Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.643179
Title: Ca²⁺-dependent conformational change in Synaptotagmin I
Author: Ciufo, Leonora Frances
Awarding Body: University of Edinburgh
Current Institution: University of Edinburgh
Date of Award: 1998
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Abstract:
This thesis reports work on synaptotagmin I, the isoform that occurs in adrenal chromaffin granules. The existence of a Ca2+-dependent conformational change in synaptotagmin was initially established by analysing the change in sensitivity of the protein to proteolytic digestion with trypsin in the presence and absence of Ca2+. An attempt was also made to investigate the Ca2+-binding activity of the protein with the lanthanide terbium (Tb3+), as a fluorescent probe for the Ca2+-binding sites but this approach proved to be of limited use, due to protein aggregation. Interaction of synaptotagmin with phospholipids was also found to be Ca2+-dependent and the protein was found to show selectivity for certain phospholipids. The interaction of the protein with calmodulin was tested using in vitro binding to immobilised calmodulin and also to calmodulin modified with a fluorescent dansyl group to act as a reporter. Both types of measurement indicated a Ca2+-dependent interaction between the proteins most probably due to affinity changes in calmodulin upon binding to Ca2+. Structural changes in synaptotagmin as a possible mechanism of action were investigated further using the biophysical techniques of circular dicheroism (CD) and light scattering. Changes in quaternary structural changes were not detectable by CD unless phospholipid vesicles were also mixed with the protein. Gel exclusion chromatography and native gel electrophoresis were used to support the data and indicated changes in quaternary structure. These techniques also suggested that the native protein, isolated from chromaffin granules, exists as a multimeric form. This study has demonstrated that synaptotagmin shows Ca2+-dependent interactions with calmodulin and phospholipids and also that it undergoes measurable structural changes in the presence and absence of Ca2+. This suggests that its action in exocytosis may be driven by a Ca2+-triggered conformational change.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.643179  DOI: Not available
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