Appendices
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Figure 2.1: Representation of P. aeruginosa azurin seen from the northern face. Residues that make up the hydrophobic patch surrounding the copper ligand histidine117 (dark grey)are shaded and numbered.At the rim of the hydrophobic patch residue 42(blue)is situated.
Figure 2.2: Cartoon depiction of the crystal packing of wild type azurin observed in the asymmetric unit (PDB entry 1E5Y).The copper atoms are depictedas large spheres,the side chains of copper ligands histidine 117 are displayed as sticks.
Figure 2.7: Crystal structure of the N42CBM M E azurin dimer (PDB entry 1JVL). The copper atoms are represented as large spheres (blue), side-chains of the residues H117 and C42 as well as the BM M E spacer are shown as sticks. The hydrogen bonded water molecules in the dimer interface connecting to H117 are indicated by small spheres (cyan). The dashed lines show the hydrogen bond network connecting the copper centres across the interface.
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Figure 2.5: Cartoon depiction of the asymmetric unit of N42C disulfide bridged azurin showing four subunits arranged in two crosslinked dimers (PDB entry 1JVO, 2.75 Å). The copper atoms are indicated with blue spheres, the C42 side chains as sticks. The average intramolecular Cu-to-Cu distance is 25.9r 0.3Å. The intermolecular Cu-to-Cu distance between two of the subunits equals 15.1 r 0.1 Å. Both Cu-to-Cu distances are indicated with dashed lines.
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Figure 4.4: Stereo tube representation of the structure of the disulfide linked dimer of N42C/M64E azurin at pH 3.1 (blue) (2.3 Å) superimposed on the crystal packing orientation of wild type azurin at pH 5.5 (beige) (1E5Y, 1.98Å). Copper atoms are depicted as spheres, the 42C-42C disulfide bond is shown in stick rendering.
Figure 4.5: Crystal structure of the N42C/M64E azurin disulfide-bridged dimer at pH 3.1. The copper centres are depicted as large spheres (dark-blue). Close-up: Interface of the dimer showing the ordered water molecules (small light-blue spheres). All residues involved in hydrogen bonding with these water molecules are shown (sticks) and labelled. The hydrogen bonds are shown as dashed lines. The letters between brackets indicate the individual protein chains. Also shown are the Cu ligands H117 (sticks).
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Figure 4.6A,B: 15N,1H HSQC spectra of H35F/N42C/M64E azurin. Black contours represent monomeric azurin, red contours the corresponding disulfide bridged dimers. A) monomer at pH 4.4, dimer at pH 4.3, B) monomer at pH 5.7, dimer at pH 5.6. Close-ups show the regions around resonances 42, 43 and 44. Residues that are shifted in the dimer are indicated with arrows.
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Figure 4.6C,D: 15N,1H HSQC spectra of H35F/N42C/M64E azurin. Black contours represent monomeric azurin, red contours the corresponding disulfide bridged dimers. C) monomer at pH 5.9, dimer at pH 7.0, D) monomer at pH 8.0, dimer at pH 8.4. Close-ups show the regions around resonances 42, 43 and 44. Residues that are shifted in the dimer are indicated with arrows.
Figure 5.6: Stereo view of the dimer interface of N42C/M64E-BMME azurin at pH 9.0 showing the experimental electron density map superimposed on the refined model and contoured at 1V. Residues 42-46, 114-118 and 121 on both subunits, as well as residue 13 in the top subunit (beige) are shown as sticks. The copper atoms are shown as spheres.
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Figure 5.5: Stereo representation of the dimer interface of the N42C/M64EBMME azurin dimer at pH 9.0 (2.25 Å). The separate crosslinked subunits are shown in blue (a) and beige (b), the connecting BMME linker is displayed in green. The sidechains of residues 11, 42, 64 and 117 and the backbone nitrogen of residues 116 are shown as sticks. Dashed lines are used to indicate the D11-E64 and CuH117 distances (grey) and the stabilizing H-bonds between G116 and E64 (blue). Copper atoms are depicted as blue spheres.
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Figure 5.7A,B: 15N,1H HSQC spectra of H35F/N42C/M64E-BMME azurin. Black contours represent monomeric azurin, red contours the corresponding BMMElinked dimers. A) monomer at pH 4.4, dimer at pH 5.0, B) monomer at pH 5.7, dimer at pH 6.6. Close-ups show the regions around resonances 42, 43 and 44. Those residues that are affected by crosslinking and which have been assigned are labelled. Shifted residues are indicated with arrows.
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Figure 5.7C,D: 15N,1H HSQC spectra of H35F/N42C/M64E-BMME azurin. Black contours represent monomeric azurin, red contours the corresponding BMME-linked dimers. C) monomer at pH 5.9, dimer at pH 7.7, D) monomer at pH 8.0, dimer at pH 8.4. Close-ups show the regions around resonances 42, 43 and 44. Those residues that are affected by crosslinking and which have been assigned are labelled.
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Figure 5.8: Proposed ET pathways between the copper centres in crosslinked dimers of N42C-BMME (left) and N42C/M64E-BMME (right) azurin. Labels a and b refer to the individual chains. The copper atoms are represented as blue spheres and the smaller grey spheres depict water molecules. Grey dashed lines indicate the predicted ET path, green dashes represent the additional H-bonds between the involved structural elements.
Figure 6.3: Stereo view of the (Zn-H117G)2-1,6-dih azurin dimer. The sidechains of residues G45, H46, C112 and M121 as well as the 1,6-dih linker are shown as sticks while both zinc atoms are depicted as spheres.
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Figure 6.6: Cartoon representations of (Zn-H117G)2-1,6-dih azurin (left) and the crystal packing of Zn-W T azurin (right) with the D-helical regions shown in blue. The zinc atoms are shown as large spheres (blue), hydrogen bonded water molecules in the protein interface of W T azurin as small spheres (light blue).
Curriculum Vitæ Thyra de Jongh was born on May 21st 1978 in Amersfoort, The Netherlands. After obtaining the ‘VWO diploma’at the Johan van Olderbarnevelt gymnasium in Amersfoort, in 1996 she began with the study of Chemistry at the University of Amsterdam. For her specialization in biochemistry she performed short-term internships at the Department of Clinical Chemistry of the Academic Medical Centre (AMC Amsterdam) and in the Biocatalysis Group (UvA). Her final year of research was conducted in the Biocatalysis Group under supervision of dr. E.G. Funhoff and Prof. B.A. Averill and concerned the investigation of the reaction mechanism of human purple acid phosphatase. In March 2001 she obtained the MSc degree in Chemistry with honours. Immediately hereafter she joined the Metalloproteins Group (MetProt) at the University of Leiden as a PhD student under supervision of Prof. G.W. Canters. 190
The research conducted here centred on the study of biological electron transfer in model systems based on the blue copper protein azurin. The work involved techniques from the fields of molecular biology, protein chemistry and spectroscopy and was carried out in colloboration with groups at the Georg-August-Universität Göttingen, Germany and the Universita di Parma, Italy. Parts of the research were presented at the international conferences ICBIC10 and ICBIC11 in Florence, Italy and Cairns, Australia respectively as well as on the annual NWO meetings for protein research in Lunteren. A presentation given here in 2004 was awarded the prize for best oral presentation. As of May 2006 Thyra has been working at the management bureau of the Leiden Institute of Chemistry. In October 2006 she will commence the MSc International Health Management programme at Tanaka Business School, Imperial College London.
Acknow ledgements Een promotieonderzoek heeft in veel opzichten wel wat weg van een zeiltocht: de blik is vrijwel voortdurend op de horizon gericht die toch steeds oneindig ver af lijkt en soms wordt, bij zwaar weer, het zicht er op je geheel ontnomen. Dat ik mijn ‘horizon’ nu toch heb bereikt had niet gekund zonder de hulp van zovelen die deze afgelopen 5 jaren elk op hun eigen manier onmisbaar zijn gebleken. Op wetenschappelijk gebied ben ik veel dank verschuldigd aan verschillende mensen: The crystals of the dimers described in Chapters 4 and 5 of this thesis were initially brought to life and nurtured in the capable hands of Davide Cavazzini in the group of Prof. Rossi in Parma. After a couple of precarious journeys between Parma, Leiden and Göttingen, these crystals eventually ended up in the lab of Prof. Einsle which resulted in their successful structure determination. Maren and Oliver: my trips to picturesque Göttingen were a pleasure. Dear Oli, I owe you my gratitude for being my personal guide into the wonderful world of PyMol. I hope you will appreciate the results shown on these pages. Hoofdstuk 6 dankt zijn bestaan voornamelijk aan de toewijding van Marike van Roon. Dear Marike and Jon, I’m looking forward to many future trips LondonCambridge and vice versa! Hoofdstuk 7 is het product van de gezamenlijke inspanning van een aantal mensen: vrijwel alle linkers werden geproduceerd in het lab van Dr. Mark Overhand dankzij het werk van Rian van den Nieuwendijk. The EPR spectra of the linked azurin complexes were enthusiastically recorded, interpreted and discussed by and with Dr. Sergei Milikysiants and Dr. Martina Huber. Gedurende mijn periode in het lab heb ik hulp gehad van twee studenten: Ruud en Elle Bartha. Helaas is het project waar jullie beiden op gewerkt hebben uiteindelijk nooit goed van de grond gekomen maar aan jullie inspanningen heeft dat zeker niet gelegen.
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The MetProt lab has throughout these past few years been a wonderful ‘melting pot’, filled with great scientists and fantastic friends that have helped me tackle many of the problems one encounters during daily lab life. It has been a pleasure working with you all. Niet alleen op wetenschappelijk gebied hebben mensen mij terzijde gestaan en ik mag dan ook vooral mijn vrienden en familie hier niet vergeten. Allereerst mijn oud-huisgenootjes, Mariken en Jocelyne;het is al weer even geleden dat we een dak deelden maar zeker in die beginperiode was het heerlijk om bij het thuis komen verhalen met jullie te kunnen uitwisselen. Heel veel plezier heb ik de afgelopen jaren ook gedeeld met de ‘Amsterdamse kliek’, of het nu om gezamenlijke vakanties ging of om gewoon een lekker ontspannen etentje en een goed gesprek, al dan niet over onze respectievelijke onderzoeken.
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Although I have shared many wonderful moments with so many in the MetProt group, I am especially grateful to those people that were there for me when I needed them most. You know who you are! To two of my former colleagues I am particularly endebted for their enduring friendship and support, both in and out of the lab: My dearest Sharm and Miguel, I couldn’t have done this without you. Tot slot een paar woorden aan hen die het allerbelangrijkst voor mij zijn: Maarten, ‘broertje’, ik ben blij jouw ‘grote zus’ te mogen wezen. Het meeste dank ik aan de onvoorwaardelijke liefde en steun van mijn ouders. Lieve mama, ik hoop dat je weet hoe trots ik er op ben je dochter te zijn. Liefste papa, ik mis je. Ik hoop dat je trots op me geweest zou zijn.
