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Dimers of Azurin as model systems for electron transfer

Jongh, Thyra Estrid de

Citation

Jongh, T. E. de. (2006, September 12). Dimers of Azurin as model systems for electron

transfer. Retrieved from https://hdl.handle.net/1887/4554

Version:

Corrected Publisher’s Version

License:

Licence agreement concerning inclusion of doctoral thesis in the

Institutional Repository of the University of Leiden

Downloaded from:

https://hdl.handle.net/1887/4554

(2)

Proefschrift

ter verkrijging van

de graad van Doctor aan de Universiteit Leiden,

op gezag van de Rector M agnificus Dr.

D.

D.

Breim er,

hoogl

eraar in de facul

teit der W iskunde en

Natuurwetenschappen en die der Geneeskunde,

vol

gens besl

uit van het Col

l

ege voor Prom oties

te verdedigen op dinsdag 12 septem ber 2006

kl

okke 13.

45 uur

door

Thyra Estrid de Jongh geboren te Am ersfoort in 1978

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Promotiecommissie

Promotor:

Prof. Dr. G.W. Canters

Co-promotor: Dr. M. Ubbink

Referent:

Prof. Dr. O. Farver

(DFU Kopenhagen)

Overige leden:

Prof. Dr. O. Einsle

(Georg-August Universität Göttingen)

Prof. Dr. M.T.M. Koper

Prof. Dr. J. Brouwer

Thyra de Jongh, Amsterdam 2006

Printed by: PrintPartners Ipskamp B.V., Enschede

Cover: Cartoon representation of a close-up of the interface between two molecules of w ild type azurin (P. aeruginosa). The copper atoms are depicted as dark blue spheres. The sidechains of residues His117 are shown in stick representation. The ordered water molecules in the crystal lattice are indicated by semi-transparent blue spheres. (This picture was generated using PyMol 0.98).

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Horizon

Een immer zinvol streven

Een doel dat nooit benaderd worden kon

M ijn horizon

Een lijn zo recht en vast getrokken

G een enkel punt dat hem bepalen kon

M ijn horizon

Z o vaak verborgen achter duist’re wolken

N ooit een die hem verdrijven kon

M ijn horizon

A ltijd aanwezig en niet te bereiken

G een mens die hem begroeten kon

M ijn horizon

G een vaster punt dat steeds beweegt

G een beter doel dat steeds weer vlucht

Ik ga er heen

U it: G edichten van een K leine Z eeman op een G rote Z ee R icor de Jongh

N ow, bring me that horizon!

C aptain Jack Sparrow, Pirates of the C aribbean

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Abbreviations

1,5-dip 1,5-di(imidazol-1-yl)pentane 1,6-dih 1,6-di(imidazol-1-yl)hexane azu azurin BMME bis-maleimidomethylether DA donor-acceptor pair DTT dithiotreitol H extinction coefficient E. coli Escherichia coli

EPR electron paramagnetic resonance e.s.e. electron self-exchange

ET electron transfer

HSQC heteronuclear single quantum correlation IPTG isopropyl-E-thiogalactopyranoside Kd dissociation constant

kese rate of electron self-exchange kET rate of electron transfer KPi potassium phosphate buffer MES 2-morpholinoethanesulfonic acid NMP N-methylpyrrolidone

NMR nuclear magnetic resonance

ox oxidised

P. aeruginosa Pseudomonas aeruginosa

red reduced

RT room temperature

TCEP tris(carboxyethyl)phosphine UV-Vis ultraviolet and visible spectroscopy

WT wild type

Amino acids

A Ala Alanine M Met Methione

C Cys Cysteine N Asn Asparagine

D Asp Aspartate P Pro Proline

E Glu Glutamate Q Gln Glutamine

F Phe Phenylalanine R Arg Arginine

G Gly Glycine S Ser Serine

H His Histidine T Thr Threonine

I Ile Isoleucine V Val Valine

K Lys Lysine W Trp Tryptophan

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Table of contents

Chapter 1:

Introduction to electron transfer in a biological context

7

Chapter 2

:

Modelling of interprotein electron transfer by covalent and non-covalent

crosslinking

25

Chapter 3

:

Electron self-exchange in N42C-B MME azurin dimers as a function of

temperature

47

Chapter 4

:

Electron transfer and complex formation in a pH sensitive dimer of

azurin

59

Chapter 5

:

Electron transfer in a crosslinked protein dimer mediated by a hydrogen-bonded network across the interface

85

Chapter 6

:

Crystal structure of a non-covalent dimer of H 117G azurin

111

Chapter 7

:

H otwiring redox proteins by ligand reconstitution using conducting

molecular wires

131

Chapter 8:

General discussion, conclusions and perspectives

159

Summary

170

Samenv

atting

174

Append

ic

es

:

179

Full colour images

180

Curriculum Vitæ

190

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6

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