Effects of Three Cardiomyopathic-Causing Mutations (D230N, D84N, and E62Q) on the Structure and Flexibility of α-Tropomyosin
Cardiac contraction at the level of the sarcomere is regulated by the thin filament (TF) composed of actin, alpha tropomyosin (TPM), and the troponin (Tn) complex (cTnT: cTnC: cTnI). The "gate-keeper" protein, α-TPM, is a highly conserved α-helical, coiled-coil dimer that spans actin and r...
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ndltd-arizona.edu-oai-arizona.openrepository.com-10150-6241012017-06-15T03:00:35Z Effects of Three Cardiomyopathic-Causing Mutations (D230N, D84N, and E62Q) on the Structure and Flexibility of α-Tropomyosin Holeman, Teryn A. Holeman, Teryn A. Tardiff, Jil C. Tardiff, Jil C. Konhilas, John P. Granzier, Henk L. Cardiomyopathy Differential Scanning Calorimetry (DSC) Flexibility Mutation Tropomyosin Cardiac contraction at the level of the sarcomere is regulated by the thin filament (TF) composed of actin, alpha tropomyosin (TPM), and the troponin (Tn) complex (cTnT: cTnC: cTnI). The "gate-keeper" protein, α-TPM, is a highly conserved α-helical, coiled-coil dimer that spans actin and regulates myosin-actin interactions. The N-terminus of one α-TPM dimer inter-digitates with the C-terminus of the adjacent dimer in a head-to-tail fashion forming the flexible and cooperative TPM-overlap that is necessary for myofilament activation. Two dilated cardiomyopathy (DCM) causing mutations in TPM (D84N and D230N) and one hypertrophic cardiomyopathy (HCM) causing mutation (E62Q), all identified in large, unrelated, multigenerational families, were utilized to study how primary alterations in protein structure cause functional deficits. We hypothesize that structural changes from a single point mutation propagate along the -helical coiled-coil of TPM, thus affecting its regulatory function. Structural effects of the mutations studied via differential scanning calorimetry (DSC) on TPM alone revealed significant changes in the thermal unfolding temperatures of both the C- and N-termini for all mutants compared to WT, indicating that mutational effects propagate to both ends of TPM, thus affecting the overlap region. Although, of note, the proximal termini to the mutation has shown more significant structural changes compared to WT. DSC analysis on fully reconstituted TF’s (Tn:TPM:Actin) revealed effects on the TPM-Actin cooperativity of activation, affecting interaction strength (thermal stability), and the rigidity of TPM moving along actin (FWHM). To characterize the resultant functional effect of these discrete changes in thermal stability and TPM rigidity, ATPase assays were used to measure actomyosin activation in the presence and absence of Ca2+. Together, these data will provide a molecular level understanding of the structural and functional deficits caused by these mutations to help elucidate the mechanisms leading to disease. 2017 text Electronic Thesis http://hdl.handle.net/10150/624101 http://arizona.openrepository.com/arizona/handle/10150/624101 en_US Copyright © is held by the author. Digital access to this material is made possible by the University Libraries, University of Arizona. Further transmission, reproduction or presentation (such as public display or performance) of protected items is prohibited except with permission of the author. The University of Arizona. |
collection |
NDLTD |
language |
en_US |
sources |
NDLTD |
topic |
Cardiomyopathy Differential Scanning Calorimetry (DSC) Flexibility Mutation Tropomyosin |
spellingShingle |
Cardiomyopathy Differential Scanning Calorimetry (DSC) Flexibility Mutation Tropomyosin Holeman, Teryn A. Holeman, Teryn A. Effects of Three Cardiomyopathic-Causing Mutations (D230N, D84N, and E62Q) on the Structure and Flexibility of α-Tropomyosin |
description |
Cardiac contraction at the level of the sarcomere is regulated by the thin filament (TF) composed of actin, alpha tropomyosin (TPM), and the troponin (Tn) complex (cTnT: cTnC: cTnI). The "gate-keeper" protein, α-TPM, is a highly conserved α-helical, coiled-coil dimer that spans actin and regulates myosin-actin interactions. The N-terminus of one α-TPM dimer inter-digitates with the C-terminus of the adjacent dimer in a head-to-tail fashion forming the flexible and cooperative TPM-overlap that is necessary for myofilament activation. Two dilated cardiomyopathy (DCM) causing mutations in TPM (D84N and D230N) and one hypertrophic cardiomyopathy (HCM) causing mutation (E62Q), all identified in large, unrelated, multigenerational families, were utilized to study how primary alterations in protein structure cause functional deficits. We hypothesize that structural changes from a single point mutation propagate along the -helical coiled-coil of TPM, thus affecting its regulatory function. Structural effects of the mutations studied via differential scanning calorimetry (DSC) on TPM alone revealed significant changes in the thermal unfolding temperatures of both the C- and N-termini for all mutants compared to WT, indicating that mutational effects propagate to both ends of TPM, thus affecting the overlap region. Although, of note, the proximal termini to the mutation has shown more significant structural changes compared to WT. DSC analysis on fully reconstituted TF’s (Tn:TPM:Actin) revealed effects on the TPM-Actin cooperativity of activation, affecting interaction strength (thermal stability), and the rigidity of TPM moving along actin (FWHM). To characterize the resultant functional effect of these discrete changes in thermal stability and TPM rigidity, ATPase assays were used to measure actomyosin activation in the presence and absence of Ca2+. Together, these data will provide a molecular level understanding of the structural and functional deficits caused by these mutations to help elucidate the mechanisms leading to disease. |
author2 |
Tardiff, Jil C. |
author_facet |
Tardiff, Jil C. Holeman, Teryn A. Holeman, Teryn A. |
author |
Holeman, Teryn A. Holeman, Teryn A. |
author_sort |
Holeman, Teryn A. |
title |
Effects of Three Cardiomyopathic-Causing Mutations (D230N, D84N, and E62Q) on the Structure and Flexibility of α-Tropomyosin |
title_short |
Effects of Three Cardiomyopathic-Causing Mutations (D230N, D84N, and E62Q) on the Structure and Flexibility of α-Tropomyosin |
title_full |
Effects of Three Cardiomyopathic-Causing Mutations (D230N, D84N, and E62Q) on the Structure and Flexibility of α-Tropomyosin |
title_fullStr |
Effects of Three Cardiomyopathic-Causing Mutations (D230N, D84N, and E62Q) on the Structure and Flexibility of α-Tropomyosin |
title_full_unstemmed |
Effects of Three Cardiomyopathic-Causing Mutations (D230N, D84N, and E62Q) on the Structure and Flexibility of α-Tropomyosin |
title_sort |
effects of three cardiomyopathic-causing mutations (d230n, d84n, and e62q) on the structure and flexibility of α-tropomyosin |
publisher |
The University of Arizona. |
publishDate |
2017 |
url |
http://hdl.handle.net/10150/624101 http://arizona.openrepository.com/arizona/handle/10150/624101 |
work_keys_str_mv |
AT holemanteryna effectsofthreecardiomyopathiccausingmutationsd230nd84nande62qonthestructureandflexibilityofatropomyosin AT holemanteryna effectsofthreecardiomyopathiccausingmutationsd230nd84nande62qonthestructureandflexibilityofatropomyosin |
_version_ |
1718458453304803328 |