede (CSE-ANL)