Crystallization of lumazine synthase from B. subtilis [Elektronische Ressource] : electron microscopic observations / Lidia Rodríguez Fernández

Sujets

• Biologie

Informations

• Publié par: technische_universitat_munchen
• Publié le: 01 janvier 2004
• Nombre de lectures: 17
• Langue: English
• Poids de l'ouvrage: 71 Mo

Extrait

Technische Universität München
Fakultät für Chemie/Abteilung Elektronenmikroskopie



Crystallization of lumazine synthase from Bacillus subtilis:
electron microscopic observations


Lidia Rodríguez Fernández


Vollständiger Abdruck der von der Fakultät für Chemie der Technischen Universität
München zur Erlangung des akademischen Grades eines

Doktors der Naturwissenschaften

genehmigten Dissertation.



Vorsitzender: Univ.-Prof. Dr. J. Buchner
Prüfer der Dissertation: 1. Univ.-Prof. Dr. S. Weinkauf
2. Ass. Prof. Dr. P. Vekilov
Univ. of Houston/ USA


Die Dissertation wurde am 24.9.2003 bei der Technischen Universität München eingereicht
und durch die Fakultät für Chemie am 16.10.2003 angenommen. Acknowledgements
The first “must” that I have to do is to thank so many people who in different ways
have contributed to the success and finalization of this work. First, I would like to
thank Prof. Dr. S. Weinkauf for giving me the opportunity to do my PhD in her group
as well as for introducing me the fascinating world of biomacromolecular crystalliza-
tion as seen by electron microscopy. I am very grateful to you, Sevil, because you al-
ways believed in me and you gave me the opportunity to go to meetings, conferences
and summer schools that so much have contributed to the development of the present
work. I would also like to thank Prof. Dr. A. Bacher for providing the model system,
lumazine synthase, as well as for allowing me to use the equipment of his department
and Prof. J. García-Ruiz and his co-workers from the University of Granada.

I would like to thank my colleagues Dr. J. Scheuring for his help in biochemistry, Dr.
M. Hanzlik in electron microscopy, especially for her patience with me during astig-
matism correction, Dr. N. Braun her support in image processing as well as Dr. L.
Northdurft, Dipl.-Chem. N. Neumaier, Dr. U. Hars-Hamdi and Marion, for the nice
atmosphere at the lab. I also thank Dr. M. Stumpf for being present at my side at
moments of “low” and “high” tension. Special thanks to Dr. M. Fischer and Dr. I.
Haase for their energetic help.

I would like to thank all these people that I have met during this time, especially Spa-
nish-speaking people: Fermín, Eva, Sofía, Paula, Bertha, Laurent, Kike, Mauricio.
Gracias por vuestra compañía y amistad, por los momentos divertidos, las risas que
nos hemos echado juntos.

Finalmente, quisiera agradecer a mi familia el respaldo que me han prestado durante
todo este tiempo: a mis padres, hermanos, a mis tías, Gini y Juani, y abuelas. También
a mi abuelo que aunque desgraciadamente no se encuentra entre nosotros, siempre me
ha apoyado.

Nicht zuletzt gilt mein herzlicher Dank Josef. Was soll ich Dir sagen? Danke für das
ständige Interesse an meiner Arbeit sowie für die vielen wissenschaftlichen
Diskussionen und, und, und... DANKE FÜR ALLES!!!

TABLE OF CONTENTS


ABSTRACT ............................................................................................................. 1I.


II. INTRODUCTION ................................................................................................... 6

1. Protein crystallization: an overview .......................................................... 7
2. Electron microscopic studies on protein crystallization and quality ...... 16
Effects of microgravity on crystal quality ................................................. 243.

III. SCOPE OF THE WORK ....................................................................................... 28

IV. METHODS .............................................................................................................. 29

1. Biochemical methods ................................................................................... 29
1.1. Expression and purification of lumazine synthase ........................................ 29
1.2. Determination of protein concentration ........................................................ 31
1.3. Gel-electrophoresis ........................................................................................32

2. Crystallization methods .............................................................................. 33
2.1. Batch crystallization ...................................................................................... 33
2.2. Crystallization in „slow mixing batch“ mode ............................................... 34
2.3. Vapour diffusion ............................................................................................ 35
2.4. Crystallization by dialysis ............................................................................. 36
2.5. Counter-diffusion technique .......................................................................... 36
2.6. Crystallization facilities ................................................................................. 37
2.6.1. Advanced Protein Crystallization Facility (APCF) ......................... 37
2.6.2. Granada Crystallization Facility (GCF) .......................................... 40

3. Microscopic methods ................................................................................... 41
3.1. Optical microscopy ........................................................................................ 41
3.2. Transmission electron microscopy (TEM) .................................................... 42
3.2.1. Negative staining ............................................................................. 42
3.2.2. Cryofixation .................................................................................... 42
3.2.3. Freeze-etching and replication ........................................................ 43
3.2.4. Cryo-electron microscopy ............................................................... 45
3.2.5. Equipment .......................................................................................46
3.3. Scanning electron microscopy (SEM) ........................................................... 47


4. Image analysis .............................................................................................. 47
4.1. Optical diffraction ......................................................................................... 47
4.2. Digitization ....................................................................................................48
4.3. Image processing ........................................................................................... 49
4.3.1. Correlation averaging ...................................................................... 50
4.3.2. Cluster analysis ............................................................................... 52
4.3.3. Point defect distribution: „Patterson analysis“ ................................ 53

V. RESULTS AND DISCUSSION ............................................................................. 56

1. Crystallization of lumazine synthase from Bacillus subtilis .................... 56
1.1. Crystallization diagram ................................................................................. 57
1.2. Influence of temperature on crystallization of lumazine synthase ................ 62
1.3. Etching and dissolution ................................................................................. 68
1.4. Defect structures observed on surfaces of lumazine synthase crystals ......... 72
1.5. Summary and discussion ............................................................................... 78

2. Effects of solution transport conditions on growth and perfection
of lumazine synthase crystals ..................................................................... 82
2.1. Search for crystallization conditions for microgravity experiments ............. 84
2.1.1. Crystallization in APCF-FID reactors ............................................. 84
2.1.2. Crystallization in Granada Crystallization Box (GCB) ................... 92
2.2. Crystallization of lumazine synthase in microgravity ................................... 93
2.2.1. ISS 7.A.1 Mission (Increment-3) .................................................... 93
2.2.1.1. Mission profile ................................................................ 93
2.2.1.2. Earth-based control experiments .................................... 95
2.2.1.3. Post-flight observations .................................................. 95
2.2.2. STS-107 Mission ............................................................................. 99
2.2.3. Andromeda Mission ........................................................................ 99
2.3. Assessment and comparison of quality of Earth- and microgravity-
grown crystals ............................................................................................... 100
2.3.1. Electron microscopic analyses ........................................................ 100
2.3.1.1. Point defect densities ...................................................... 100
2.3.1.2