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Chapter 6: Further characterisation of Mycobacterial chaperonins Section 6.1: Testing the ability of M. tuberculosis Cpn60.1 to function in M. smegmatis Fig 6.1: A representation of the cloning process into pMsGroEL1 213 Fig 6.2: Biofilm assay results after 5 days of incubation 215 Fig 6.3: Masses of 4 day old biofilms 216 Section 6.2: Testing the existence of double ring M. tuberculosis Cpn60.2 by attempting to make single ring mutants Fig 6.4: Sequence alignment between GroEL and Cpn60.2 222 Fig 6.5: Complementation assay of Cpn60.2 mutants 224 Fig 6.6: SDS-PAGE of Cpn60.2 mutants showing 225 Fig 6.7: Complementation assay of GroEL-E461A mutant in glucose 227 Section 6.3: Testing the hypothesis that phosphorylation is needed for improved oligomerisation Fig 6.8: A schematic representation of the process used to obtain pSUES 234 Fig 6.9: SDS-PAGE gel of pSUEH and pSUES 235 Fig 6.10: Complementation results of the T/A60.2 and T/E60.2 mutants 237 Fig 6.11: SDS-PAGE gel of Cpn60.2 WT, T/A, and T/E mutants 238 Fig 6.12: Results of the WT Cpn60.2, T/A60.2, and T/E60.2 mutants without His-tag 240 Fig 6.13: SDS-PAGE gel of Cpn60.2 WT, T/A, and T/E mutants without His-tags 241 X
List of tables Chapter 1: Introduction Table 1.1: Classes of molecular chaperone families 15 Chapter 3: Expression and complementation analysis of M. tuberculosis chaperonins in E. coli Table 3.1: Summary of the complementation data in MGM100 110 Table 3.2: Summary of the complementation data in MGM100 with pRARE 115 Table 3.3: Summary of the complementation data in Tab21 119 Table 3.4: Summary of the complementation data of ATG-Cpn10 128 Chapter 4: Construction and analysis of chimeric chaperonins Table 4.1: The list of chimeric chaperonins made 151 Table 4.2: Complementation data of imprecise domain swaps 163 Table 4.3: Complementation data of precise domain swaps 174 Table 4.4: Summary of data obtained from apical domain swaps 182 Chapter 6: Further characterisation of Mycobacterial chaperonins Table 6.1: List of residues and mutations for Cpn60.2 mutants 221 XI
Page 1 and 2: FUNCTIONAL AND STRUCTURAL CHARACTER
Page 3 and 4: Abstract Proteins are involved in a
Page 5 and 6: Dedicated to Mum, Dad, and Sharique
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: Fig 4.1: Schematic representation o
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
Page 63 and 64: Chapter 1 main difference between t
Page 65 and 66: Chapter 1 1.5.3.3 Prefoldin It has
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Chapter 1 result the substrate prot
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Chapter 1 Fig 1.18: The structures
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Chapter 1 6- If the released peptid
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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
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Chapter 2 Materials and Methods: 2.
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Chapter 2 pET-cpn10- cpn60.1 Deriva
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Chapter 2 2.3 Primers: Below is a l
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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
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Chapter 2 - Colony/culture 1μl - S
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Chapter 2 The PCR cycling parameter
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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
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Chapter 3 Background As discussed a
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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
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Chapter 3 3.3 Complementation and e
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Chapter 3 of MGM100. The use of Tab
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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
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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
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Chapter 4 of GroEL (fig 4.7). This
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Chapter 4 (a) Dilutions 10 -1 10 -2
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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
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Chapter 5 Background As discussed i
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Chapter 5 macromolecules at equilib
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Chapter 5 5.1 Purification of JNL 5
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Chapter 5 (a) 1 2 3 4 5 6 7 8 9 10
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Chapter 5 (a) A B C D E (b) 1 2 3 4
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Chapter 5 5.2 AUC analysis of JNL U
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Chapter 5 (a) 1 JNL in Buffer 5 0.9
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Chapter 5 (a) (b) 100 mM P i 75 mM
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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
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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
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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
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Tyagi, N. K., W. A. Fenton & A. L.
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Yamamori, T. & T. Yura, (1982) Gene
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