Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.793000
Title: From disorder to order : the importance of context in protein folding and binding mechanisms
Author: Altomar Testa Furtado de Mendonca, Carolina
ISNI:       0000 0004 8501 091X
Awarding Body: University of Cambridge
Current Institution: University of Cambridge
Date of Award: 2019
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Abstract:
Anfinsen's seminal work has shown that the information required for a protein to fold into its specific three-dimensional structure is encoded into its amino acid sequence. The protein structure was believed to determine its activity, meaning that a protein needs to fold in order to function. More recently, intrinsically disordered proteins (IDPs) have been shown to represent a significant portion of the proteome. Despite the lack of a predefined structure, they still play important roles in cellular function, challenging the structure-function paradigm. Proteins are largely studied in isolated conditions, but in a cellular environment they are part of a vastly more complex system. The work presented here aims to shed light on how context can influence folding and binding mechanisms. First, we used SasG - a bacterial protein that defies the disorder prediction with its unique sequence composition and unusual structure - as a template to investigate co-translational folding, and how the presence of the ribosome can affect its folding mechanism. SasG in vitro translation was investigated utilising force profile experiments. We showed that both the G52 and E-G52 constructs can fold very early, when still inside the vestibule of the ribosome. Moreover, our results suggest that non-native interactions can also provide sufficient force to release the stall sequence. Next we employed protein members of the BCL-2 family - involved in controlling the cell death mechanism - to understand what encodes a coupled folding and binding reaction. Although displaying a variety of conformations, some IDPs can fold upon binding to a partner protein. Promiscuous binding is a great advantage of disordered molecules, as multiple IDPs are able to bind and fold to the same partner protein. This raises the question of what orchestrates a coupled folding and binding reaction: the IDP or the partner protein? Using phi-value analysis we studied four IDP-partner protein complexes, composed of alternative pairs of BCL-2 family members. In the bimolecular context, the disordered protein dictates the transition state interactions. Therefore, analogous to Anfinsen's postulate, the folding pathway is encoded by the protein that folds, even when binding to another macromolecule is required. Finally, studies of the BCL-2 member BID on its full-length context showed that it cannot interact with its partner A1 unless it is cleaved (tBID). These results provide insights on the role of tBID as a player during programmed cell death and hence why the pathway of cleaving BID with caspase is energetically favourable.
Supervisor: Clarke, Jane Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.793000  DOI:
Keywords: Protein Folding ; Co-translational Folding ; Intrinsically Disordered Proteins
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