mohammad tabish ahmed - eTheses Repository - University of ...

More documents

Recommendations

Info

Chapter 5 Discussion As discussed in the introduction, the accepted mechanism by which chaperonins function involves the assembly of stable ringed structures to provide a central cavity for protein folding. Substrate proteins can fold within this central cavity without being influenced by other cellular components (Ellis, 1994). Mycobacterial chaperonins however have been shown to be structurally very unstable and posses very weak ATPase activity (Qamra et al., 2004, Kumar et al., 2009). The original aim of this section of the project was to analyse and compare the oligomeric and ATP hydrolysing properties of two the Mycobacterial chaperonin chimeras AHC and JNL. AHC however proved to be very difficult to remake and as such only JNL could be further analysed. Once JNL was purified using ion exchange chromatography, samples were run in the AUC using two different buffers. As discussed above, extensive work in the lab had shown that under high salt conditions and in the presence of ATP, Cpn60.2 formed large tetradecameric structures in vitro (Elsa Zacco, Master’s thesis, 2010). Since the equatorial domain of JNL was from Cpn60.2, it was expected that JNL would also structurally behave like Cpn60.2. The results that were obtained confirmed this hypothesis. In the presence of low salt and no ATP (buffer 5), JNL was shown to be primarily monomeric. A point to keep in mind is that none of the JNL samples were analysed using MS, and as such the results obtained from AUC can only be compared with those of Cpn60.2. Elsa Zacco had shown that in buffer 5, Cpn60.2 was also monomeric in nature. In the presence of high salt buffer and ATP, as 203
Chapter 5 expected, evidence of larger oligomeric structures of JNL was seen. As MS could not be done, the AUC data was compared with that of Cpn60.2. Due to similar results being obtained with Cpn60.2, it is reasonable to infer that the presence of higher oligomeric structures of JNL also includes tetradecamers. Interestingly, when the AUC data in buffer 5 of JNL is compared with that of Cpn60.2, there are signs of slightly higher oligomeric structures, possibly trimers or tetramers, present in the JNL sample. This was consistent with what was observed on the native gel. As was speculated, this may be due to the presence of the apical domain of GroEL. Exactly how this interaction takes place is unclear. However it does suggest that the apical domain also plays a role in the structural stability of a chaperonin. As discussed above the ATPase activity of Mycobacterial chaperonins was shown to be very low when compared with that of GroEL (Kumar et al., 2009, Qamra et al., 2004). The second objective of this study was to analyse the ATPase activity of JNL. As it is known that the protein folding cycle of stable chaperonins such as GroEL is dependent on its ATPase activity, it was expected that in conditions that support oligomerisation, the ATPase activity of JNL would rise. The results that were obtained from the ATPase assays confirmed this hypothesis. It was shown that in buffer 5 when large oligomers are not being formed, the ATPase activity of JNL and Cpn60.2 is extremely low when compared to that of GroEL. However, in buffer A where both Cpn60.2 and JNL show the presence of larger oligomers, their ATPase activity rises considerably. However, even under these conditions the ATPase activity of both Cpn60.2 and JNL is only approximately 30% of that of GroEL. 204
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 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
Page 63 and 64:
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
Page 75 and 76:
Chapter 1 Recent work has suggested
Page 77 and 78:
Chapter 1 (A) cpn10 cpn60.1 cpn60.2
Page 79 and 80:
Chapter 1 1998). Cpn60.1 however ha
Page 81 and 82:
Chapter 1 1.6 Aims of the project T
Page 83 and 84:
Chapter 2 Materials and Methods: 2.
Page 85 and 86:
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
Page 91 and 92:
Chapter 2 29R-EL-60.2 groEL 432-412
Page 93 and 94:
Chapter 2 Biofilm base media: 13.6g
Page 95 and 96:
Chapter 2 Preparation of bacterioph
Page 97 and 98:
Chapter 2 then resuspended in 200µ
Page 99 and 100:
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
Page 107 and 108:
Chapter 2 2.6 Protein analysis 2.6.
Page 109 and 110:
Chapter 2 2.6.2- Native gel Reagent
Page 111 and 112:
Chapter 2 - Diluted primary antibod
Page 113 and 114:
Chapter 2 supernatant was decanted
Page 115 and 116:
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
Page 121 and 122:
Chapter 3 20 mins 40 mins 60 mins 8
Page 123 and 124:
Chapter 3 (a) 60 mins 90 mins 120 m
Page 125 and 126:
Chapter 3 For this experiment, MGM1
Page 127 and 128:
Chapter 3 When IPTG was not include
Page 129 and 130:
Chapter 3 The results from these co
Page 131 and 132:
Chapter 3 3.2.1 The effect of chang
Page 133 and 134:
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
Page 141 and 142:
Chapter 3 1 2 3 4 5 6 7 8 95 kDa 72
Page 143 and 144:
Chapter 3 it only digests the paren
Page 145 and 146:
Chapter 3 72 kDa 1 2 3 4 5 55 kDa 1
Page 147 and 148:
Chapter 3 30°C/26°C 37°C MGM100
Page 149 and 150:
Chapter 3 Fig 3.12: Comparison of t
Page 151 and 152:
Chapter 3 AI90/pACYC-Ptrc-GroESL-Sa
Page 153 and 154:
Chapter 3 1 2 3 4 5 6 7 1000 bp 600
Page 155 and 156:
Chapter 3 10 -1 10 -2 10 -3 10 -4 1
Page 157 and 158:
Chapter 3 this observation. The res
Page 159 and 160:
Chapter 3 expression. However, from
Page 161 and 162:
Chapter 4 Construction and analysis
Page 163 and 164:
Chapter 4 Background In the last ch
Page 165 and 166:
Chapter 4 Experimental design To ma
Page 167 and 168:
Chapter 4 Cpn60.2 7 8 9 Cpn60.1 1 2
Page 169 and 170:
Chapter 4 The full list of all the
Page 171 and 172: 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: Chapter 5 1 2 3 Corrected values Av
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 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.
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
show all

mohammad tabish ahmed - eTheses Repository - University of ...

You also want an ePaper? Increase the reach of your titles

Delete template?

Save as template?