Use this URL to cite or link to this record in EThOS: https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.754823
Title: The properties and function of tropomyosin dimers in muscle regulation
Author: Mackenzie, Cassidy
ISNI:       0000 0004 7427 8400
Awarding Body: University of Kent
Current Institution: University of Kent
Date of Award: 2017
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Abstract:
Myosin binding to actin, and thus muscle contraction, is regulated by Tropomyosin (Tpm), Troponin (Tn) and calcium (Ca2+). Tpm, is an α-helical coiled-coil dimer, which exists as a homo- or heterodimer. Two major isoforms of Tpm are found in striated muscle, α and β. Though it is known that different dimers exist, the mechanism by which they form and exchange is not fully understood. The thermal stability and exchange between dimers was explored with the use of circular dichroism and SDS PAGE densitometry analysis. Homodimers showed little exchange to form heterodimers at temperatures up to 20 °C . Dimer stability at these temperatures reduces the need for chemical cross-linking samples. While extensive exchange was seen at 37 °C . Reverse exchange of WT and mutant (E54K - dilated cardiomyopathy mutant) containing heterodimers to form homodimers did not show the same extent of exchange, suggesting a dimer preference. Results showed the ability to determine dimer content of a solution with the use of polyacrylamide gels and chemical cross-linking. The thermal melting curves of Tpm highlighted a significant destabilisation of β Tpm against the α isoform. Tpm heterodimer containing E54K mutant showed a decreased thermal stability. Notable differences were seen not only for isoforms and homo- and heterodimers, but also for buffer conditions and protein tags. Increased salt concentrations led to an increase in thermal stability. Crosslinking dimers increased thermal stability, whereas addition of His-tags led to a decrease in thermal stability. Changes in thermal stability highlighted the need for caution when tagging or cross-linking the protein. Dimer exchange on actin provided conflicting results between SDS PAGE cosedimentation assays and pyrene fluorescence cosedimendation assays, which highlighted limitations of cross-linking Tpm and using fluorescent labels. The stiffness of Tpm dimers was explored using atomic force microscopy (AFM) to image Tpm particles. Significant differences were seen between the relatively stiff α Tpm and less stiff β isoform. Changes in stiffness of Tpm affect its ability to cooperatively activate the thin filament, and provides insight into the assembly of dimers in vivo.
Supervisor: Geeves, Michael ; Xue, Wei-Feng Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.754823  DOI: Not available
Keywords: Q Science
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