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Chapter 1 Table 1.1: Classes of molecular chaperone families. The examples shown are but a few of the known chaperones and are by no means the only example. Information obtained from Pete Lund, personal communication. Bacteria Mitochondria (m) / chloroplasts (c) Archaea Eukaryotic cytosol Endoplasmic reticulum Function Hsp100 ClpA ClpB ClpX HSP78 (m) ClpC (c) ClpB (c) Absent HSP104; absent from cytosol of animals. Absent Disassembly of oligomers and aggregates. Hsp90 HtpG TRAP1 (m) HSP90 (c) Absent HSP90 HSP82 HSP83 GRP94 In eukaryotes: regulation of assembly of steroid receptors and signal transduction proteins; protein folding and degradation. In prokaryotes the function of HtpG is less clear, although there is evidence that it facilitates protein folding in stressed E. coli. Hsp70 DnaK Hsc66 Grp75 (m) HSP70 (absent from many archaea) HSP70, HSC70, SSB, SSA BiP Prevention of aggregation of unfolded/newly translated/newly imported proteins; many other functions such as clathrin uncoating, regulation of heat shock response. Chaperonin/ Hsp60/Cpn60 GroEL (also referred to as Cpn60) HSP60 (m) Rubisco sub-unit binding protein (c) Thermosome, TF55, CCT CCT (also called TRiC) Absent Folding of newly translated and newly imported proteins. A vital part of the heat shock response. 15
Chapter 1 Hsp40 DnaJ CbpA DjlA Ydj1 (m) Cdj1 (c) HSP40 (absent from many archaea) HSP40 Sis1 Zuo1 Sec63 Binding of unfolded proteins, stimulation of ATPase activity of HSP70 (HSP70 cochaperone). Very heterologous group as many proteins possess “DnaJ-domain”. sHsps (small heat shock proteins) IbpA IbpB HSP22 (m) HSP21(c) HSP16.5 HSP26 Alphacrystallin Absent Stabilisation of unfolded proteins, prevention of protein aggregation. Cochaperonin GroES HSP10 Absent Absent Absent Essential for function of type I chaperonins. Prefoldin Absent Absent Prefoldin GimC Prefoldin GimC Absent Prevents protein aggregation; functions with type II chaperonins but not essential. Trigger factor Tig Absent (m); Present (c) Absent Absent Absent Assists folding of nascent ribosome-bound polypeptide. Nascent polypeptideassociated complex Absent Absent MTH177 NAC Absent Interacts with and probably assists folding of nascent ribosome-bound polypeptide. ER chaperones Absent Absent Absent Absent Calnexin Calreticulin ERp53 HSP47 Folding of ER glycosylated proteins. HSP47 is specific for collagen chains. 16
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: Chapter 1 1.4.1 Classes of molecula
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
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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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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.
Page 295:
Yamamori, T. & T. Yura, (1982) Gene
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