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Chapter 1 For this review, the practical and theoretical information of the Hsp60 family of chaperones will be discussed in detail after some of the other chaperone families, as it is the Hsp60 family of chaperones that is being studied in this project. Fig 1.6: A general model for chaperone assisted protein folding within a cell. This figure shows just some of the routes a newly translated polypeptide chain can take to reach its native state. Image taken from Hartl & Hayer-Hartl, 2002. 19
Chapter 1 1.4.3.1 The ribosome associated factor (trigger factor) The trigger factor is a 48kDa protein encoded by the tig gene, which is highly conserved in the eubacteria. In 1998 it was shown to bind with the large subunit of the ribosome (Lill et al., 1988). It is now known to bind to the L23 and L29 proteins near the exit channel of the ribosome where it interacts with nascent polypeptides (Blaha et al., 2003, Kramer et al., 2002, Lakshmipathy et al., 2007). It has also been shown to be associated with up to 90% of all ribosomes in the cytosol (Patzelt et al., 2002). The structure of the trigger factor can be seen in figure 1.7. The trigger factor functions in an ATP independent manner and recognises nascent chains using hydrophobic interactions. It forms complexes with chains as small as 57 residues in length (Hesterkamp et al., 1996). As has already been discussed, the next step in the process involves either allowing the protein to fold (if it is small enough to fold without further support) or if it is unable to do so, then it is passed on to the next set of chaperones like the HSP70 (DnaK) chaperone family (section 1.4.3.2). It has been shown that both TF and DnaK cooperate in chaperoning unfolded proteins. In E. coli this was achieved with the help of ∆tig mutants which resulted in DnaK transiently binding to 40% of nascent polypeptides instead of the usual 15% (Genevaux et al., 2004, Deuerling et al., 1999, Teter et al., 1999). It is important to note that this deletion showed no defects in growth or protein folding in the mutant cells, however, a tig dnaK double mutant or tig dnaK dnaJ triple mutant exhibited synthetic lethality (Deuerling et al., 1999, Teter et al., 1999). In 2004 Genevaux et al also showed that both DnaK and TF could be deleted and the cells would still survive, but this was only achieved at a low temperature (20°C), showing the importance of these chaperones during normal growth conditions. However, research done on gram positive bacteria B. 20
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: Chapter 1 obtained. These revertant
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
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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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