DARWIN Digitale Dissertationen German Version Strich

FU Berlin
Digitale Dissertation

Uwe Müller :
Macromolecular Crystallography at Atomic Resolution: Synthetic Nucleic acid fragments and bacterial Cold-shock proteins
Makromolekulare Kristallographie bei atomarer Auflösung:Synthetische Nukleinsäurefragmente und bakterielle Kälteschockproteine

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Abstract

Crystal Structure of ALAwt and ALAC70
The acceptor stem of tRNAAla from E. coli contains the main identity element for specific aminoacylation by it´s cognate alanyl-tRNA-Synthetase (AlaRS). It is known, that the presence of the G3*U70 wobble base pair, which is fully conserved within all kingdoms of life, is essential for this process. The major goal of this work was the structure analysis of the heptameric microhelix ALAwt as well as of the inactive mutant ALAC70, in order to gain insight into the structural basis of recognition. Additionally, this work was aimed to provide information about the behavior of a single G*U wobble base pair within a Watson-Crick double helix.

Crystal Structure of a 3´-DNA-RNA-5´ Hybrid from HIV-1
During initiation of minus-strand synthesis by HIV-1 reverse transcriptase a 3´-DNA-RNA-5´ junction is formed involving the 3´-end of tRNAlys,3. The HIV-RT-associated RNase H cleaves the. RNA template strand specifically, opposite the newly synthesized DNA strand. The crystal structure at 1.9 Å resolution of an eight-base pair hybrid duplex representing the junction has been performed to identify global or local structural perturbations which may be recognized by HIV-RT RNase H. In the crystal HIVChi is present as two independent copies in the asymmetric unit. Both junction octamers adopt A-type conformation throughout their entire length. This is clearly apparent from the C3´-endo sugar pucker present in all 32 nucleotides and from the average helical parameters.

Crystal Structure of Bc-Csp and the Bc-CspR3E mutant
The crystal structure of the cold-shock protein Bc-Csp and it´s R3E-mutant was refined close to atomic resolution. Both proteins crystallize isomorphously in the tetragonal space group I41 with two molecules present in the asymmetric unit. These dimers are different from the CspB dimer. The three dimensional structures of Bc-Csp monomer in comparison with CspB are highly identical. There are only some slight variations of main chain geometry within flexible loop regions. These differences are further discussed in terms of an increased thermostability of the Bc-Csp protein. One region of special interest is the site of the R3E and E46A mutation converting two residues of the sequence of the mesophilic CspB to the thermophilic Bc-Csp protein sequence. Bc-Csp has the ability of forming a salt bridge between R3 and E46 and involving interactions with the side chain of residue K5. However the Bc-CspR3E mutant is 6 kJ/mol less stable than wildtype Bc-Csp. destabilization when compared to wildtype Bc-Csp.

Table of Contents

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Titelseite und Inhaltsverzeichnis
1. Einleitung
1.0. Die Bedeutung der Auflösung bei Kristallstrukturanalysen
1.1. Der Akzeptorstamm der tRNAAla aus Escherichia coli
1.2. DNA-RNA Hybridstrukturen im Replikationszyklus des HIV-Virus
1.3. Das Kälteschockprotein Bc-Csp aus Bacillus caldolyticus

2. Materialien und Methoden
2.1. Hybridisierung und Reinigung der Nukleinsäuren
2.2. Kristallisation der Nukleinsäuren
2.3. Kristallisation von Bc-Cspund Bc-CspR3E
2.4. Das Diffraktionsexperiment
2.5. Die Methode des Isomorphen Ersatzes der tRNAAla-Helix
2.6. Die Methode des Molekularen Ersatzes bei HIVChi und Bc-Csp
2.7. Detektion und Dekonvolution von Fehlordungsstrukturen
2.8. Strukturverfeinerung von HIVChi mit XPLOR-V3.8
2.9. Verfeinerung von ALAwt, ALAC70 und Bc-Csp mit SHELXL-97

3. Ergebnisse und Diskussion
3.1. Kristallstrukturanalyse von ALAwt und ALAC70
3.2. Kristallstrukturanalyse eines 3´-DNA-RNA-5´-hybrids aus HIV-I
3.3. Kristallstrukturanalyse von Bc-Csp und der Bc-CspR3E-Mutante
3.4. Besonderheiten der Kristallstrukturanalyse biologischer Makromoleküle bei atomarer Auflösung

4. Zusammenfassung
4.1. Kristallstrukturanalyse von ALAwt und ALAC70
4.2. Kristallstrukturanalyse eines 3´-DNA-RNA-5´ Hybrids aus HIV-I
4.3. Kristallstrukturanalyse von Bc-Csp und der Bc-CspR3E-Mutante

5. Summary
5.1. Crystal Structure of ALAwt and ALAC70
5.2. Crystal Structure of a 3´-DNA-RNA-5´ Hybrid from HIV-1
5.3. Crystal Structure of Bc-Csp and the Bc-CspR3E mutant

6. Literaturverzeichnis
6.1. Publikationsliste

7. Anhang
7.1. Abbildungsverzeichnis
7.2. Tabellenverzeichnis
7.3. Nukleinsäurebausteine
7.4. Standard-Aminosäuren

Danksagung
Curriculum Vitae
Eidesstattliche Erklärung

More Information:

Online available: http://www.diss.fu-berlin.de/1999/38/indexe.html
Language of PhDThesis: german
Keywords: nucleic acid and protein structure
DNB-Sachgruppe: 30 Chemie
Date of disputation: 28-Jun-99
PhDThesis from: Department Biologie, Chemie, Pharmazie, Freie Universität Berlin
First Referee: Prof. Dr. Udo Heinemann
Second Referee: Prof. Dr. Hans Hartl
Contact (Author): umue@mdc-berlin.de
Contact (Advisor): heinemann@mdc-berlin.de
Date created:29-Jun-99
Date available:07-Jul-99

 

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