movimiento de atracción y formación del complejo

Movimiento de Atraccióny

Formación del Complejo

Long-range interactions (global charges)

-+

+++ ---

Short-range interactions (local charges)

Reorientation

Collision

Sliding along themembrane surface

Free diffusion

Interaction between Plastocyanin and PSI

Sliding along themembrane surface

Collision

Reorientation

Free diffusion

Electrostatic Protein-Protein Interactions

Long-range (global charges)Monopole-monopole interactions

Short-range (local charges)Dipole-dipole interactionsMonopole-dipole interactions

Dipole-Dipole Interaction of Plastocyanin with PSI

--

+ -

--+ -

--+

-

--

+ -

--

+

-

Heme P62H87

Y83

Cytochrome c6 Plastocyanin

Frazão et al. (1995) Structure 3, 1159-1169


R67



P62Heme H87


Cytochrome f

Plastocyanin


Mutagénesis Dirigida

Análisis Funcional de las Interacciones entre Proteínas

Lys33(35)His87(92)

Leu12(14)

Arg88(93)

Tyr83(88)

Lys58(62)

His57(61)

Asp49(54)AnabaenaPlastocyanin

Molina-Heredia et al. (2001) J. Biol. Chem. 276, 601-605

WTK33E K58A D49K

+-

Reactivity towards photosystem I

Surface electrostatic potential

Anabaena Plastocyanin

Con

stan

te d

e ve

loci

dad

Fuerza iónica

Efecto de la fuerza iónica sobre las interacciones entre proteínas

Val25

Arg64Lys66

Asp72

Lys62Lys29

hydrophobicpatch

Cytochrome c6 from AnabaenaTarget residues for site-directed mutagenesis

chargedarea


Anabaena Cytochrome c6

WT V25E K62E

R64E K66E D72K


Anabaena Plastocyanin


Anabaena


Cytochrome c6Plastocyanin

Anabaena


De la Rosa et al. (2002) Bioelectrochemistry 55, 41-45

Orientación de los Centros Redoxy

Disociación del Complejo

Plastocyanin-PSI Complex

E. Myshkin et al. (2002) Biophys J. 82, 3305

Cyt c6Pc

PS I

b6f

PSI-driven Electron Transfer

Fd

light

Green algaC. reinhardtii

CyanobacteriumPhormidium laminosum

Cytochrome f

Higher plantBrassica rapa

Electrostatic complex between cytochrome f and plastocyanin

Ubbink et al. (1998) Structure 6, 323-335

Ubbink et al. (1998) Structure 6, 323-335

Electron transfer pathway from cytochrome f to plastocyanin

Crowley et al. (2002) submitted

Chemical shift map of Ana-cytc6 in the presence of cytf

Residues are coloured according to increasing Bind from blue to red

Prolines and the haem group are in grey and green, respectively

Complejo INADH-quinonaóxido-reductasa Complejo II

Succinato-quinonaóxido-reductasa

Complejo IIIQuinol-citocromo cóxido-reductasa

Complejo IVCitocromo c-O2

óxido-reductasa

citocromo c

Cadena respiratoria mitocondrial

Complejo III (o citocromo bc1)Quinol-citocromo c óxido-reductasa

Cyt c

Cyt c1

Cyt c

Cyt c1

Interacción entre el complejo bc1 (inmerso en la membrana) y el citocromo c (soluble)

Interacción entre los citocromos f y c

Cyt c1

Cyt c

Cyt c1

Cyt c

Hemo c

Cyt c1

Hemo c1Cyt c

Zona de contacto entre los hemos f y c

Main conformational changes between the oxidized (red) and reduced (blue) forms of Monoraphidium cytochrome c6

De la Rosa et al. (2002) Bioelectrochemistry 55, 41-45

Centro de Investigaciones

Científicas

Isla de la Cartuja

Sevilla

Universidad de Sevilla

http://transientprot.ibvf.csic.es

Workshop on Weak Protein-Protein Interactions

Seville, SpainJuly 9-13, 2003

Target residues for mutagenesisTarget residues for mutagenesisof cytochrome of cytochrome cc66

AnabaenaSynechocystisMonoraphidium



1

1

1

31

31

32

59

59

62

303031

585861

868589

D L A LG K A V F D GN C A AC H AGGGN N V I P D H T

L K K AD L E K Y - - GMY S A E A I I AQ V T N G KN - AML KMAD L E AN - - G KN S V A A I V AQ I T N G - N G AML Q K A A I E Q F L D G KN S V A A I V AQ I T N G - N G AM

P A F G L KK P E Q I E V A A Y V L G K AD A

- AD S V N G A K I F S AN C AS C H AGG KN L Q AQ T- AD L AH G K A I F AGN C A AC H N GG L N A I N P S K TE A

DWKP G KG I S DDS DD ME D V A A Y V L D Q A E KGWP G F KG R I S D S D ME D V A A Y V L D Q A - AGN KW

FFRRRR

KKVV

KK DD

Molina-Heredia et al. (1999) J. Biol. Chem. 274, 33565 De la Cerda et al. (1999) J. Biol. Chem. 274, 13292

0 100 200Cytochrome c6 (M)

kob

s x 1

0-4 (

s-1)

0

2

4

6

Anabaena

Molina-Heredia et al. (1999) JBC 274, 33565

WT

D72K

V25AV25EK29HK62ER64EK66E

WT

0 100 200 300

0

2

4

8

6

Cytochrome c6 (mM)

kob

s x 1

0-4 (

s-1)

Anabaena

Molina-Heredia et al. (1999) JBC 274, 33565

V25A

D72K

Synechocystis Plastocyanin

De la Cerda et al. (1997) Biochemistry 36, 10125

0.2 0.3 0.4 0.5 0.6

Square root of ionic strength (M1/2)

0.5

1.0

1.5

2.0

0k o

bs x

10-

3 (s

-1)

WT

D47R

D47A



0.2 0.3 0.4 0.5 0.6


0.5

1.0

1.5

2.0

0k o

bs x

10-

3 (s

-1)

D44A

D44K

WT



0.2 0.3 0.4 0.5 0.6


0.5

1.0

1.5

2.0

0k o

bs x

10-

3 (s

-1)

WT

D44R/D47R

D44A/D47A

ReducedOxidised

Cytochrome c6 from Monoraphidium

Bioreactor forgrowing cells

movimiento de atracción y formación del complejo

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