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fulfil different functions within the cell. In both cases Cpn60.2 is predicted to be the main house-keeping chaperonin. Interestingly, while most chaperonins occur as stable complexes with a characteristic double ring structure of 14 subunits, the Mycobacterial chaperonins are very unstable and appear to form much smaller complexes. Given that the large structures formed by most chaperonins are considered essential to their mechanism of action, it is unclear why the oligomers are so much less stable in Mycobacteria. In this study I present detailed functional and oligomeric analysis of the M. tuberculosis chaperonins. Using various biological techniques, including complementation assays, site directed mutagenesis, and domain swap experiments; I have been able to demonstrate that both chaperonins have evolved to fulfil alternate functions within the cell. I have shown that while Cpn60.2 is fully functional in E. coli, Cpn60.1 is not. However, M. tuberculosis Cpn60.1 was able to function in M. smegmatis and complement in an assay of biofilm formation for the loss of its endogenous chaperonin. Domain swap experiments between Cpn60.2 and E. coli GroEL have provided further evidence to support the hypothesis that they function in a similar manner. Using analytical ultracentrifugation on purified proteins, I have been able to show that Cpn60.2 and its functional chimera do form larger oligomeric complexes in vitro under certain conditions and in the presence of ATP.
Dedicated to Mum, Dad, and Sharique Thank you for everything!
Page 1 and 2: FUNCTIONAL AND STRUCTURAL CHARACTER
Page 3: Abstract Proteins are involved in a
Page 7 and 8: Index of contents Chapter 1: Introd
Page 9 and 10: 2.4.5: Bacteriophage P1 Transductio
Page 11 and 12: 4.1.5: Chimeras DHF & GEI 162 4.2:
Page 13 and 14: List of illustrations Chapter 1: In
Page 15 and 16: Fig 4.1: Schematic representation o
Page 17 and 18: List of tables Chapter 1: Introduct
Page 19 and 20: SDW STPKs TAE TEMED TBS Sterile dis
Page 21 and 22: Chapter 1 1.1 Protein Folding The g
Page 23 and 24: Chapter 1 Fig 1.1: The energy lands
Page 25 and 26: Chapter 1 To try and escape from th
Page 27 and 28: Chapter 1 Fig 1.2: Pathways regulat
Page 29 and 30: Chapter 1 As protein folding in Myc
Page 31 and 32: Chapter 1 1.4 The molecular chapero
Page 33 and 34: Chapter 1 1.4.1 Classes of molecula
Page 35 and 36: Chapter 1 Hsp40 DnaJ CbpA DjlA Ydj1
Page 37 and 38: Chapter 1 obtained. These revertant
Page 39 and 40: Chapter 1 1.4.3.1 The ribosome asso
Page 41 and 42: Chapter 1 (A) (B) Fig 1.7: Structur
Page 43 and 44: Chapter 1 complete the reaction cyc
Page 45 and 46: Chapter 1 1.4.3.4 The small Hsps On
Page 47 and 48: Chapter 1 1.4.3.5 Hsp90 family of c
Page 49 and 50: Chapter 1 Fig 1.11: Structure of Ht
Page 51 and 52: Chapter 1 1.4.3.6 Hsp100 family of
Page 53 and 54: Chapter 1 Fig 1.13: The different r
Page 55 and 56:
Chapter 1 encoded by more than one
Page 57 and 58:
Chapter 1 Fig 1.15: The illustratio
Page 59 and 60:
Chapter 1 (Rommelaere et al., 1993,
Page 61 and 62:
Chapter 1 Fig 1.16: Octameric struc
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Chapter 1 main difference between t
Page 65 and 66:
Chapter 1 1.5.3.3 Prefoldin It has
Page 67 and 68:
Chapter 1 result the substrate prot
Page 69 and 70:
Chapter 1 Fig 1.18: The structures
Page 71 and 72:
Chapter 1 6- If the released peptid
Page 73 and 74:
Chapter 1 1.5.4.3 GroEL substrates
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Chapter 1 Recent work has suggested
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Chapter 1 (A) cpn10 cpn60.1 cpn60.2
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Chapter 1 1998). Cpn60.1 however ha
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Chapter 1 1.6 Aims of the project T
Page 83 and 84:
Chapter 2 Materials and Methods: 2.
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Chapter 2 pET-cpn10- cpn60.1 Deriva
Page 87 and 88:
Chapter 2 2.3 Primers: Below is a l
Page 89 and 90:
Chapter 2 Mut EL.3 Rev groEL 1401-1
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Chapter 2 29R-EL-60.2 groEL 432-412
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Chapter 2 Biofilm base media: 13.6g
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Chapter 2 Preparation of bacterioph
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Chapter 2 then resuspended in 200µ
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Chapter 2 - 10X BSA (depending on t
Page 101 and 102:
Chapter 2 - Colony/culture 1μl - S
Page 103 and 104:
Chapter 2 The PCR cycling parameter
Page 105 and 106:
Chapter 2 tubes were placed on ice
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Chapter 2 2.6 Protein analysis 2.6.
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Chapter 2 2.6.2- Native gel Reagent
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Chapter 2 - Diluted primary antibod
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Chapter 2 supernatant was decanted
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Chapter 2 2.6.6- Analytical ultrace
Page 117 and 118:
Chapter 3 Background As discussed a
Page 119 and 120:
Chapter 3 the -35 region of the trp
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Chapter 3 20 mins 40 mins 60 mins 8
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Chapter 3 (a) 60 mins 90 mins 120 m
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Chapter 3 For this experiment, MGM1
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Chapter 3 When IPTG was not include
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Chapter 3 The results from these co
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Chapter 3 3.2.1 The effect of chang
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Chapter 3 3.2.2 Using pRARE plasmid
Page 135 and 136:
Chapter 3 3.3 Complementation and e
Page 137 and 138:
Chapter 3 of MGM100. The use of Tab
Page 139 and 140:
Chapter 3 3.3.1 Using pET plasmid i
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Chapter 3 1 2 3 4 5 6 7 8 95 kDa 72
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Chapter 3 it only digests the paren
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Chapter 3 72 kDa 1 2 3 4 5 55 kDa 1
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Chapter 3 30°C/26°C 37°C MGM100
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Chapter 3 Fig 3.12: Comparison of t
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Chapter 3 AI90/pACYC-Ptrc-GroESL-Sa
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Chapter 3 1 2 3 4 5 6 7 1000 bp 600
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Chapter 3 10 -1 10 -2 10 -3 10 -4 1
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Chapter 3 this observation. The res
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Chapter 3 expression. However, from
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Chapter 4 Construction and analysis
Page 163 and 164:
Chapter 4 Background In the last ch
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Chapter 4 Experimental design To ma
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Chapter 4 Cpn60.2 7 8 9 Cpn60.1 1 2
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Chapter 4 The full list of all the
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Chapter 4 4.1 Imprecise domain swap
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Chapter 4 4.1.1 Chimera AEC This is
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Chapter 4 (a) Dilutions 10 -1 10 -2
Page 177 and 178:
Chapter 4 of GroEL (fig 4.7). This
Page 179 and 180:
Chapter 4 (a) Dilutions 10 -1 10 -2
Page 181 and 182:
Chapter 4 4.1.5 Chimeras DHF & GEI
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Chapter 4 4.2 Precise domain swaps
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Chapter 4 intermediate domains were
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Chapter 4 1 2 800 kDa 480 kDa 146 k
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Chapter 4 Dilutions 10 -2 10 -3 10
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Chapter 4 is important to note that
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Chapter 4 Summary the complementati
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Chapter 4 The other aspect of this
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Chapter 4 caused due to lack of exp
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Chapter 4 structures were not expec
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Chapter 4 Name Construct Blue -Cpn6
Page 203 and 204:
Chapter 5 Background As discussed i
Page 205 and 206:
Chapter 5 macromolecules at equilib
Page 207 and 208:
Chapter 5 5.1 Purification of JNL 5
Page 209 and 210:
Chapter 5 (a) 1 2 3 4 5 6 7 8 9 10
Page 211 and 212:
Chapter 5 (a) A B C D E (b) 1 2 3 4
Page 213 and 214:
Chapter 5 5.2 AUC analysis of JNL U
Page 215 and 216:
Chapter 5 (a) 1 JNL in Buffer 5 0.9
Page 217 and 218:
Chapter 5 (a) (b) 100 mM P i 75 mM
Page 219 and 220:
Chapter 5 In this experiment GroEL
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Chapter 5 1 2 3 Corrected values Av
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Chapter 5 expected, evidence of lar
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Chapter 5 In summary, based on the
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Chapter 6 In this chapter I will be
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Chapter 6 of Mscpn60.1 can indirect
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Chapter 6 can produce products with
Page 233 and 234:
Chapter 6 As the group in Pittsburg
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Chapter 6 Fig 6.3: Masses of 4 day
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Chapter 6 than 7 days, so there is
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Chapter 6 are required to facilitat
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Chapter 6 Fig 6.4: Sequence alignme
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Chapter 6 To ensure that the lack o
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Chapter 6 6.2.3 Complementation ana
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Chapter 6 Discussion The double rin
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Chapter 6 Section 6.3: Testing the
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Chapter 6 Experimental design For t
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Chapter 6 EcoRV groEL (1.6kb) HindI
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Chapter 6 6.3.2 Complementation res
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Chapter 6 1 2 3 4 72 kDa 55 kDa Fig
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Chapter 6 10 -1 10 -2 10 -3 10 -4 1
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Chapter 6 Discussion The results th
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Summary The main aim of this study
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together, these results strongly su
Page 267 and 268:
Basha, E., K. L. Friedrich & E. Vie
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Cehovin, A., A. R. Coates, Y. Hu, Y
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Diaz-Acosta, A., M. L. Sandoval, L.
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Fux, C. A., J. W. Costerton, P. S.
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Gupta, P., S. Mishra & T. K. Chaudh
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Hoffmann, A., F. Merz, A. Rutkowska
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Ito, K., Y. Akiyama, T. Yura & K. S
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Krzewska, J., G. Konopa & K. Libere
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Lin, Z., D. Madan & H. S. Rye, (200
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Mogk, A. & B. Bukau, (2004) Molecul
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Patzelt, H., G. Kramer, T. Rauch, H
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Rommelaere, H., M. Van Troys, Y. Ga
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Strickland, E., B. H. Qu, L. Millen
Page 293 and 294:
Tyagi, N. K., W. A. Fenton & A. L.
Page 295:
Yamamori, T. & T. Yura, (1982) Gene
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