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Molecular architecture of Caveolin-3 and the investigation of an interaction with the Ryanodine receptor

Whiteley, Gareth

[Thesis]. Manchester, UK: The University of Manchester; 2012.

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Abstract

The muscle-specific membrane protein, Caveolin-3, is a building block of caveolae a type of specialised lipid raft. Caveolin-3 is proposed to play a central role in variety of cellular functions both structural and functional, from cell signalling to cholesterol homeostasis. Caveolin-3 has also been implicated in processes involved in targeting membrane proteins to the plasma membrane, as well as mediating a host of cell signalling processes. Initial attempts were made to express full-length Caveolin-3 in E.coli. However, more success was achieved in expressing and purifying domains of Caveolin-3. To produce purified full-length Caveolin-3 the baculovirus expression system was employed and we report here that the expression of Caveolin-3 in insect (Sf9) cells leads to the formation of caveolae comparable in size to those observed in native vesicles. We subsequently purified the recombinant Caveolin-3 and determined, using multi-angle laser light scattering, that the isolated protein forms an oligomer with a molecular mass of ~200-220kDa. Using negative-stain transmission electron microscopy in conjunction with single particle analysis we have determined the first three-dimensional structure for Caveolin-3 with data converging to suggest that it forms a nonamer. The 9-fold symmetric three-dimensional Caveolin-3 volume is toroidal, ~16.5nm in diameter and 5.5nm thick, and is characterised by an outer rim of protein connected to a central ‘cone-shaped’ domain. Labelling studies revealed that the C-terminal domain of each of the contributing Caveolin-3 monomers associate to form the central cone density. There is also evidence to suggest that Caveolin-3 is associated with a range of proteins involved in excitation-contraction coupling. Having identified multiple potential caveolin-binding motifs within the Ryanodine Receptor, one of the key protein components of excitation-contraction coupling, we have purified the skeletal isoform of the Ryanodine Receptor (Ryanodine Receptor-1) from sheep calf muscle and using several biophysical techniques probed whether there is an interaction between Caveolin-3 and Ryanodine Receptor-1. Co-immunoprecipitation experiments indicated that the two proteins do indeed interact, but functional studies for analysis of binding characteristics were inconclusive. In conclusion, this thesis describes both the successfully purification and structural determination of Caveolin-3, generating the first 3D data for any of the caveolin proteins, as well as work aimed at understanding its functional relationship with Ryanodine Receptor-1.

Layman's Abstract

The shape of a protein found in the heart

Bibliographic metadata

Type of resource:
Content type:
Administered thesis
Form of thesis:
Traditional
Type of submission:
Doctoral level ETD - final
Thesis title:
Molecular architecture of Caveolin-3 and the investigation of an interaction with the Ryanodine receptor
Degree type:
Doctor of Philosophy
Degree programme:
PhD Medicine (Cardiovascular Sciences)
Publication date:
2012-10-09T11:45:08
Institution:
The University of Manchester
Location:
Manchester, UK
Total pages:
291
Abstract:
The muscle-specific membrane protein, Caveolin-3, is a building block of caveolae a type of specialised lipid raft. Caveolin-3 is proposed to play a central role in variety of cellular functions both structural and functional, from cell signalling to cholesterol homeostasis. Caveolin-3 has also been implicated in processes involved in targeting membrane proteins to the plasma membrane, as well as mediating a host of cell signalling processes. Initial attempts were made to express full-length Caveolin-3 in E.coli. However, more success was achieved in expressing and purifying domains of Caveolin-3. To produce purified full-length Caveolin-3 the baculovirus expression system was employed and we report here that the expression of Caveolin-3 in insect (Sf9) cells leads to the formation of caveolae comparable in size to those observed in native vesicles. We subsequently purified the recombinant Caveolin-3 and determined, using multi-angle laser light scattering, that the isolated protein forms an oligomer with a molecular mass of ~200-220kDa. Using negative-stain transmission electron microscopy in conjunction with single particle analysis we have determined the first three-dimensional structure for Caveolin-3 with data converging to suggest that it forms a nonamer. The 9-fold symmetric three-dimensional Caveolin-3 volume is toroidal, ~16.5nm in diameter and 5.5nm thick, and is characterised by an outer rim of protein connected to a central ‘cone-shaped’ domain. Labelling studies revealed that the C-terminal domain of each of the contributing Caveolin-3 monomers associate to form the central cone density. There is also evidence to suggest that Caveolin-3 is associated with a range of proteins involved in excitation-contraction coupling. Having identified multiple potential caveolin-binding motifs within the Ryanodine Receptor, one of the key protein components of excitation-contraction coupling, we have purified the skeletal isoform of the Ryanodine Receptor (Ryanodine Receptor-1) from sheep calf muscle and using several biophysical techniques probed whether there is an interaction between Caveolin-3 and Ryanodine Receptor-1. Co-immunoprecipitation experiments indicated that the two proteins do indeed interact, but functional studies for analysis of binding characteristics were inconclusive. In conclusion, this thesis describes both the successfully purification and structural determination of Caveolin-3, generating the first 3D data for any of the caveolin proteins, as well as work aimed at understanding its functional relationship with Ryanodine Receptor-1.
Layman's abstract:
The shape of a protein found in the heart
Keyword(s):
Thesis main supervisor(s):
Thesis advisor(s):
Funder(s):
Degree grantor:
Language:
en

Institutional metadata

University researcher(s):
Academic department(s):

Record metadata

Manchester eScholar ID:
uk-ac-man-scw:175631
Created by:
Whiteley, Gareth
Created:
9th October, 2012, 10:45:09
Last modified by:
Whiteley, Gareth
Last modified:
6th April, 2017, 08:06:06

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