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Chapter 5 Based on the results obtained here and from chapters 3 & 4, it becomes apparent that under appropriate conditions, Cpn60.2 and its chimera JNL can not only function in E. coli, but also form higher oligomeric structures and hydrolyse ATP. This is in contrast to the data obtained by Kumar et al., and Qamra et al. It is however important to note that all the experiments done in this chapter were in vitro and used conditions that were in effect forcing the chaperonins to oligomerise. It still does not explain the chaperonin cycle that takes place in vivo. It is possible that M. tuberculosis chaperonins have evolved to be less energy dependent. This reduced dependency on energy could result in their unstable nature. However, since Cpn60.2 and JNL can both function in E. coli, even though they show bands corresponding to monomers on a native gel, it can be speculated that the disassembled and assembled states of the chaperonins are transient. It is possible that under conditions that do not require high concentrations of functional chaperonins, the Mycobacterial chaperonins are primarily in an unassembled monomeric/dimeric state. When the conditions within the cell change to the point where more functional chaperonins are needed, then higher concentrations of oligomeric chaperonins are present. How this is achieved within the cell is unclear. It is possible that the oligomerisation of Mycobacterial chaperonins may be substrate dependent. It can also be speculated that under stressful conditions, such as heat shock, the highly crowded nature of the cells and the over-expression of the heptameric Cpn10 (or GroES in E. coli) may provide the Mycobacterial chaperonins with the scaffolding needed to assemble into ringed structures. To prove the presence of large oligomeric structures in vivo, further experiments would need to be done. One possibility is cross-linking experiments. Cross-links are ionic or covalent bonds that link interacting polypeptide chains to each other and is used as a probe for protein-protein interaction. In context to Mycobacterial chaperonins, it could be used to see if they are forming tetradecameric structures in vivo. 205
Chapter 5 In summary, based on the results obtained here and the results obtained from the previous result chapters, it is very probable that within the cell where there are various crowding agents and different salt concentrations, the Mycobacterial chaperonins form higher oligomeric structures and hydrolyse ATP in the substrate folding reactions. 206
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FUNCTIONAL AND STRUCTURAL CHARACTER
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Abstract Proteins are involved in a
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Dedicated to Mum, Dad, and Sharique
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Index of contents Chapter 1: Introd
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2.4.5: Bacteriophage P1 Transductio
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4.1.5: Chimeras DHF & GEI 162 4.2:
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List of illustrations Chapter 1: In
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Fig 4.1: Schematic representation o
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List of tables Chapter 1: Introduct
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SDW STPKs TAE TEMED TBS Sterile dis
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Chapter 1 1.1 Protein Folding The g
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Chapter 1 Fig 1.1: The energy lands
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Chapter 1 To try and escape from th
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Chapter 1 Fig 1.2: Pathways regulat
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Chapter 1 As protein folding in Myc
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Chapter 1 1.4 The molecular chapero
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Chapter 1 1.4.1 Classes of molecula
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Chapter 1 Hsp40 DnaJ CbpA DjlA Ydj1
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Chapter 1 obtained. These revertant
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Chapter 1 1.4.3.1 The ribosome asso
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Chapter 1 (A) (B) Fig 1.7: Structur
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Chapter 1 complete the reaction cyc
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Chapter 1 1.4.3.4 The small Hsps On
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Chapter 1 1.4.3.5 Hsp90 family of c
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Chapter 1 Fig 1.11: Structure of Ht
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Chapter 1 1.4.3.6 Hsp100 family of
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Chapter 1 Fig 1.13: The different r
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Chapter 1 encoded by more than one
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Chapter 1 Fig 1.15: The illustratio
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Chapter 1 (Rommelaere et al., 1993,
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Chapter 1 Fig 1.16: Octameric struc
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Chapter 1 main difference between t
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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
Page 173 and 174: Chapter 4 4.1.1 Chimera AEC This is
Page 175 and 176: 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
Page 183 and 184: Chapter 4 4.2 Precise domain swaps
Page 185 and 186: Chapter 4 intermediate domains were
Page 187 and 188: Chapter 4 1 2 800 kDa 480 kDa 146 k
Page 189 and 190: Chapter 4 Dilutions 10 -2 10 -3 10
Page 191 and 192: Chapter 4 is important to note that
Page 193 and 194: Chapter 4 Summary the complementati
Page 195 and 196: Chapter 4 The other aspect of this
Page 197 and 198: Chapter 4 caused due to lack of exp
Page 199 and 200: Chapter 4 structures were not expec
Page 201 and 202: 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
Page 221 and 222: Chapter 5 1 2 3 Corrected values Av
Page 223: Chapter 5 expected, evidence of lar
Page 227 and 228: Chapter 6 In this chapter I will be
Page 229 and 230: Chapter 6 of Mscpn60.1 can indirect
Page 231 and 232: Chapter 6 can produce products with
Page 233 and 234: Chapter 6 As the group in Pittsburg
Page 235 and 236: Chapter 6 Fig 6.3: Masses of 4 day
Page 237 and 238: Chapter 6 than 7 days, so there is
Page 239 and 240: Chapter 6 are required to facilitat
Page 241 and 242: Chapter 6 Fig 6.4: Sequence alignme
Page 243 and 244: Chapter 6 To ensure that the lack o
Page 245 and 246: Chapter 6 6.2.3 Complementation ana
Page 247 and 248: Chapter 6 Discussion The double rin
Page 249 and 250: Chapter 6 Section 6.3: Testing the
Page 251 and 252: Chapter 6 Experimental design For t
Page 253 and 254: Chapter 6 EcoRV groEL (1.6kb) HindI
Page 255 and 256: Chapter 6 6.3.2 Complementation res
Page 257 and 258: Chapter 6 1 2 3 4 72 kDa 55 kDa Fig
Page 259 and 260: Chapter 6 10 -1 10 -2 10 -3 10 -4 1
Page 261 and 262: Chapter 6 Discussion The results th
Page 263 and 264: Summary The main aim of this study
Page 265 and 266: together, these results strongly su
Page 267 and 268: Basha, E., K. L. Friedrich & E. Vie
Page 269 and 270: Cehovin, A., A. R. Coates, Y. Hu, Y
Page 271 and 272: Diaz-Acosta, A., M. L. Sandoval, L.
Page 273 and 274: 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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