Recent Advances in Biomolecular NMR Lucia Banci CERM University of - - PowerPoint PPT Presentation

Recent Advances in Biomolecular NMR

Lucia Banci CERM – University of Florence

Recent Advances in Biomolecular NMR

• Protonless NMR

for the characterization of Unfolded proteins, Large protein assemblies, Paramagnetic systems

• In cell NMR

For studying biomolecules in a cellular context

• Combination of Solution and Solid State

NMR

For characterization of dynamic proteins and large aggregates

• Mechanistic Systems Biology

To describe and understand biological processes at molecular level

Why protonless NMR?

1H 13C 15N

Inverse (i.e. through 1H) detection

• f heteronuclei was a major

advanchement!!

Properties of 1H (high gH, ..)  high 1H sensitivity / large dipolar interactions / efficient relax processes (paramagnetic and large)  relatively low chemical shift dispersion (unfolded systems)

13C direct detection

…with increase in sensitivity, (high B0, cryo!) direct detection of heteronuclei (low  nuclei) becomes accessible

Isotopic enrichment necessary anyway

13C direct detection

is a complementary tool

1H 13C 15N

13C direct detection, protonless NMR

A complementary tool for challenging systems

• paramagnetic proteins
• very large proteins
• parts of proteins affected by

exchange processes

• unfolded systems
• high salt concentrations

1H 13C 15N

C´ direct detection – The experiments

Set of exclusively heteronuclear experiments based on C´ and Ca detection for sequence specific assignment of a protein

More complete information   automation Solution & solid state NMR   common/complementary

C´ direct detection - IPAP

SUM DIFF IP AP

Set-up on 13C-15N labeled Alanine Nielsen N.C., Thøgersen H., Sørensen O.W., J. Am. Chem. Soc., 1995, 117, 11365-6 Ottiger M., Delaglio F., Bax A., J. Magn. Reson., 1998, 131, 373-378 Andersson P., Weigelt J., Otting G., J. Biomol. NMR, 1998, 12, 435-441 Duma L., Hediger S., Lesage A., Emsley L., J. Magn. Reson, 2003, 164, 187-195 Hu K., Eletsky A. Pervushin K., J. Biomol. NMR, 2003, 26, 69 Bertini I., Felli I.C., Kümmerle R., Luchinat C., Pierattelli R., J. Biomol. NMR, 2004, 30, 245-251

Info on the spiltting!!  RDC!!!

C´ direct detection – CON-IPAP

d(13C´)

CON C´i-Ni+1

Transfer pathway: F1(CO)  F3(N,t1)  F1(CO,t2) Correlations observed: N i -C´i-1

CON-IPAP - The delays are:  = 9 ms, 1 = 25 ms,  = t1(0). The phase cycle is: 1 = x,-x; 2 = 2x,2(-x); 3 = 4x,4(-x); IPAP(IP) = x; IPAP(AP) = - y; rec = x,(-x),x,(-x),(-x),x,(-x),x. Quadrature detection in the F1 dimension is obtained by incrementing 1 in a States-TPPI manner.

d(15N)

C´ direct detection – CON-IPAP

161 out of the 163 expected correlations are resolved

CON-IPAP 600 MHz Prototype cryoprobe

• ptimized for

13C sensitivity

(S/N 1400:1) Reduced monomeric SOD (15 kDa)

Bermel, W.; Bertini, I.; Felli, I. C.; Kümmerle, R.; Pierattelli, R. J.Magn.Reson. 2006, 178, 56-64.

C´ direct detection –CACO-IPAP

d(13C´)

CACO C´i-Ca

i

d(13Ca)

Transfer pathway: F1(Ca, t1)  F1(CO,t2) Correlations observed: Ca

i -C´i

CACO-IPAP - The delays are:  = 9 ms. The phase cycle is: IPAP (IP)= x,-x and rec = x,-x; IPAP (AP)= -y, y and rec = x, -x. Quadrature detection in the F1 dimension is obtained by incrementing 1 in a States-TPPI manner.

C´ direct detection –CBCACO-IPAP

d(13Ca,b) d(13Ca) d(13C´)

CBCACO C´i-Ca

i

C´i-Cb

i

Transfer pathway: F1(Ca/b, t1)  F1(Ca, t2)  F1(CO,t3) Correlations observed: Cb

i - Ca i -C´i, Ca i - Ca i -C´i

CBCACO-IPAP - The delays are:  = 9 ms, 1 = 8 ms. The phase cycle is: 1 = x,-x; 2 = 8x,8(-x); 3 = 2y,2(-y); IPAP(IP) = 4(x),4(-x); IPAP(AP) = 4(-y),4(y); rec = x,(-x),(-x),x,(-x),x,x,(-x). Quadrature detection in the F1 and F2 dimensions is obtained by incrementing 1 and 3 in a States-TPPI manner.

C´ direct detection – S3E

d(13Ca,b) d(13C) d(13C´)

CCCO

d(13Ca,b,..)

C´i-Cb

i

C´i-C

i

C´i-Cd

i

Transfer pathway: F1(Cali, t1)  F1(Ca, t2)  F1(CO,t3) Correlations observed: Cali

i - Ca i -C´i, Ca i - Ca i -C´i

CCCO-IPAP - The delays are:  = 9ms,  = t1(0). The phase cycle is: 1 = x, -x; 2 = 2x, 2(-x); IPAP(IP) = 4x, 4(-x); IPAP(AP) = 4(-y),4y; rec = x, (- x), (-x), x, (-x), x, x, (-x). Quadrature detection in the F1 and F2 dimensions is obtained by incrementing 1 and 2 respectively in a States-TPPI manner.

C´ detection - Assignment strategy

d(13C´) d(13Ca)

CACON C´i-Ca

i

d(13C´)

CBCACON

d(13Ca,b)

C´i-Ca

i

C´i-Cb

i

d(13C) d(13C´)

CCCON

d(13Ca,b,..)

C´i-Cb

i

C´i-C

i

C´i-Cd

i

Spin system identification

CACO, CBCACO, CCCO-IPAP

CACO-IPAP CBCACO-IPAP CCCO-IPAP 600 MHz Cryoprobe

• ptimized for

13C sensitivity

(S/N 1400:1) 16 scans 2-3.5 hours

the majority of the 13C spin systems could be assigned

Bermel W., Bertini I., Duma L., Felli I.C., Emsley L., Pierattelli R., Vasos P.R., Angew. Chem., 2005, 44, 3089- 3092 Bermel, W., Bertini, I., Felli, I. C., Kümmerle, R., Pierattelli, R. J.Magn.Reson. 2006, 178, 56-64.

C´ detection - Assignment strategy

d(13C´) d(13C´)

C´i-C´i-1 C´i-C´i+1 COCON C´i-C´i

d(13C´)

C´i-Ca

i

C´i-Ca

i+1

CANCO

d(13C´)

CBCANCO

d(13Ca,b)

C´i-Ca

i

C´i-Cb

i

C´i-Cb

i+1

d(13Ca)

C´i-Ca

i+1

Sequential assignment

C´ detection - Assignment strategy CBCACON-IPAP CCCON-IPAP

Dd(13C’) Dd(13C’)

@600 MHz CPTXO (S/N 1400:1) on 1.5 mM 13C, 15N labeled reduced monomeric

• SOD. CBCACON-IPAP, 16 scans, 3 days, CCCON-IPAP, 32 scans, 4.5 days.

96 % of the 13C resonances could be identified

Bermel W., Bertini I., Felli I.C., Kümmerle R., Pierattelli R., JMR, 2006, 178, 56-64

C´ detection - Assignment strategy PRO 74 LYS 75

Bermel, W., Bertini, I., Felli, I. C., Kümmerle, R., Pierattelli, R. J.Magn.Reson. 2006, 178, 56-64.

Intrinsically disordered proteins - IDPs!

Folded Aggregated

One of the powerful applications of 13C direct detection NMR

Synuclein 140 AA IDP Cu(I)Zn(II)SOD 153 AA Well folded

... Reduction in 1H chemical shifts

178 177 176 175 174 173 172 171 170 145 140 135 130 125 120 115 110 105

15N chemical shift

13C chemical shift

HN

i-Ni

C´i-1-Ni

Schwarzinger S., Kroon G.J., Foss T.R., Chung J., Wright P.E., Dyson H.J., J. Am. Chem. Soc. 2001, 123, 2970-2978 9,2 9,0 8,8 8,6 8,4 8,2 8,0 7,8 7,6 7,4 145 140 135 130 125 120 115 110 105

15N chemical shift 1H chemical shift

Zhang, H., Neal, S., Wishart, D.S., J. Biomol. NMR 2003, 25, 173-195

13C carbonyl direct detection – IDPs

CON of intrinsically unfolded a-synyclein

All residues assigned (N,C´,Ca,Cb)

Bermel W., Bertini I., Felli I.C., Lee Y.M., Luchinat C., Pierattelli R., J. Am. Chem. Soc., 2006, 128, 3918-3919

Prolines are visible

Intrinsically unfolded a-synyclein

Bermel W., Bertini I., Felli I.C., Lee Y.M., Luchinat C., Pierattelli R., J. Am. Chem. Soc., 2006, 128, 3918-3919

3D CBCACON-IPAP 3D COCON-IPAP

Strips from the 3D COCON-IPAP

Sequence specific assignment

Interphase Prophase Prometaphase Metaphase Anaphase Telophase Cytokinesis Metaphase Anaphase

Securin inhibitor of separase

Securin Intrinsically disordered protein (IDP!) 202 AA (>10% PROs)

Securin – Intrinsically disordered protein

Intrinsically unfolded human securin

PRO (N) 22 corr obs GLY (N) 11 corr obs

Observed well resolved peaks: HSQC: 122 68% of the expected 60% of the whole protein CON: 165 82% of the expected 82% of the whole protein

GLY (N) 9 corr obs

Securin – 202 AA, 24 PRO

Csizmok V., Felli I., Tompa P., Banci L., Bertini I., J. Am. Chem. Soc., 2009, 130, 16873-16879

Intrinsically unfolded human securin

193, out of the 201 expected, spin patterns are identified (96%) in CBCACON-IPAP. Correlations observed: Ca

i,C´i,Ni+1

Cb

i, C´i,Ni+1

Csizmok V., Felli I., Tompa P., Banci L., Bertini I., J. Am. Chem. Soc., 2009, 130, 16873-16879

Securin – 202 AA, 24 PRO

a-helical secondary structure propensity for the stretch D150-F159

Csizmok V., Felli I., Tompa P., Banci L., Bertini I., J. Am. Chem. Soc., 2009, 130, 16873-16879

Assignment and chemical shift analysis of securin

D150-F159, E113-S127 and W174-L178

Csizmok V., Felli I., Tompa P., Banci L., Bertini I., J. Am. Chem. Soc., 2009, 130, 16873-16879

Human securin - other NMR observables

 Can one implement all the tricks to reduce experimental time?

• Longitudinal relaxation enhancement
• Reduction in datapoints acquired in indirect dimensions

Decrease the recycle delay 13C direct detection – Speeding up?

Diercks, T.; Daniels, M.; Kaptein, R. J.Biomol.NMR 2005, 33, 243-259. Deschamps, M.; Campbell, I. D. J.Magn Reson. 2006, 178, 206-211. Schanda, P.; Brutscher, B. J.Am.Chem.Soc. 2005, 127, 8014-8015. Müller, L. J.Biomol.NMR 2008, 42, 129-137. Bermel, W., Bertini I., Felli I.C., Pierattelli, R., J. Am. Chem. Soc., 2009, 131, 15339-15345

Longitudinal relaxation enhancement

1H-start, 1H-flip

13C direct detection – Speeding up

Bermel, W., Bertini I., Felli I.C., Pierattelli, R., J. Am. Chem. Soc., 2009, 131, 15339-15345

(Hflip)CACO-IPAP ~2 min

3 days ½ day

(Hflip)CANCO -IPAP

Reduction in datapoints acquired in indirect dimensions 13C direct detection – Speeding up

In-cell NMR spectroscopy

• In-cell NMR allows the characterization of biomolecules

inside living cells.

• It relies on high resolution NMR experiments to obtain

information at atomic resolution on biomolecule structure, folding and interactions.

• It has a high biological relevance, as the biomolecules are

monitored in a cellular environment.

In-cell NMR spectroscopy

In-cell NMR: what’s new?

• With traditional in vivo NMR, few small metabolites were

monitored in living cells. With high resolution in-cell NMR a biomacromolecule, such as a protein is characterized at atomic level.

• To overcome the sensitivity limit of NMR, the protein

needs to be concentrated, often above the physiological levels (that is why “in-cell” and not “in vivo”, but the cells are alive!).

• Uniform or selective isotopic labelling of the molecule of

interest with NMR-active nuclei is needed to record heteronuclear multidimensional NMR experiments.

Non-specific effects:

• The microscopic viscosity slows down the tumbling rate of the protein,

increasing its rotational correlation time tc, and increasing the linewidth

• f the protein NMR signals.
• Weak interactions with small cellular components have the same effect.
• Crowding and ionic strength can cause small chemical shift differences

between in-cell and in vitro.

• P. Selenko, G. Wagner, Journal of Structural Biology, 158 (2007).

Effects of the cellular environment

The cellular environment can have protein-specific and/or general, non specific effects

Protein-specific effects:

• Some proteins can interact strongly with high molecular weight cellular

components (e.g. DNA, heat-shock proteins, folding machineries, cell membranes).

• This is a protein-dependent effect, which can cause line broadening up to

the complete loss of the NMR signals.

• P. Selenko, G. Wagner, Journal of Structural Biology, 158 (2007).

Effects of the cellular environment

• Different cells have been and are used: bacteria, oocytes

and mammalian cells. Different techniques are exploited to

• btain high protein concentration: overexpression and

injection/insertion.

• Prokaryotic cells are more commonly used. Indeed, the

bacterial cytoplasm is a good model of the eukaryotic one, in terms of molecular crowding, pH and redox potential.

• Eukaryotic cells have been used to monitor protein

interactions with specific cellular components, such as

• kinases. They have the machineries and chaperones for

the correct maturation of eukaryotic proteins.

Different living organisms

Prokaryotes: E. coli cells

NMR in E. coli cells allows to investigate the folding state of a nascent protein, to monitor maturation steps which do not require specific chaperones, to have insights on interactions with the environment, and to investigate the structural features and interaction network of a protein, Interactions can be studied by sequentially expressing two or more proteins, with only one labelled.

• Pros: fast growing, easy to handle, good overexpression, ease
• f labelling.
• Cons: no cellular compartments, no machineries for protein

maturation, difficult to insert external proteins or small molecules.

• E. coli cells allow for overexpression of the protein of interest.

The strategy to obtain selective isotopic labelling of the protein consists of switching the culture medium before the induction of the protein with a medium containing isotopically labelled nutrients.

Protein expression in E. coli cells

13C 15N

Biomass growth in LB medium Protein expression in M9 minimal medium

• 1. Cell centrifugation
• 2. Cell resuspension
• 3. Induction
• 1. Cell centrifugation
• 2. Cell resuspension

Transformed cells NMR tube The figure shows a protocol adapted from: Z. Serber et al, Nature Protocols, vol. 1 n° 6 (2006).

• E. coli sample preparation

In cell NMR on eukaryotic cells allows the characterization of an eukaryotic protein in its true environment, and therefore it is more physiologically relevant. The effects of post-translational modifications and localization in different cellular compartments can be monitored directly.

• Pros: more physiologically relevant, correct protein maturation
• ccurs, sub-cellular localization of the protein can be monitored.
• Cons: slower to grow, more difficult to handle than bacterial

cells, difficult to obtain a high amount of protein inside the cells.

Eukaryotic cells

Two kinds of cells have been used: mammalian cells strains (e.g. CHO, HeLa), and Xenopus laevis oocytes.

Protein insertion in eukaryotic cells

For mammalian cell cultures, protein insertion is achieved by using cell- penetrating peptides to deliver a fusion protein, or porins to permeabilize the cells. To insert the protein into X. laevis oocytes the microinjection technique is used.

• Fig. From: D.S. Burz, A. Shekhtman, Nature, 458 (2009).
• P. Selenko, G. Wagner, J Struct Biol, 158 (2007).
• K. Inomata et al, Nature, 458 (2009).

Virtually any solution NMR pulse sequence can be used for in-cell NMR experiments, BUT:

• The low sensitivity of NMR requires high protein

concentration, not always obtained;

• The viability of the cell sample is limited to few hours;

NMR pulse sequences

Therefore fast and sensitive experiments are often needed:

• Fast pulsing experiments: 2D SOFAST-HMQC, 3D

BEST-triple resonance experiments;

• Sparsed sampling experiments: non-uniform

sampling, projection spectroscopy.

The 1H-15N SOFAST-HMQC(1) is often used for in-cell NMR. It is the fast equivalent of the 1H-15N HMQC. The selective 1H pulse excites only the amide protons, allowing faster longitudinal relaxation between the scans: shorter interscan delays. The pulse can be set at the Ernst angle α (120° instead of 90°), to maximize sensitivity.

SOFAST-HMQC

Schanda,P., Kupce,E., and Brutscher,B., J. Biomol. NMR 33, 199-211 (2005).

Application: In-cell NMR of hSOD1

• Human superoxide dismutase 1

(hSOD1): 32 kDa dimeric enzyme.

• The mature form binds one

copper ion and one zinc ion per monomer (Cu,Zn-hSOD1). It has an intramolecular disulfide bridge.

• The in vivo copper loading and

disulfide formation involves the copper chaperone for SOD (CCS).

Banci, L., Barbieri, L., Bertini, I., Cantini, F., Luchinat, E., Nascent SOD1 analyzed by in-cell NMR, Submitted

• Solution NMR assignment serves as the starting points of SSNMR data

assignment

• SSNMR assignment can help to resolve some uncertainties in solution NMR

assignment of partially unfolded species

A Cross-talk between Solid-State (SS) and Solution NMR Apo SOD1 – a partially disordered molecule

Hints on the structures of fibril-ready states

Average Structural Differences between solid-state and solution (b propensity) Apo dimer chemical shifts

(13C,15N) + X-ray

structure

Pintacuda et al

• Angew. Chem. 2007

Banci et al, Proc. Natl. Acad. Sci. 2009 Banci et al, Biochemistry 2003 Banci et al

• Eur. J. Biochem. 2002

Solid-state (crystals/microcrystals) Solution-state

Cu, Zn dimer chemical shifts

(13C, 15N)

Apo dimer/monomer chemical shifts

(1H, 13C, 15N)

Cu, Zn dimer chemical shifts

(13C, 15N)

aid for assigning loops starting point of assignment

TALOS +

comparison comparison

aid for assignment

TALOS +

Banci L., et al, J. Am. Chem. Soc., 2011, 133, 345–349

NMR spectra of ApoSOD1 in Microcrystals and Solution

• Similarity in spectral patterns permits the integrative analysis of both

SSNMR and solution NMR data Red : 13C-13C 2D TOCSY spectrum of apoSOD1 in solution Blue : 13C-13C 2D DARR spectrum of apoSOD1 in microcrystals

d2 (13C) /ppm d1 (13C) /ppm Banci L., et al, J. Am. Chem. Soc., 2011, 133, 345–349

Hot Spots for ApoSOD1 Amyloidosis

Loop IV Loop VII A/L (crystals) -> B (solution)

• In solution loops IV and VII gain transiently high β-propensity
• SSNMR and solution NMR are complementary methods
• SSNMR facilitates the use of solution NMR data for understanding

the mechanism of amyloidosis at residue specific level

Banci L., et al, J. Am. Chem. Soc., 2011, 133, 345–349 B (crystal) -> A/L (solution)

Mechanistic Systems Biology

Mechanistic Systems Biology

Complex living systems should be studied in their integral state

Functional processes need to be described based on the 3D structural and dynamic interactions of the various players. …A system-wide perspective requires the identification of all the players in the studied process and within the “system” under analysis Proteins must be framed within their cellular context

Structure of a mitochondrion

ATP Syntase

O2 flows in and over 50 kg/day

• f ATP are

produced

A relatively small-scale, physiologically central system:

The InterMembrane Space of the Mitochondrion

Mitochondria derive from parassitic Gram-negative bacteria: they contain 1000 proteins but only 15 are produced in situ The large majority of them must be imported, including those involved in copper trafficking

COX17

CcO copper chaperone

CuB

subunit 1 of CcO

CuA

subunit 2 of CcO

Cytc

cytochrome c

Tim

mitochondrial import Traslocase Inner Membrane

Some mitochondrial pathways involved in protein import and copper transport

COX11

CcO assembly protein ctaG

SCO

Synthesis of CcO

Mia40

Mitochondrial intermembrane space Import and Assembly protein 40

ALR

Augmenter of Liver Regeneration

Banci, Bertini, Cefaro, Ciofi Baffoni, Gallo, Sideris, Tokatlidis Nat Struct Mol Biol 2009

Partially Reduced Mia40 Oxidized Mia40

E0 < -0.34 V 2 mM DTT CPC motif CX9C O2 E0 -0.20 V

Solution structure of Mia40, a key protein in IMS protein import

cytosol

OM

Mia40

IM

matrix

Reduced apoCox17 is unstructured

apoCox172S-S

IMS

SH SH SH SH SH SH SH SH SH SH SH SH

Cox17 mitochondrial import

Banci L, Bertini I, Cefaro C, Cenacchi L, Ciofi-Baffoni S, Felli I C, Gallo A, Gonnelli L, Luchinat E, Tokatlidis K, PNAS, 2010

Cox17 is unfolded in the cytoplasm

detected in living cells The protein folding state depends on the cellular compartment

cytosol OM

SH SH SH SH SH SH

IM matrix IMS Mitochondria Mia40

SH SH S S S S

apoCox172SH

Hb-Cb

Cb 36.4 ppm Cb 39.5 ppm

1H 13C

2D 1H -13C HSQC apoCox172SH1S-S

Cb 28.0 ppm

Hb-Cb

Cb 29.5 ppm

SH

apoCox176SH

SH SH SH SH SH Hb-Cb (all)

Cb 25.3 ppm

13C

2D 1H-13C HSQC apoCox174SH

1H

Mitochondrial Oxidative Folding Mechanism by

13C NMR

Cox17 partially reduced Cox17 fully reduced Cox17 fully oxidized

Cox17 in mitochondria has a CHCH fold

Cox17 Mia40

Intermolecular Mia40-Cox17 disulphide

Solution Structure

• f the Cox17-Mia40

intermediate A hydrophobic cleft on Mia40 is the interaction site for Cox17

Banci, Bertini, Cefaro, Ciofi, Gallo, Sideris, Tokatlidis Nature Struct Mol Biol 2009

Upon intermolecular S-S bond formation, the first helix is formed

The first step in Cox17 folding

Cox171S-S Cox172S-S

+ O2

Disulphide formation

Banci, Bertini, Cefaro, Ciofi Baffoni, Gallo, Sideris, Tokatlidis Nat Struct Mol Biol 2009 S S S S H H

• r GSSG

Final steps in the maturation of Cox17

Then the first intramolecular S-S bond and the second helix are formed O2 can now rapidly form the second disulphide bond

Oxidative folding reaction between Mia40 and Cox17

Mia40 N N C Matrix IMS Cytoplasm TOM C N

O2

CHCH C C N Mia40- CHCH

H2O2

CHCH1S-S CHCH2S-S CHCH C N

Banci L, Bertini I, Cefaro C, Cenacchi L, Ciofi-Baffoni S, Felli I C, Gallo A, Gonnelli L, Luchinat E, Tokatlidis K, PNAS, 2010

Oxidative folding processes in IMS

Mia40 acts as a chaperon A general folding process for CHCH proteins

COX17

CcO copper chaperone

Mia40

Mitochondrial intermembrane space Import and Assembly protein 40

ALR

Augmenter of Liver Regeneration

Schematic overview of mitochondrial pathways

Banci L, Bertini I, Calderone V, Cefaro C, Ciofi-Baffoni S, Gallo A, Tokatlidis K PNAS, 2011

N C

Hydrophobic residues in the N-terminus are the crucial molecular mediators to efficiently guide the electron transfer process from Mia40 to FAD in ALR FAD ALR ALR’ There are two splicing variants for ALR: in mitochondria there is the long one N-term domain

Structure of lf-ALR

Banci L, Bertini I, Calderone V, Cefaro C, Ciofi-Baffoni S, Gallo A, Tokatlidis K PNAS, 2011

Hydrophobic interactions between Mia40 and the N- terminal domain of ALR mediate efficient electron transfer from Mia40 to FAD in ALR Mia40 ALR ALR’

Structural model of lf-ALR/Mia40 complex based on NMR interaction data

S-S S-S

FAD FAD

S-S S-S

N N + S S S S HS SH

FAD FAD

S-S

N N

+2e- +2e-

lfALR oxidized Mia402S-S lfALR reduced S-S S-S S-S

Banci L, Bertini I, Calderone V, Cefaro C, Ciofi-Baffoni S, Gallo A, Tokatlidis K PNAS, 2011

+2e- +2e-

S S S S +

Mia403S-S

Efficient transfer: The reaction of sf-ALR with MIA40 proceeds to completion at 0.5:1 molar ratio

Electron shuttling mechanism

Cu-Cox172S-S

IMS Matrix

CCO Cox11 Sco1 Sco2

Cu Cu Cu Cu Cu Cu Cu Cu

Copper incorporation in CcO

Banci, Bertini, Ciofi-Baffoni, Karit, Kozyreva, Palumaa, Nature, 2010

Towards systems biology of copper

Mia40

Mitochondrial intermembrane space Import and Assembly protein 40

ALR

Augmenter of Liver Regeneration

CytC

cytochrome c

e-

CcO

Cytochrome c Oxidase Complex IV

COX17

CcO copper chaperone

COX11

CcO assembly protein ctaG

CuB

subunit 1 of CcO

SCO

Synthesis of CcO

CuA

subunit 2

• f CcO

Cu(I) Cu(I) Cu(I) Cu(I)

e- e- e-

Schematic overview of mitochondrial pathways

Mia40

Mitochondrial intermembrane space Import and Assembly protein 40

ALR

Augmenter of Liver Regeneration

CytC

cytochrome c

e-

CcO

Cytochrome c Oxidase Complex IV

COX17

CcO copper chaperone

COX11

CcO assembly protein ctaG

CuB

subunit 1

• f CcO

SCO

Synthesis

• f CcO

CuA

subunit 2 of CcO

Cu(I) Cu(I) Cu(I) Cu(I)

e- e- e-

Tim

mitochondrial import Traslocase Inner Membrane

CHCH

coiled coil-helix- coiled coil-helix

e- e-

CIAPIN1 Cytokine Apoptosis Induced inhibitor 1

e-

Schematic overview of mitochondrial pathways

CERM – Magnetic Resonance Center

950*

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