Structural characterisation of the catalytic core of the class III adenylyl cyclase Rv0386 from mycobacterium tuberculosis [Elektronische Ressource] / Christina Pancevac

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Informations

• Publié par: ruprecht-karls-universitat_heidelberg
• Publié le: 01 janvier 2006
• Nombre de lectures: 25
• Langue: English
• Poids de l'ouvrage: 6 Mo

Extrait

Dissertation

submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences



Structural Characterisation of the Catalytic Core of
the Class III Adenylyl Cyclase Rv0386 from
Mycobacterium tuberculosis







Christina Pancevac
Master of Science in Engineering Biology
Heidelberg, 2006





Dissertation
submitted to the
Combined Faculties for the Natural Sciences and for Mathematics
of the Ruperto-Carola University of Heidelberg, Germany
for the degree of
Doctor of Natural Sciences















Presented by
Christina Pancevac,
Civilingenjörsexamen i teknisk biologi,
Master of Science in Engineering Biology,
born in Göteborg, Sweden
Oral-Examination:……………………………………………….







Structural Characterisation of the Catalytic Core of
the Class III Adenylyl Cyclase Rv0386 from
Mycobacterium tuberculosis















Referees: Prof. Dr. Irmgard Sinning
Prof. Dr. Heiner Schirmer

TABLE OF CONTENTS

Acknowledgements i
Abstract ii
Zusammenfassung iii
Publication and Conference Presentations iv
List of Figures v
List of Tables vii
Abbreviations viii

1. INTRODUCTION……………………………………………………………………….....1

1.1 Adenylyl Cyclases...................................................................................................1
1.1.a Intracellular Signals................................................................................................ 1
1.1.b Adenylyl Cyclase Signaling Pathway in Mammals ............................................... 2
1.2 Classification of Adenylyl Cyclases ...................................................................... 4
1.2.a AC Class I .............................................................................................................. 4
1.2.b AC Class II............................................................................................................. 5
1.2.c AC Class III............................................................................................................ 6
1.3 Regulation of Class III Adenylyl Cyclases ......................................................... 10
1.3.a Adenylyl Cyclase Class III Enzyme Cycle .......................................................... 10
1.3.b Regulation of Mammalian Adenylyl Cyclases..................................................... 12
1.3.c Regulation of ACs from M. tuberculosis ............................................................. 18
1.4 Structural Characterisation of Class III ACs.................................................... 23
1.4.a Structures of Mammalian ACs............................................................................. 23
1.4.b AC Structures from Trypanosoma brucei ............................................................ 28
1.5 AC Rv0386 from M. tuberculosis ........................................................................ 28
1.5.a Overview and Domain Organisation.................................................................... 28
1.6 Tuberculosis..........................................................................................................30
1.6.a History and Epidemiology ................................................................................... 30
1.6.b Treatment ............................................................................................................. 31
1.6.c Pathogenesis ......................................................................................................... 31
1.6.d Intracellular Lifestyle of M. tuberculosis............................................................. 32
1.7 Goal of this Study ................................................................................................. 35


2. MATERIAL and METHODS……………………………………………………………36

2.1 Cloning of the Catalytic Domain of Rv0386 ........................................................ 36
2.1.a N-His CHD of Rv0386......................................................................................... 36
2.1.b C-His CHD of Rv0386 37
2.1.c Rv0386 CHD C168S.................................................................................... 38 (1-168)
2.1.d and Rv0386 CHD Cys168Ser ...................................... 39 (1-172) (1-172)
2.1.e Interface Mutants: V132A and E98A................................................................... 40
2.2 Expression and Purification of the CHD of Rv0386 ........................................... 41
2.2.a Overexpression..................................................................................................... 41
2.2.b Over expression of Rv0386 CHD , Rv0386 CHD Cys168Ser and (1-172) (1-172)
Rv0386 CHD Cys168Ser ......................................................................................... 41 (1-168)
2.2.c Purification of C-His CHD of Rv0386................................................................. 42
2.2.d Purification of C-His CHD of Rv0386 as Monomer............................................ 43
2.2.e Purification of N-Hi ................................................................ 43
2.2.f Purification of Rv0386 CHD Cys168Ser and Rv0386 CHD Cys168Ser(1-172) (1-168)
…………………………………………………………………………………...44
2.2.g Production of Selenomethionine Substituted Protein........................................... 45
2.3 Activity Studies of the Catalytic Domain of Rv0386 47
2.4 Biophysical Characterisation................................................................................47
2.4.a Dynamic Light Scattering .................................................................................... 47
2.4.b Mass Spectrometry............................................................................................... 47
2.4.c Analytical Ultracentrifugation.............................................................................. 48
2.5 Crystallisation, Data Collection and Structure Determination ......................... 48
2.5.a Crystallisation....................................................................................................... 48
2.5.b Crystal Freezing and Data Collection .................................................................. 50
2.5.c Data Processing.................................................................................................... 51
2.5.d Molecular Replacement........................................................................................ 52
2.5.e Single Anomalous Dispersion (SAD) 53
2.5.f Refinement ........................................................................................................... 55
2.5.g Data Analysis and Interpretation.......................................................................... 57
2.6 Small Angle X-ray Scattering (SAXS) 57
2.6.a Introduction to SAXS........................................................................................... 57
2.6.b Theory of SAXS................................................................................................... 59
2.6.c Programs used for Processing and Analysis of SAXS Data ................................ 63


3.RESULTS………………………………………………………………………………….69

3.1 Purification and Biophysical Characterisation of the Catalytic Domain of
Rv0386................................................................................................................................. 69
3.1.a Purification of C-His CHD of Rv0386................................................................. 69
3.1.b Purification of N-Hi ................................................................ 71
3.1.c Purification of SeMet Substituted Rv0386 CHD ................................................. 72
3.1.d Activity Studies.................................................................................................... 74
3.1.e Purification of Rv0386 C-His CHD as Monomer................................................ 76
3.1.f Degradation Test of CHD Rv0386....................................................................... 78
3.1.g Mass Spectrometry............................................................................................... 78
3.1.h Dynamic Light Scattering .................................................................................... 79
3.2