1 Movimiento de Atracción Formación del Complejoy Formación del Complejo

2 - + - Long-range interactions (global charges)Short-range interactions (local charges)

3 Collision Reorientation Sliding along the membrane surface Free diffusion

4 Electrostatic Protein-Protein InteractionsLong-range (global charges) Monopole-monopole interactions Short-range (local charges) Dipole-dipole interactions Monopole-dipole interactions

5 D i p o l e - I n t r a c f P s y w h S - + - + - + - + - +

6 Cytochrome c6 Plastocyanin H87 Heme P62 Y83Frazão et al. (1995) Structure 3,

7 Cytochrome c6 Plastocyanin R67Frazão et al. (1995) Structure 3,

8 Cytochrome c6 Plastocyanin Heme H87 P62Frazão et al. (1995) Structure 3,

9 Plastocyanin Cytochrome f Frazão et al. (1995) Structure 3,

10 Análisis Funcional de las Interacciones entre ProteínasMutagénesis Dirigida Análisis Funcional de las Interacciones entre Proteínas

11 Anabaena Plastocyanin Lys33(35) His87(92) Lys58(62) Leu12(14)Arg88(93) Tyr83(88) Lys58(62) His57(61) Asp49(54) Anabaena Plastocyanin Molina-Heredia et al. (2001) J. Biol. Chem. 276,

12 - + Anabaena Plastocyanin K33E K58A WT D49KSurface electrostatic potential Reactivity towards photosystem I

13 Efecto de la fuerza iónica sobre las interacciones entre proteínasConstante de velocidad Fuerza iónica

14 Cytochrome c6 from Anabaena Target residues for site-directed mutagenesis Lys29 Lys62 hydrophobic patch charged area Val25 Asp72 Arg64 Lys66 Molina-Heredia et al. (1999) J. Biol. Chem. 274,

15 Anabaena Cytochrome c6 WT V25E K62E R64E K66E D72KMolina-Heredia et al. (1999) J. Biol. Chem. 274,

16 Anabaena PlastocyaninMolina-Heredia et al. (2001) J. Biol. Chem. 276,

17 Anabaena Molina-Heredia et al. (2001) J. Biol. Chem. 276,

18 Anabaena Plastocyanin Cytochrome c6Molina-Heredia et al. (2001) J. Biol. Chem. 276,

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

20 Orientación de los Centros Redox Disociación del Complejoy Disociación del Complejo

21 Plastocyanin-PSI ComplexE. Myshkin et al. (2002) Biophys J. 82, 3305

22 PSI-driven Electron TransferFd light PS I b6f Cyt c6 Pc

23 Cytochrome f Green alga C. reinhardtii CyanobacteriumPhormidium laminosum Higher plant Brassica rapa Cytochrome f

24 Electrostatic complex between cytochrome f and plastocyaninUbbink et al. (1998) Structure 6,

25 Electrostatic complex between cytochrome f and plastocyaninUbbink et al. (1998) Structure 6,

26 Electron transfer pathway from cytochrome f to plastocyaninUbbink et al. (1998) Structure 6,

27 Chemical shift map of Ana-cytc6 in the presence of cytfResidues are coloured according to increasing Bind from blue to red Prolines and the haem group are in grey and green, respectively Crowley et al. (2002) submitted

28 Cadena respiratoria mitocondrialComplejo I NADH-quinona óxido-reductasa Complejo II Succinato-quinona Complejo III Quinol-citocromo c Complejo IV Citocromo c-O2 citocromo c

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

30 Interacción entre el complejo bc1 (inmerso en la membrana) y el citocromo c (soluble)Cyt c Cyt c1 Cyt c Cyt c1

31 Interacción entre los citocromos f y cCyt c1 Cyt c

32 Zona de contacto entre los hemos f y cCyt c1 Hemo c Cyt c Hemo c1

33

34 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

35 Protein-Protein InteractionsCentro de Investigaciones Científicas Isla de la Cartuja Sevilla Universidad de Sevilla Workshop on Weak Protein-Protein Interactions Seville, Spain July 9-13, 2003

36

37 Target residues for mutagenesis of cytochrome c6Anabaena 1 31 32 59 62 30 58 61 86 85 89 D L A G K V F N C H I P T E Y - M S Q W R Synechocystis Monoraphidium Anabaena Synechocystis Monoraphidium Anabaena Synechocystis Monoraphidium Molina-Heredia et al. (1999) J. Biol. Chem. 274, 33565 De la Cerda et al. (1999) J. Biol. Chem. 274, 13292

38 Anabaena 100 200 Cytochrome c6 (mM) kobs x 10-4 (s-1) 2 4 6 D72K WT100 200 Cytochrome c6 (mM) kobs x 10-4 (s-1) 2 4 6 Anabaena Molina-Heredia et al. (1999) JBC 274, 33565 WT D72K V25A V25E K29H K62E R64E K66E

39 Anabaena 100 200 300 2 4 8 6 Cytochrome c6 (mM) kobs x 10-4 (s-1) D72KWT 100 200 300 2 4 8 6 Cytochrome c6 (mM) kobs x 10-4 (s-1) Anabaena Molina-Heredia et al. (1999) JBC 274, 33565 D72K V25A

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

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

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

43 Cytochrome c6 from MonoraphidiumReduced Oxidised

44 Cytochrome c6 from MonoraphidiumReduced Oxidised

45 Bioreactor for growing cells