Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.588751
Title: Novel methods to study the amyloid structure and formation of Ig fold domains
Author: Petrik, Eva
Awarding Body: University of Leeds
Current Institution: University of Leeds
Date of Award: 2012
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Abstract:
The correct formation of a stable tertiary structure is essential for most proteins to fulfil their function. Besides the loss of function, the misfolding of the polypeptide chain can lead to aggregation which has a potentially lethal outcome for living systems. Under certain conditions proteins can form highly organised, elongated aggregates called amyloid fibrils. More than 30 human proteins are involved in such diseases which are characterised by the formation of amyloid deposits in vivo. Although several properties of amyloid aggregates are similar, the monomeric proteins that form amyloid fibrils in disease do not share a common characteristic amino acid sequence or secondary structure motif. Only two of the disease-causing proteins, β2-microglobulin (β 2m) and immunoglobulin (Ig) light chain (LC), belong to the same fold family. Whilst the aggregation mechanism of ~2m has been extensively studied, our knowledge of LC polymerisation is limited. Comparison of the molecular changes that lead to aggregation of these structurally related proteins provides a unique opportunity to learn more about amyloid formation. In this thesis the work presented in Chapter 3 describes the development of a high yield expression, purification and refolding protocol to .establish the production of two K LC variable domains, SMA and LEN. These two variants show different thermodynamic stability and ability to form amyloid aggregates despite their highly similar primary sequences. Out of the 8 amino acid differences in their sequences two (P40L and Y96Q) have been shown previously to play crucial roles in destabilising LEN. Together with the wild-type proteins the two single mutants and the double mutant (P40LIY96Q) of LEN were also generated and characterised. After the optimisation of conditions under which amyloid-like fibrils could be generated from these proteins in vitro, 1H_15N HSQC NMR experiments were conducted to investigate the main structural changes that occur under amyloidogenic conditions. Due to the size and insolubility of amyloid fibrils the study of the structural properties of these aggregates by conventional biochemical and biophysical methods is challenging. Moreover, it is even more difficult to characterise structurally amyloid assembly intermediates using structural methods. As a consequence, it is essential to develop new approaches to achieve a better understanding of the structural molecular mechanism of amyloid formation. To address two of the main questions about this phenomenon - what are the conformational changes of monomers during the early aggregation stage and what is the nature of protein- protein interactions within early oligomers/fibres - the use of non-natural amino acids to provide new probes of structure formation was chosen. The studies on this subject are detailed in Chapter 4 and 5. First, by introducing a cross-linking group into the sequence of the monomers side chains proximal to each other within fibrils and oligomers at different aggregation states can be captured and identified. In principal, therefore, this approach could provide more detailed information about the conformation properties of the polypeptide chains during amyloid assembly, as well as within the fibrils themselves. For this project, the non-natural amino acid p-benzoyl-l-phenylalanine (pBpa) was utilised. Chapter 4 of this thesis details the development of a method for the efficient in vivo insertion of pBpa into the sequence of β2m. After designing and generating a library of 20 mutants, the expressed variants were characterised and their ability to forms fibrils of different morphology was assessed. The results showed that the presence of the non-natural amino acid did not affect the folded conformation of the monomer or the ability to form long-straight or worm-like fibrils. The process of the optimisation of the cross-linking protocol and the first results of the cross-linked fibrils are also presented in Chapter 4. Chapter 5 introduces the development of an approach to study the conformational changes of the monomer in situ prior to aggregation. Using the previously optimised expression system for the incorporation of non-natural amino acids, β 2m variants containing p-cyanophenylalanine (CNP) were produced. This non-natural amino acid can be used as a FRET probe together with Trp. Experiments demonstrating that this amino acid pair is sensitive enough to detect the conformation changes of the monomer induced by different solution conditions are discussed, together with the future potentials of this method for measurement of intra- and inter-molecular distances within the fibrils and oligomers. These approaches open up exciting future prospects for the study of the architecture and formation of amyloid aggregates with potential extension to light chains and other amyloid forming proteins.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.588751  DOI: Not available
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