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microscopy, phospholamban, SERCA, sarcolipin, electron, crystal, oligomer

Glaves, John Paul J

Supervisor and department: Young, Howard S Biochemistry

Examining committee member and department: Glover, JN Mark Biochemistry Marassi, Francesca Sanford Burnham Medical Research Institute Young, James Physiology Fliegel, Larry Biochemistry

Department: Department of Biochemistry

Specialization:

Date accepted: 2011-09-29T03:29:19Z

Graduation date: 2011-11

Degree: Doctor of Philosophy

Degree level: Doctoral

Abstract: In 2007, heart disease was the second leading cause of death in Canadians. In heart muscle cells, calcium Ca2+ is released from the sarcoplasmic reticulum SR during contraction and must be replenished for relaxation to occur. Three proteins restore Ca2+ to the SR following contraction: the sarcoendoplasmic reticulum ATPase SERCA and its regulatory binding partners, phospholamban PLB and sarcolipin SLN. The importance and sensitivity of SERCA regulation has been highlighted by identified human mutations in PLB and SLN that lead to heart disease and heart failure. This thesis aims to provide upstream biochemical insights into regulatory complexes formed between SERCA and PLB and between SERCA and SLN for the development of heart disease therapies. The structural information suggests novel interactions between SERCA and its regulatory binding partners.We studied SERCA-PLB and SERCA-SLN complexes using electron microscopy EM of two-dimensional co-crystals. Cryo-EM of SERCA and PLB co-crystals revealed a novel mode of binding between the PLB pentamer and SERCA. The binding site involved the third transmembrane helix of SERCA and represented a site distinct from the SERCA binding site for monomeric PLB. Cryo-EM of SERCA and SLN co-crystals suggested that SLN can also bind the third transmembrane helix of SERCA. The functional mutation of PLB affected co-crystal frequency and the structure of PLB in the projection maps. The co-crystal frequency was directly related to the functional state of PLB. The structure of PLB was also influenced by phosphorylation, but the interaction was maintained between the phosphorylated PLB pentamer and SERCA. Combined, the results support an active, functional interaction between pentamers of PLB and SERCA. The function of the pentameric interaction is proposed to deliver monomeric PLB to its inhibitory interaction with SERCA at a different binding site.

Language: English

DOI: doi:10.7939-R3D700

Rights: Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only. Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the author's prior written permission.





Author: Glaves, John Paul J

Source: https://era.library.ualberta.ca/


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University of Alberta Cryo-electron microscopy of SERCA interacting with oligomeric phospholamban and oligomeric sarcolipin by John Paul Glaves A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Doctor of Philosophy Biochemistry ©John Paul Glaves Fall 2011 Edmonton, Alberta Permission is hereby granted to the University of Alberta Libraries to reproduce single copies of this thesis and to lend or sell such copies for private, scholarly or scientific research purposes only.
Where the thesis is converted to, or otherwise made available in digital form, the University of Alberta will advise potential users of the thesis of these terms. The author reserves all other publication and other rights in association with the copyright in the thesis and, except as herein before provided, neither the thesis nor any substantial portion thereof may be printed or otherwise reproduced in any material form whatsoever without the authors prior written permission. Dedicated to my grandfather Joseph Glaves, your heart was stronger than it let on. Abstract In 2007, heart disease was the second leading cause of death in Canadians.
In heart muscle cells, calcium (Ca2 ) is released from the sarcoplasmic reticulum (SR) during contraction and must be replenished for relaxation to occur.
Three proteins restore Ca 2 to the SR following contraction: the sarco(endo)plasmic reticulum ATPase (SERCA) and its regulatory binding partners, phospholamban (PLB) and sarcolipin (SLN). The importance and sensitivity of SERCA regulation has been highlighted by identified human mutations in PLB and SLN that lead to heart disease and heart failure.
This thesis aims to provide upstream biochemical insights into regulatory complexes formed between SERCA and PLB and between SERCA and SLN for the development of heart disease therapies.
The structural information suggests novel interactions between SERCA and its regulatory...





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