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Chapter 6 10 -1 10 -2 10 -3 10 -4 10 -5 10 -6 10 -7 1 2 3 4 Fig 6.7: Complementation assay of GroEL-E461A mutant in glucose. Cells were grown on LB plates at 30°C with 0.2% glucose and 0.1 mM IPTG. 1- MGM100/ pTrc-GroES-GroEL-E461A 2- MGM100/ pTrc-GroES-GroEL 3- MGM100/ pTrc-GroES-Cpn60.2 4- MGM100/ pTrc99a 227
Chapter 6 Discussion The double ring structure of chaperonins like GroEL play a major functional role, although single ringed chaperonins are also known to occur in nature that are functional (Nielsen & Cowan, 1998). In either case, a ringed structure (either double or single) is needed to allow substrates to fold within a central cavity. Work done in our lab has already proven the existence of Cpn60.2 tetradecameric structures under certain conditions in vitro (Elsa Zacco, Master’s thesis 2010, University of Birmingham). It was also shown in the last chapter that JNL forms large tetradecameric structures under the same conditions in vitro. However, it becomes a lot more difficult to prove the existence of tetradecameric Cpn60.2 structures in vivo due to its unstable nature. The main reason why it is believed Cpn60.2 does form ringed structures in vivo is because it can function in E. coli MGM100, both with Cpn10 and GroES. As has been stated before, both these cochaperonins are known to exist as heptameric structures (Hunt et al., 1996, Roberts et al., 2003). In section 3.7 this was confirmed by deleting the cochaperonin GroES from the plasmid and showing that Cpn60.2 cannot function in E. coli MGM100 without it. The results that have been obtained here are a little difficult to interpret. It cannot be claimed that Cpn60-E458A proves that the Cpn60.2 forms a double ringed structure, since the same result could not be obtained with GroEL with the same mutation. The GroEL-E461A complementation data was a little surprising. This mutation had no affect on the functional ability of the chaperonin, although similar work done on GroEL where a E461K mutation was made resulted in the cells being heat sensitive (Sewell et al., 2004). However a point to note here is that this mutation alone did not result in the formation of SR1 either. The results obtained here can however support the possibility that the second mutation was able to 228
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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
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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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Page 197 and 198: Chapter 4 caused due to lack of exp
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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 and 224: Chapter 5 expected, evidence of lar
Page 225 and 226: Chapter 5 In summary, based on the
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: Chapter 6 6.2.3 Complementation ana
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.
Page 275 and 276: Gupta, P., S. Mishra & T. K. Chaudh
Page 277 and 278: Hoffmann, A., F. Merz, A. Rutkowska
Page 279 and 280: Ito, K., Y. Akiyama, T. Yura & K. S
Page 281 and 282: Krzewska, J., G. Konopa & K. Libere
Page 283 and 284: Lin, Z., D. Madan & H. S. Rye, (200
Page 285 and 286: Mogk, A. & B. Bukau, (2004) Molecul
Page 287 and 288: Patzelt, H., G. Kramer, T. Rauch, H
Page 289 and 290: Rommelaere, H., M. Van Troys, Y. Ga
Page 291 and 292: 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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