Molecular chaperones are proteins that interact with and aid in stabilization and activation of other proteins. Chaperones help proteins attain their three dimensional conformation, without forming a part of the final structure. Many of the chaperones are stress proteins known as Heat shock proteins (Hsps). Their expression is upregulated in response to various kinds of stress such as heat stress, oxidative stress etc., which threaten the protein homeostasis, by structurally destabilizing cellular proteins, and increasing the concentration of aggregation-prone folding intermediates. The Hsps are classified according to their molecular weight into Hsp40, Hsp60, Hsp70, Hsp90, Hsp100, and the small Hsp families. Some of them are constitutively expressed and play a fundamental role in de novo protein folding. They further aid in proteome maintenance by assisting in oligomeric assembly, protein trafficking, refolding of stress denatured protein, preventing protein aggregation and protein degradation. Heat shock protein 90 (Hsp90) are one of the important representatives of this class of proteins. Hsp90 are highly conserved class of molecular chaperones. They are found in bacteria, eukaryotes, but not in archaea. In contrast to the eukaryotes which require a functional cytoplasmic Hsp90 for viability, the bacterial counterpart (HtpG) is typically nonessential. Hsp90 is an ATP dependent chaperone. Hsp90 form dimers, with each protomer consisting of three functional domains: N- terminal, ATP binding domain, Middle domain and C-terminal domain. Hsp90 is a dynamic protein, and undergoes an elaborate conformational cycle during its ATPase cycle, which is essential for its chaperoning activity. The Hsp90 chaperone cycle is regulated by interaction with diverse cochaperones. Hsp90 interacts with specific set of substrate proteins. Many of these substrate proteins function at the heart of several cellular processes like signalling, cell cycle, apoptosis. Studies from protozoans like Leishmania, Plasmodium, Trypanosoma etc. have also implicated the role of Hsp90 in their growth and stage transitions. Thus, selective inhibition of Hsp90 has been explored as an intervention strategy against important human diseases such as cancer, malaria and other protozoan diseases. The ATP binding N-terminal domain (NTD), has been explored as the target domain for inhibition of Hsp90 using competitive inhibitors of ATP. Several chemical classes of Hsp90 inhibitors are known, including ansamycins, macrolides, purines, pyrazoles, and coumarin antibiotics. However, many inhibitors are observed to be toxic, less soluble and unstable. Hence, there is a requirement for new approach to design inhibitors which are more soluble and less toxic and serve as effective therapeutic drugs.inhibitors are observed to be toxic, less soluble and unstable. Hence, there is a requirement for new approach to design inhibitors which are more soluble and less toxic and serve as effective therapeutic drugs. The work presented in this thesis mainly concerns with the structural studies and biochemical and biophysical characterization of Hsp90 from two different sources viz. Dictyostelium discoideum, a cellular slime mould and a plant source Oryza sativa (rice). The structural analyses of these two proteins have been carried out by X-ray crystallography. Though yeast has been explored extensively as a model system to understand the different roles of Hsp90, it lacks the various signalling pathways essential for growth and development present in case of higher eukaryotes. D. discoideum has been employed as a model system to understand multicellular development, which occurs in response to starvation induced stress. D. discoideum has the advantages due to its ease of manipulation. The organism's genome also shows many signalling pathway for growth and differentiation that are conserved between D. discoideum and mammals. With this motivation, we have studied several structural aspects of the cytosolic isoform of Hsp90 from D. discoideum called HspD. HspD was also observed to play a role in the multicellular development of D. discoideum. It has been demonstrated that the treatment of D. discoideum with inhibitors like Geldanamycin or Radicicol causes an arrest in the multicellular development at the mound stage, and the few which escaped this arrest gave rise to abnormal fruiting bodies. A subset of the proteins involved in this mound arrest phenotype, were observed to have homologs in humans, which are clients of Hsp90. Therefore, a structural perspective of HspD can aid in better understanding of the role of this protein in the organism, as well as, elucidate any structural differences observed as compared to other species, which may have an impact on its activity. Studies on the physiological role of Hsp90 in plants began much later as compared to fungi and humans. In plants Hsp90 are involved in various abiotic stress responses. In addition, their roles have also been implicated in plant growth and development, innate immune response and buffering genetic variations. However, the molecular mechanisms of these various actions are not clearly understood. Also, the structural aspects of plant Hsp90 are yet to be explored. The structure of the NTD of Hsp90 from barley is the only one available from a plant source till now. We have initiated the studies on rice Hsp90 with the objective to understand the mechanism of Hsp90 in plants, which may aid in improving stress tolerance in plants. The thesis has been divided into five chapters. The first chapter introduces the various aspects of Hsp90 protein. The chapter starts with a general overview of concept of molecular chaperones and describes briefly the different classes of molecular chaperones. This is followed by a detailed description of different aspects of Hsp90 with main emphasis on the structure and its conformational flexibility. The chapter describes the association of Hsp90 with other accessory proteins like cochaperones and its interaction with its substrate proteins and explains the functional significance of Hsp90 as a drug target and the need for the development of new class of inhibitors, followed by the significance of the study of Hsp90 in the two model systems (D. discoideum and rice) chosen to be studied. The second chapter gives a brief overview of the principles behind the different experimental methods employed during the course of this research, which includes the tools of X-ray crystallography and other biochemical and biophysical techniques employed for the characterization of the protein. Chapter 3 describes the crystal structure of NTD of Hsp90 from D. discoideum. The structure of NTD was solved in two different native (ligand-free) forms viz. monoclinic and hexagonal. The two forms differed in local structural rearrangement of a segment of NTD known as the lid region. The lid region in the hexagonal form showed a shift in its position as compared to the other solved structures of NTD. The structure of NTD was also solved in complex with various ligands which include ADP, substrate analogs and an inhibitor molecule. A comparison of all the structures showed that the overall structure is well-conserved. One of the crystal structures of NTD showed a heptapeptide (part of the vector) bound at the active site. The peptide was observed to make several complementary interactions with the residues of the ATP binding pocket and retain several interactions which the nucleotide makes with the NTD. The NTD showed subtle conformational differences when compared with the NTD of Hsp90 from yeast. Chapter 4 details the structural and functional characteristics of full length Hsp90 protein from D. discoideum. Due to the large size and flexibility, the full length protein did not crystallize in spite of several attempts. Hence, HspD was studied using different solution studies like Small Angle X-ray Scattering (SAXS) and Dynamic Light Scattering (DLS). Both the studies showed the presence of higher oligomers. The SAXS data showed the presence of tetramers and hexamers while, the addition of the ligand shifts the protein from a dimer to a higher oligomer as observed from DLS studies. The chapter also describes the study of interaction of HspD with a cochaperone protein p23. The interactions were studied using ITC, which showed a strong binding. The ATPase activity was also evaluated in the presence of increasing concentrations of p23, which was observed to decline with increasing concentrations of p23. In chapter 5, we describe the biochemical characterization of Hsp90 from Oryza sativa (rice) and the crystallographic analysis of its NTD. Binding of the rice Hsp90 to ATP and an inhibitor were studied by fluorescence. The ATPase activity of rice Hsp90 was checked by radioactive assay and the protein was observed to be active. The NTD of rice Hsp90 crystallized as a monomer in complex with a substrate analog AMPPCP and the structure was determined.

Thesis (Ph.D.)--University of Alabama at Birmingham, 2007. / Title from PDF title page (viewed on Sept. 17, 2009). Includes bibliographical references.

No description available.

published_or_final_version / abstract / Physiology / Doctoral / Doctor of Philosophy

The heat shock response is a highly conserved genetic mechanism which is induced by a wide range of environmental stimuli. Although intensively studied in both prokaryotes and eukaryotes, no regulatory mechanism has been identified by which the environmental stimuli affect expression of the heat shock genes. In addition, although many inducers of the heat shock response are known to cause DNA damage, the role of heat shock in repair of DNA damage remains unclear. Mutants of Escherichia coli defective in the recA, uvrA, and xthA genes are more sensitive to heat than wild type. However, these mutants are able to develop thermotolerance, suggesting that thermotolerance is an inducible response capable of repairing heat-induced DNA damage independent of recA, uvrA, and xthA. Thermotolerance itself is shown to depend on the dnaK gene, directly linking the E. coli heat shock response to thermotolerance. In addition, the dnaK mutant is sensitive to heat and H₂O₂, but not to UV suggesting that the DnaK protein may function to protect cells from the specific DNA damage caused by heat and H₂O₂. An E. coli grpE mutant was found to be substantially more resistant to 50°C heat treatment than wild type. However, grpE⁻ cells have the same H₂O₂ and UV sensitivity as wild type. This implies that the conditions, for which a grpE mutation is beneficial, are unique to heat exposure and are not caused by H₂O₂ or UV exposure. Furthermore, heat shock protein synthesis occurs sooner in the grpE mutant than in wild type, indicating that the grpE gene product of E. coli may act as a negative regulator of the heat shock response. An adenyl cyclase deletion mutant of E. coli (cya) failed to exhibit a heat shock response even after 30 min. at 42°C. Furthermore, a presumptive cyclic AMP receptor protein (CRP) binding site exists within the promoter region of the E. coli htpR gene. Together, these results suggest that the cya gene may regulate the heat shock response, through cyclic AMP, by directly affecting the level of expression of the heat shock sigma factor.

Exposure of cells to environmental or chemical stressors will initiate the heat shock response, which is mediated by heat shock proteins. Heat shock proteins are molecular chaperones which are classified by size into six main families: HSP100, HSP90, HSP70, HSP60, HSP40 and the small heat shock proteins (sHsps). The sHsp family members bind to denatured proteins and maintain them in a folding competent state such that they may be refolded by other molecular chaperones. <br /><br /> The present study examined the expression and function of two amphibian sHsps, namely, <em>Rana catesbeiana</em> HSP30 and <em>Xenopus laevis</em> HSP27. Initially, an antisense riboprobe was produced to study the mRNA accumulation of <em>Rana hsp30</em> in cultured tongue fibroblast (FT) cells. Results showed that <em>Rana hsp30</em> mRNA was optimally induced when maintained at 35&deg;C for 2 h. An antibody to the recombinant <em>Rana</em> HSP30 protein was also produced in order to study HSP30 protein accumulation in <em>Rana</em> FT cells. Analysis showed that <em>Rana</em> HSP30 was heat-inducible and accumulated maximally at 4 h when maintained at 35&deg;C and then allowed to recover at 22&deg;C for 2 h. Immunocytochemical analysis indicated that <em>Rana</em> HSP30 protein was present primarily in the nucleus, with diffuse localization in the cytoplasm. Additional immunocytochemical analysis showed that <em>Rana</em> HSP30 remained in the nucleus following heat stress and extended periods of recovery. <br /><br /> The molecular chaperone function of <em>Rana</em> HSP30 was also studied. Recombinant <em>Rana</em> HSP30 was found to inhibit the heat induced aggregation of various target proteins including citrate synthase, luciferase and malate dehydrogenase. Also, no major difference was detected between <em>Rana</em> HSP30 and <em>Xenopus</em> HSP30C in the inhibition of heat-induced aggregation of target proteins. <br /><br /> This study also examined the expression and function of <em>Xenopus laevis</em> HSP27. Analysis of the putative amino acid sequence of the <em>Xenopus hsp27</em> cDNA revealed that it had an identity of 71% with chicken, 65% with zebrafish, 63% with human and 53% with topminnow. Most of the identity was located within the &alpha;-crystallin domain of the protein. Interestingly, <em>Xenopus</em> HSP27 shared only a 19% identity with 2 other <em>Xenopus</em> sHsps, HSP30C and HSP30D. <br /><br /> Western blot analysis using an anti-<em>Xenopus</em> HSP27 antibody revealed that HSP27 was not detectable in cultured kidney epithelial cells. However, examination of early <em>Xenopus</em> embryos revealed that HSP27 was first detected in tadpole embryos (stage 44). Heat-inducible HSP27 was also first detected at this stage. The accumulation pattern of <em>Xenopus</em> HSP27 protein was distinct from <em>Xenopus</em> HSP30, which was heat-inducible at midtailbud stage 26, approximately two and a half days earlier in development. <br /><br /> Analysis of recombinant HSP27 by native pore exclusion limit electrophoresis showed that it formed high molecular weight, multimeric complexes. The molecular chaperone function of HSP27 was assessed by means of thermal aggregation assays employing citrate synthase, luciferase and malate dehydrogenase. <em>Xenopus</em> HSP27 inhibited the heat-induced aggregation of all of these target proteins. This study has revealed that <em>Xenopus</em> HSP27 is a member of the HSP27 subfamily of small heat shock proteins in <em>Xenopus</em> and distinct from the HSP30 family. The accumulation of HSP27 under constitutive and stress-inducible conditions is developmentally regulated. Finally, this sHsp appears to function as a molecular chaperone.

M.Sc. / Heat shock proteins (HSP) are a group of highly conserved proteins induced in pro- and eukaryotes by a wide variety of environmental stresses such as heat shock (HS) and oxidative injury. HSP are classified into families according to their apparent molecular mass and respective inducers. Induction of HSP is primarily regulated on transcriptional level through multiple copies of a conserved cis-acting heat shock element (HSE) in the promoter region of all hsp genes to which the heat shock transcription factor (HSF) binds. Members of the HSP family function collectively as molecular chaperone systems, and fulfil essential roles under normal conditions and provide protection and adaptation during and following stress. The induction of HSP following stress and the subsequent protection confer HSP the potential application in stress therapy and in biomarking of stress. During a previous study in which the effect of Mycobacterium tuberculosis (M.tb) on the stress response of peripheral blood moncytes (PBM) from different donors was investigated, it was observed that different individuals from different South African populations showed differential a HSP synthesis in response to M.tb. This compelled us to investigate the following: Variation in HSP synthesis in peripheral blood monocytes (PBM) from different individuals in response to the classical HSP inducer, HS. The most appropriate technique to study HSP expression on protein level. HSP synthesis was studied in PBM from 36 individuals (European (E): n=22; non-Europeans (nE): n=14) using biometabolic labelling. Three techniques were compared in the determination of HSP expression in six donors in terms of HSP synthesis, which is measured by biometabolic labelling, and accumulation of hsp70 that were measured by Western blot analysis and flow cytometry. Results obtained are : European (E) and non-European (nE) populations differed significantly (p < 0.05) from each other in spite of a prominent variation in HSP synthesis within donors ; Flow cytometry is the technique of choice for the analysis of HSP levels, since it allows fast and safe measurement of HSP levels in single cel populations within a mixed population. Data from flow cytometry correlate with Western blot analysis, but not with biometabolic labelling. The means and ranges for different HSP synthesis in different populations reported in this study, set a standard for the use of HSP as biomarker of pa environmental stress for populations inhabiting southern Africa. Efficient measurement of HSP expression as biomarker of stress can therefore be implemented in routine analysis of environmental stress, as well as investigations concerning the implications of HSP in pathology.

Os genes de choque térmico groES e groEL de Caulobacter crescentus foram isolados utilizando-se os genes homólogos de E.coli como sonda e por sequenciamento demonstrou-se que estes genes estão organizados na forma de um operon em um fragmento de DNA de aproximadamente 2,5 kb, contendo também sua região regulatória. \"Northern blots\" de RNA total de células crescidas a 300C ou submetidas a choque térmico mostraram a presença de um único RNA de tamanho aproximado de 2,3kb, altamente induzido por choque térmico, permanecendo em altos níveis mesmo após longos períodos de choque térmico. Amostras de RNA total de células sincronizadas, de diferentes estágios do ciclo celular de Caulobacter, foram também analisadas mostrando que os níveis do mRNA groESL variam durante o ciclo, apresentando um máximo na célula prédivisional. Análises através de \"Western blot\" mostraram uma pequena variação nos níveis da proteína GroEL ao longo do ciclo celular, sendo os tempos 60 e 120 minutos, respectivamente, os pontos de mínimo e máximo acúmulo da proteína concordando com os resultados obtidos em \"N orthern blots\". O mesmo tipo de análise foi feito com extratos totais obtidos a partir uma população mista de células crescidas a 300C e submetidas a choque térmico, observando-se o acúmulo da proteína até 60 minutos depois do choque térmico, com aumento da ordem de 5 vezes nos níveis de GroEL, níveis estes que diminuem lentamente a partir deste ponto. Os inícios de transcrição foram determinados em experimentos de \"primer extension\" utilizando-se RNA total de células incubadas 300C e de células submetidas a diferentes condições de choque térmico. Dois possíveis sítios de início de transcrição foram determinados nas posições -119 e -88 do ATG da metionina iniciadora de groES, sendo as regiões -10 e -35 dos promotores correspondentes (P 1 e P2) identificadas. Somente a transcrição iniciando a partir de P2, que apresenta características de um promotor transcrito pelo &#963;32, aumenta durante o choque térmico. Fusões de transcrição com o vetor repórte placZ/290 e a região 5\' regulatória do operon groESL foram construídas para identificar as sequências responsáveis pelo controle por choque térmico e pelo controle temporal. Fusões de transcrição contendo deleções na região 5\' do operon mostraram que sequências a montante do promotor P2 não são necessárias para a indução por choque térmico ou para o controle temporal. Fusões de transcrição contendo mutações sítio-dirigidas na repetição invertida, encontrada a 3\' do promotor P2, antes do gene groES, revelaram que este elemento, conhecido como CIRCE, regula negativamente a expressão de groESL a 300C e mutações neste elemento levam à perda do controle temporal deste operon. / The heat shock genes groES and groEL of Caulobacter crescentus were isolated using the homologous genes of E.colí as a probe. DNA sequence analysis has shown that these genes are organized as an operan in a fragment of about 2.5kb, which includes the 5\' regulatory region. Northern blot analysis of total RNA from cells grown at 300C or heat shocked treated has shown the presence of a single mRNA species for groESL, of approximately 2.3kb in size, which presented increased leveis even after long periods of heat shock. Samples of total RNA from synchronized cells, corresponding to different stages of the Caulobaaer cell cycle, were also analysed, showing that the amount of groESL mRNA varies during the cycle, with maximum leveis in predivisional cells. Western blot analysis of GroEL leveis in Caulobaaer has shown that the amount of the protein decreases during the first 60 minutes of C.crescentus cell cycle and then starts to increase again. These results corroborate the data obtained with Northern blot analysis. A similar experiment was performed after exposing a mixed population of C.crescentus cells to different times of heat shock at 400C. Western blot of extracts of these cells showed a fivefold increase in the leveis of GroEL after 60 minutes of heat shock, which then begins to decrease. Primer extension experiments were performed using total RNA from cells incubated at normal growth temperature or after heat shock treatment. Two possible transcription start sites were determined at positions -119 and -88 from the ATG of the groES initiator methionine and the -10 and -35 regions of the corresponding promoters (P 1 and P2) were identified. Only trancription initiating from the P2 promoter, which has caracteristics of a &#963;32 promoter, I ncreases during heat shock .Transcription fusions with the reporter vector placZ/290 and the 5\' regulatory region of the groESL operan were contructed in order to identify the sequences responsible for heat shock and cell cycle contral. Deletion analysis in the 5\' region of the operon showed that no sequences upstream of the P2 promoter are necessary for heat shock induction or for temporal contral. Site-directed mutagenesis in the inverted repeat found 3\' of the P2 promoter, in front of the groES gene, revealed that this element, also known as CIRCE, negatively regulates groESL expression at 300C and mutations in it lead to loss of temporal control of this heat shock operon.

by Lam Wai Yip. / Thesis (M.Phil.)--Chinese University of Hong Kong, 1995. / Includes bibliographical references (leaves 184-195). / Contents --- p.I - IV / Abstract --- p.V / Abbreviations --- p.VI / List of Tables and Figures --- p.VII - XV / Chapter Chapter One: --- Introduction / Part I / Chapter 1.1 --- Human genome project --- p.1 / Chapter 1.2 --- Progress of human genome project --- p.2 / Chapter 1.3 --- Human heart cDNA sequencing --- p.3 / Chapter 1.4 --- Significance of the human heart cDNA library project --- p.5 / Chapter 1.5 --- Homology search tools for cDNA sequences alignment --- p.5 / Part II / Chapter 1.6 --- Investigation of a human heart cDNA clone A076 --- p.7 / Chapter 1.7 --- General introduction of Heat Shock Proteins (HSPs) --- p.7 / Chapter 1.7.1 --- Definition of HSP --- p.8 / Chapter 1.7.2 --- Discovery of HSP --- p.10 / Chapter 1.7.3 --- Transcriptional regulation of heat shock genes --- p.11 / Chapter 1.7.4 --- Nomenclature of HSPs --- p.13 / Chapter 1.7.5 --- HSP110 --- p.13 / Chapter 1.7.6 --- HSP90 --- p.14 / Chapter 1.7.7 --- HSP70 --- p.15 / Chapter 1.7.8 --- HSP60 --- p.17 / Chapter 1.7.9 --- Ubiquitin - HSP8 --- p.19 / Chapter 1.7.10 --- HSP27 --- p.20 / Chapter 1.8 --- The theme of this thesis --- p.28 / Chapter Chapter Two: --- Method and Materials / Chapter 2.1 --- The human heart cDNA library --- p.29 / Chapter 2.2 --- Plating out the cDNA library --- p.29 / Chapter 2.3 --- DNA amplification --- p.31 / Chapter 2.4 --- DNA sequencing reaction - Cycle sequencing reaction --- p.32 / Chapter 2.5 --- Operation of the A.L.F. DNA sequencer --- p.33 / Chapter 2.5.1 --- Preparation of the gel cassette --- p.33 / Chapter 2.5.2 --- Preparation of the acrylamide gel --- p.34 / Chapter 2.5.3 --- Fitting the gel cassette into the electrophoresis unit --- p.35 / Chapter 2.5.4 --- Settings of electrophoresis --- p.36 / Chapter 2.6 --- Comparison of DNA sequences to databases --- p.37 / Chapter 2.7 --- Programming for sending cDNA sequences to NCBI --- p.38 / Chapter 2.8 --- Storage of sequence data --- p.39 / Chapter 2.9 --- Synthesis and purification of primers --- p.40 / Chapter 2.10 --- Connection of cDNA clones using Polymerase Chain Reaction (PCR) --- p.41 / Chapter 2.11 --- Purification of DNA fragment from agarose gels by GENECLEAN´ёØ --- p.42 / Chapter 2.12 --- "Preparation of competent Escherichia coli for transformation (Hanahan, 1986)" --- p.43 / Chapter 2.13 --- Transformation of Plasmid into Competent Escherichia coli --- p.44 / Chapter 2.14 --- "Small scale preparation of plasmid DNA (Sambrook et al.,1989" --- p.45 / Chapter 2.15 --- Large scale plasmid preparation by QIAGEN´ёØ --- p.46 / Chapter 2.16 --- DNA sequencing reaction - Unicycle sequencing reaction --- p.48 / Chapter 2.17 --- Synthesis of Radiolabeled DNA probe --- p.49 / Chapter 2.18 --- "Isolation of genomic DNA from human blood cells (Thomas A. Ciulla, 1988)" --- p.51 / Chapter 2.19 --- Southern blotting --- p.52 / Chapter 2.20 --- Prehybridization and hybridization procedure for Southern blot analysis --- p.54 / Chapter 2.21 --- "AGPC-RNA extraction method (Chomczynski and Sacchi 1987, modifed)" --- p.56 / Chapter 2.22 --- Electrophoresis of RNA through gels containing formaldehyde --- p.58 / Chapter 2.23 --- First-Strand cDNA synthesis --- p.59 / Chapter 2.24 --- Use of T7 RNA polymerase to direct expression of the cloned hsp27b gene (A076&B490) --- p.60 / Chapter 2.25 --- "Sodium Dodecyl Sulphate Polyacrylamide Gel Electrophoresis (Laemmli, 1970)" --- p.61 / Chapter 2.26 --- Staining of the Gel by the Commassie Blue Method --- p.63 / Chapter Chapter Three: --- Results / Part I / Chapter 3.1 --- Sample results of Sequencing a few clones --- p.64 / Chapter 3.2 --- A Catalogue of 497 cDNA clones obtained from human heart cDNA sequencing --- p.71 / Chapter 3.3 --- Submission of novel sequences to genbank --- p.81 / Chapter 3.4 --- A Catalogues of genes that are expressed in the adult human heart --- p.83 / Chapter 3.5 --- The use of the programmes to assist the sending and receiving of sequence data E-mail message --- p.90 / Chapter 3.5.1 --- The use of the SENDMAIL.EXE programme --- p.91 / Chapter 3.5.2 --- "The use of the EDITBLN.EXE, ALLFILE.EXE and DATABASE.EXE" --- p.95 / Part II / Chapter 3.6 --- DNA sequence profiles of cDNA clones A076 and B490 --- p.105 / Chapter 3.7 --- Ligation of cDNA clones using Polymerase Chain Reaction (PCR) --- p.112 / Chapter 3.8 --- Cloning of the PCR product A076&B490 into the pAED4 expression vector --- p.117 / Chapter 3.9 --- Unicycle sequencing of the subcloned insert A076&B490 --- p.121 / Chapter 3.10 --- Southern hybridization of hsp27b (A076&B490) --- p.125 / Chapter 3.11 --- Results of RT-PCR and PCR --- p.127 / Chapter 3.12 --- Expression pAED4-A076&B490 in E.coli --- p.133 / Chapter Chapter Four: --- Discussion / Part I / Chapter 4.1 --- EST characterization --- p.138 / Chapter 4.2 --- Further investigation --- p.140 / Chapter 4.3 --- Disadvantage of randomly picked cDNA sequencing --- p.141 / Chapter 4.4 --- Problem of GenBank database searching --- p.141 / Part II / Chapter 4.5 --- The DNA sequence of A076 and B490 --- p.143 / Chapter 4.6 --- Ligation of HSP27B by using PCR --- p.144 / Chapter 4.7 --- Analysis of the DNA and protein sequence ofhsp27b (A076&B490) --- p.145 / Chapter 4.8 --- Southern hybridization of human hsp27b --- p.153 / Chapter 4.9 --- "RT-PCR and PCR of first strand cDNA with primers A076-ATG, A076-mid and oligo dT" --- p.153 / Chapter 4.10 --- Expression of human hsp27b --- p.154 / Chapter 4.11 --- The possible roles of human hsp27b --- p.156 / Chapter 4.12 --- Further analysis --- p.160 / Appendix I --- p.161-182 / Appendix II --- p.183 / References --- p.184-195

Healthy cells continuously produce proteins to accomplish various functions, including immune responses, reaction catalyses, transmitting signals, structural supports and molecular transport. Protein needs to fold correctly into three-dimensional shape in order to function well, using the information stored in the amino acid sequence. Proteins may fold spontaneously in solution, but the situation in living cells can be complicated. Cells are filled with nucleic acids and proteins thus they are usually in a stressful environment. Under such circumstances, proteins can be unfolded or misfolded, leading to non-function or even toxicity. Cells employ molecular chaperones to solve protein folding problems. Among the many types of chaperones, heat shock proteins of approximately 70KDa (Hsp70s) act as a hub, because its functions feed into other members of the chaperone network. Hsp70s help to stabilize nascent polypeptides, facilitate cross-membrane translocation, refold the misfolded proteins, and guide non-recoverable denatured proteins to degradation. Hsp70s have explicit role in cancer cells, because elevated metabolism requires increased Hsp70s’ activity to avoid apoptosis and ensure survival. Hsp70s also help to prevent neurodegenerative diseases, and decreased level of Hsp70s is found in age-related symptoms and diseases. In general, it is well understood what Hsp70s can do, but little is known how Hsp70s do the job. Hsp70s are present and highly conserved in all living species, comprised of two structural domains. The nucleotide binding domain (NBD) binds and hydrolyzes ATP, while the substrate binding domain (SBD) binds and releases hydrophobic peptides. Although Hsp70s are known to act as an allosteric molecular machine, the details are elusive about how the domains are regulated. Besides, how nucleotide binding affects the Hsp70s’ function, and how ATP hydrolysis is performed are also unknown. In this thesis, I first introduce salient background on the Hsp70 subject, then explore previously unclear aspects of Hsp70 allosteric regulation and catalytic activity in two chapters describing my dissertation research, and finally conclude with my perspectives on future directions.