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http://hdl.handle.net/10603/19955
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| DC Field | Value | Language |
|---|---|---|
| dc.coverage.spatial | Microbiology | en_US |
| dc.date.accessioned | 2014-06-30T09:16:26Z | - |
| dc.date.available | 2014-06-30T09:16:26Z | - |
| dc.date.issued | 2014-06-30 | - |
| dc.identifier.uri | http://hdl.handle.net/10603/19955 | - |
| dc.description.abstract | n | en_US |
| dc.format.extent | 111p. | en_US |
| dc.language | English | en_US |
| dc.relation | - | en_US |
| dc.rights | university | en_US |
| dc.title | Structural-biochemical studies of protein (mis) folding and aggregation | en_US |
| dc.title.alternative | The ability of proteins to accomplish diverse biological functions with great specificity is dependent on the attainment of the native conformation. When the proteins, under some conditions, fail to fold correctly, this failure can result in misfolding accompanied with aggregation, which is known to lead to a group of disorders known as the protein conformational disorders or PCDs. Several theories have been put forward to explain the underlying basis of protein conformational disorders. According to the "loss of function" hypothesis, the depletion of the normal biological function of the protein due to misfolding and aggregation is the key step leading to the disease, whereas the "gain-of toxic activity" hypothesis suggests that the crucial element is the toxicity of the aggregates themselves. The current interest in the topic of protein aggregation arises from several considerations: knowledge of the molecular basis of protein misfolding and aggregation is expected to aid in the understanding ofthe physicochemical aspects of protein folding; it is expected to shed light on the molecular and biochemical basis of the PCDs that have an immense social impact; it is also expected to impact directly on the recombinant protein industry. Protein aggregation and protein folding are understood to be in kinetic competition and the structural factors playing relevant roles in one process are also thought to be involved in the other one. The present work relates mainly to understanding and manipulating the aggregative behavior shown by polypeptide chains either during, or after, structure formation, or during the destruction of formed structures, as occurs e.g., upon exposure of proteins to destabilizing influences such as high temperatures. One of the main issues addressed is the extent to which the native structure of the aggregating polypeptide survives within an aggregate. Persistence of native structure within aggregates may be expected to impact fundamentally on the success of efforts designed to recover folded proteins from aggregates (e.g those formed during overexpression of recombinant proteins) through partial unfolding, especially where yields from processes involving complete unfolding- refolding are known to be poor. Views differ as to the likely persistence of native- structure, and the little evidence that does exist currently points to possibilities of both (a) large elements of native-like structure surviving, with only a small section of a protein chain participating in aggregation, and (b) complete destruction of native-like Abstract structure, concomitant with the formation of non-native structures such as intermolecular beta sheets as seen in the formation of amyloid fibres. One would like to examine the conditions under which these two situations obtain. It is important to appreciate, however, that discerning native-like structural content in aggregates is not easily accomplished. For one thing, aggregates are notoriously intractable to routine structural-biochemical investigations. For another, even with the design and application of novel, and protein-specific strategies, culling out any evidence of native-like structure from a morass of polypeptide chains is not easy. Recognizing that different proteins may throw up different answers, we have designed and applied a number of novel methods to answer the question "How native-like is a polypeptide chain within an aggregate?" This thesis is divided into seven chapters with references listed at the ends of each chapter. Chapter 1 gives a brief introduction to protein misfolding and aggregation, discussing the interactions thought to mediate protein aggregation besides highlighting the role of protein aggregation studies. Chapter 2 provides details of general materials and methods common to all aspects of this study. Each of the remaining chapters then has a brief 'introduction' section followed by an 'experimental strategies and design' section. Due to obvious problems that arise in studying protein aggregates (as discussed in section 1.4.1 ), we have attempted to develop different approaches to address core issues pertaining to different types of protein aggregates. Therefore, in the experimental strategies and design section of each of chapters 3-7, we have individually discussed the problem and the approach that we have followed to address the problem. The materials and methods unique to each chapter are also discussed in this section. In chapter 3, we have examined amorphous aggregates of bovine carbonic anhydrase (BCA). We have investigated the extent to which there is survival of native- like three-dimensional structure during the thermal unfolding-associated aggregation of bovine carbonic anhydrase (BCA). Given the intractability of aggregates to direct structural investigations, we have addressed the question tangentially, through use of a non-sequence-specific protease, subtilisin, to probe peptide bonds that remain physically accessible and chemically amenable to scission in native and aggregated BCA chains. Briefly, proteolytic digests of the thermally aggregated BCA and native BCA were compared. SDS-PAGE analysis revealed several bands of the aggregated BCA that survive the proteolytic attack, indicating that sizeable sections of folded structure (that manage to bury peptide bonds away from scission) must exist within the aggregates. A Abstract number of bands obtained through digestion of aggregates were similar in size to those obtained through digestion of control samples of native BCA. Pairs of size-matched polypeptides were subjected to N-terminal sequencing. Remarkably, the N-termini of size-matched fragments were discovered to be either identical, or proximal, between bands derived through digestion of native and aggregated BCA. All but one of these N- termini map onto the surface of BCA's native structure. The fact that very nearly the same sites in aggregated and native BCA chains turn out to be susceptible to proteolysis by a non-specifically acting protease, establishes that sizeable sections of the polypeptide chain ofBCA somehow survive within heated BCA chains (as they unfold and associate to form amorphous aggregates) in native-like structural format, with structured and protease-resistant subdomains intact. In chapters 4 and 5, we have tried to examine whether amino acid sequences with very high aggregation propensities (derived from known amyloidogenic peptides) have the ability to coerce, or persuade stably folded domains present with them in genetic fusion to deposit into aggregates. The question addressed is whether native-like structure can survive in sections of a polypeptide chain in which other parts of the same chain engage in amyloid formation. We addressed these issues through creation of a genetic fusion of (i) an extremely aggregation-prone polypeptide sequence (an amyloidogen) with (ii) the sequence of an extremely structurally-stable, naturally- occuring protein domain; preferably one imbued with an easily monitored spectroscopic characteristic that is widely accepted to be diagnostic of a folded, native state. We then examined the behaviour of these fusion constructs. In chapter 4, we describe studies of a construct genetically fusing the green fluorescent protein with an amyloidogenic protein, retroCspA. The aggregation behavior of genetic fusion constructs of enhanced green fluorescent protein (EGFP) with amyloid- forming retroCspA, was examined from a structural-biochemical viewpoint. In the fusion construct with retroCspA at its N-terminus, intracellular aggregation was observed with a failure of EGFP to fold into a structured and fluorescent form, both within overexpressing cells, and upon purification. However, our purpose was to obtain a construct containing folded EGFP, upon purification to enable us to observe changes in this construct, if any, with the aggregation of the chain through the retroCspA section of Abstract the chain. This situation could be obtained in a fusion placing retroCspA (the amyloidogen) at the C-terminal end of folded EGFP. To begin with, the fusion protein aggregated into urea- and guanidine hydrochloride (GdnCI)-insoluble aggregates within overexpressing cells that, however, displayed green fluorescence, indicating a retention of native-like structure within the EGFP domain of at least some of the fusion construct. Extraction of the fusion protein from inclusion bodies was achieved using a combination of heat, GdnCI and high salt (1M NaCI). Following refolding, the affinity purified protein turned out to form soluble as well as insoluble aggregates, with negligible adoption of monomeric forms. Both soluble and insoluble aggregates could be shown to contain populations of native-like EGFP domains, displaying green fluorescence, as well as all structural characteristics of folded EGFP. Electron micrographs of insoluble aggregates revealed that they consisted of fibrillar forms, and no amorphous aggregates. Remarkably, the fibrillar material shows an unusual morphology which has never been reported for amyloid protein forms; all fibrils are decorated more or less evenly with globular beads emanating from the fibrils in all directions, on their surfaces. We speculate that the folded EGFP domain which cannot be incorporated into amyloids associates into the globular structures observed to coat the amyloid fibrils, while the retro-CspA domain forms the bulk of the proteinaceous material constituting the fibres. In chapter 5 we examine the behaviour of a fusion of a thermophilic cellulase, Cei12A (from the thermophilic bacterium Rhodothermus marinus) with a well-known amyloidogen, Alzheimer's amyloid 13 peptide1-42• Our studies with the Cei12A-13 amyloid fusion suggest that the highly thermostable Cei12A in the fusion maintains its structure (within aggregates oflow association number) despite the presence of highly aggregation prone sequences at its C-terminus that could theoretically have invaded the cellulase domain to pull the same into amyloid forms. Studies with the soluble fusion suggest that on account of the fusion, there exist two different populations in solution: there being monomers and soluble multimers. These multimers are not seen in the native Cei12A (without the 13 amyloid structures), suggesting that these multimers are likely to be the result of the association between 13 amyloid components of the fusion. The 13 amyloid fragments after excision from the fusion, deposit into amyloid aggregates on account · their high aggregating propensity. The Cei12A resists structural distortions in the fusion Abstract and does not participate in 'runaway' aggregation. Somehow the A(31_42 gets truncated in the cell, even before purification. After purification too, the truncations continue leading to a reduction in the length of the polypeptide. The truncations could be caused either by contaminating proteases or by some proteolytic activity associated with the cellulase. In chapter 6, we describe the behavior of a genetic fusion of glutathione S transferase (GST) with triose phosphate isomerase (TIM) derived from the hyperthermophile, Pyrococcus .furiosus. We studied this fusion construct of GST with PfuTIM, in an attempt to create a 'designer' soluble aggregate; a meshwork of protein with two multimerization faculties i.e. with GST forming dimeric structures and TIM forming tetrameric structures. The soluble fusion protein comprises many populations of varying sizes as assessed by gel filtration chromatography and SDS-PAGE. These primarily arise due to the individual abilities of the GST to dimerize and the PfuTIM to tetramerize. The presence of free GST/ TIM further adds to the complexity feasible in the meshwork. In theory, a number of distinct possibilities exist, which are detailed in the chapter. Experimentally, we have tried to correlate the sizes of different populations with predictable schemes of association of different species, such as cleaved GST, cleaved PfuTIM and the GST-PfuTIM fusion construct. Our studies show that that the soluble fusion protein retains both GST and the PfuTIM in the folded state within soluble aggregates. Further, the fusion protein can be caused to aggregate thermally through the unfolding and intermolecular reassociation of the GST component of the fusion, and this insoluble aggregate retains PfuTIM in its native structure, as evidenced by a novel gel electrophoresis-based technique. This last result, in particular, augurs well for the conception of a new design for the creation of immobilized enzymes - in which a stable protein is fused with a protein of relatively lower stability. If, upon heating, one component of the fusion remains native in the precipitate resulting from aggregation, this component may presumably perform its enzymatic function in the aggregated state. Of course, in the present instance, difficulties relating to assaying hyperthermophile PfuTIM (owing to the need for a supporting hyperthermophile enzyme) prevented us from actually demonstrating the 'immobilized enzyme' status of PfuTIM. Even so, we believe that this work lays the groundwork for future studies of this nature. Abstract In chapter 7, we describe the behavior of a 12 residues-mutated variant of wild type human prion protein (residues 90-231). This mutant was selected previously by a phage- display library screening approach, by another investigator. We created the desired 12- residue mutation (from position 10 1-112) in the wild type prion protein (WtPrP) 90-231 sequence by recombinant DNA methods and expressed and purified this mutant (MutPrP). The objective of the study was to investigate the role of these mutations located in the N- terminal region of WtPrP in its misfolding and aggregation. Comparative studies were done for both the WtPrP and MutPrP. The results highlight the role of mutations in theN-terminal region of the human prion protein in causing the conformational switching from a-helical to ~-sheet structure. As is well known, prion protein aggregation into amyloids is believed to involve an intermediate species that is rich in ~-sheet structure. The present study shows that such a beta sheet-dominated conformation of WtPrP [which is known to be generated through refolding of prion protein in acidic buffer (pH 4.0) under reducing conditions] also tends to be naturally-formed by a (12 residues altered) mutant form of PrP under non- reducing conditions at pH 10. This suggests a role ofthe 101-112 region in the PrP (alpha helix to beta sheet) conformation switch, relevant to disease. | en_US |
| dc.creator.researcher | Sharma, Swati | en_US |
| dc.subject.keyword | Microbiology | en_US |
| dc.description.note | - | en_US |
| dc.contributor.guide | Guptasarma, Purnananda | en_US |
| dc.publisher.place | Delhi | en_US |
| dc.publisher.university | Jawaharlal Nehru University | en_US |
| dc.publisher.institution | Institute of Microbial Technology | en_US |
| dc.date.registered | n.d. | en_US |
| dc.date.completed | 2006 | en_US |
| dc.date.awarded | n.d. | en_US |
| dc.format.dimensions | - | en_US |
| dc.format.accompanyingmaterial | None | en_US |
| dc.type.degree | Ph.D. | en_US |
| dc.source.inflibnet | INFLIBNET | en_US |
| Appears in Departments: | Institute of Microbial Technology | |
Files in This Item:
| File | Description | Size | Format | |
|---|---|---|---|---|
| 01_title.pdf | Attached File | 18.14 kB | Adobe PDF | View/Open |
| 02_dedication.pdf | 13.12 kB | Adobe PDF | View/Open | |
| 03_contents.pdf | 30.89 kB | Adobe PDF | View/Open | |
| 04_acknowledgements.pdf | 78.18 kB | Adobe PDF | View/Open | |
| 05_abstract.pdf | 275.2 kB | Adobe PDF | View/Open | |
| 06_chapter 1.pdf | 1.15 MB | Adobe PDF | View/Open | |
| 07_chapter 2.pdf | 3.04 MB | Adobe PDF | View/Open | |
| 08_chapter 3.pdf | 3.45 MB | Adobe PDF | View/Open | |
| 09_chapter 3.pdf | 3.49 MB | Adobe PDF | View/Open | |
| 10_chapter 4.pdf | 1.6 MB | Adobe PDF | View/Open | |
| 11_chapter 6.pdf | 1.73 MB | Adobe PDF | View/Open | |
| 12_chapter 7.pdf | 2.68 MB | Adobe PDF | View/Open |
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