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Chapter 5 (a) 1 2 3 4 5 6 7 8 9 10 72 kDa 55 kDa 17 kDa 11 kDa (b) 1 2 3 4 5 6 7 8 9 10 72 kDa 55 kDa 17 kDa 11 kDa Fig 5.3: SDS-PAGE analysis of JNL from second ion exchange column, and purified JNL (a) Lane 1: Prestained marker (Fermentas); Lane 2: Tube 15; Lane 3: Tube 16; Lane 4: Tube 17; Lane 5: Tube 18; Lane 6: Tube 19; Lane 7: Tube 20; Lane 8: Tube 21; Lane 9: Tube 22; Lane 10: Tube 23 (b) Lane 1: Prestained marker (Fermentas); Lane 2: Unbound 1; Lane 3: Unbound 2; Lane 4: Unbound 3; Lane 5: Tube 10; Lane 6: Tube 11; Lane 7: Tube 12; Lane 8: Tube 13; Lane 9: Tube 14; Lane 10: Concentrated JNL 193
Chapter 5 5.2 AUC analysis of JNL Using AUC (analytical ultracentrifugation), attempts were made to determine the oligomeric state of JNL. The JNL protein sample was run in buffer 5 and buffer A at 20°C and 40,000g for 20 hours. As discussed above, it was shown that under high salt buffer conditions with ATP, M. tuberculosis Cpn60.2 displays the presence of higher oligomeric structures in the AUC and this was confirmed to include the presence of tetradecameric structures by MS (fig 5.4a and 5.4b). As such, it was expected that JNL would also oligomerise in buffer A. On the graphs obtained from the AUC data, the increase in sedimentation coefficient (S) value denotes an increase in oligomeric structures. For Cpn60.2 a sedimentation coefficient of around 13 represents the tetradecameric structure. With that in mind it can be seen from the results obtained here that there is evidence of some large oligomers in buffer A but not in buffer 5 (fig 5.5a and 5.5b). However, since sedimentation velocity experiments depend not only on the molecular mass but also on the shape of a molecule, it becomes difficult to obtain the precise molecular mass measurements from the S value. Although we were unable to analyse the structure further with MS, by analogy with Cpn60.2 we expect this to include tetradecamers. This result was as expected and is consistent with what has been observed with Cpn60.2. Since it is possible to obtain JNL at higher oligomeric forms, it was decided that ATPase assays would be done to see if its ability to hydrolyse ATP is dependent on its ability to oligomerise. For this reason, ATPase assays were done in buffer A and buffer 5 for JNL, Cpn60.2, GroEL (positive control), and BSA (negative control). 194
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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
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: Chapter 5 (a) A B C D E (b) 1 2 3 4
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 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
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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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