The structural basis of mechanosensitivity in cardiac proteins

Thesis

Mechanosensitive proteins represent a regulatory mechanism for mechanical stress within cells. Their ability to convert mechanical force into a biochemical response en- ables the cell to actively respond to its internal and external mechanical environment. Despite most proteins being exposed to forces in one form or another, the mechanism of mechanosensitivity remains largely undefined structurally. Single molecule force spectroscopy techniques provide accurate correlations between mechanical force input and the resultant extension of a given protein. However, current methods are unable to directly associate these global changes in conformation to rearrangements of molecular interactions. In this thesis I apply a variety of mass spectrometry- based approaches to capture structural signatures of mechanosensitivity in two cardiac proteins, filamin C and talin. For filamin C I use HDXMS along with collision-induced unfolding ion mobility (CIU-IMS) and native mass spectrometry to identify key dynamic structures in the domain 20 insert and the donated β- strand, that represent initial steps in the mechano-unfolding pathway. In a disease- related variant of filamin C, I describe the pathogenic progression of hypertrophic cardiomyopathy from clinical discovery of the mutation (W2164C) to a concerted molecular interaction switch. This results in significant conformational changes surrounding domain 20 which indicates an altered unfolding pathway. I establish a similar study of a second cardiac mechanosensitive protein: talin. In this case I investigate the mechanically vulnerable R3 domain. Again using our repetoire of mass spectrometry techniques, I am able to characterise the structures and dynamics of R3’s 4-helix bundle. I identify a hydrophobic core which can be further stabilised by introducing more hydrophobic residues. Furthermore, I distinguish a gradient of thermodynamic stability between the 4 helices which I correlate to a proposed unfolding pathway. Finally, I move from the zero-force regime to probe the force perturbed conformation of talin’s R3 domain. I do so through an ongoing development of a hybrid methodology that encompasses contraction-expansion microfluidics and HDXMS. The workflow uncovers that the hydrophobic core of the protein is exposed under force unfolding, further evidencing our proposed unfolding pathway made from the zero-force data. Together these findings further our understanding of the structures and dynamics of mechanosensitive proteins and how they functionally unfold under physiological forces.

Authors: Holden, E

• DOI: 10.5287/ora-wrmovqzxk
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: mechanosensitive; mass spectrometry; hydrogen deuterium exchange; cardiomyopathy
• Subjects: Biophysics; Biochemistry