Actin’s C-terminus contributes to interactions with actin-binding proteins (ABPs) in cellular processes. Historical resolution constraints have prevented a complete understanding of how actin’s C-terminus mediates actin’s interactions, but evidence suggests that it undergoes conformational shifts upon ABP binding. Actin’s C-terminus also shifts in response to actin’s nucleotide state, guiding nucleotide-dependent ABP binding. Previously, we proposed that C-terminal shifts regulate actin’s functions through allosteric communication networks. To further examine the role of C-terminal shifts in actin communication networks and understand how C-terminal modifications influence actin’s functional state, we utilized in silico modeling of actin filaments crosslinked by N,N′-para-phenylenebismaleimide (PBM). Our modelling examined how constraining C-terminal flexibility with PBM crosslinks affects the conformation of distant structural elements and shifts internal communication networks critical for actin’s functions. We present evidence that disruption of the stabilizing F375–R116 interaction by PBM-crosslinks reshapes allosteric communication networks through the protomer, altering nucleotide cleft architecture and nucleotide dynamics. We therefore propose that actin’s C-terminus acts as a nexus for actin structural changes, shifting in response to stimuli, and propagating changes that allow actin to adopt different functional conformations that influence ABP binding.

Two common types of cardiovascular disease are hypertrophic cardiomyopathy (HCM) and dilated cardiomyopathy (DCM) which occur from changes to sarcomere contractile mechanisms and activity. Actin amino acid substitutions R312C and R312H have been found in HCM and DCM patients, respectively. Previously, we observed that R312C/H variants display both hyperactivity and hypoactivity in vitro, contradicting traditional characterizations of HCM- and DCM-causing variants. Here, we further characterized R312C/H actin variants in vitro and conducted in silico modelling to better understand the mechanisms differentiating HCM and DCM. Our results suggest that R312C/H substitutions cause structural changes that differentially impact actomyosin activity. A gradient of altered interactions with regulatory proteins troponin, tropomyosin, and the C0C2 domains of myosin-binding protein C was also observed, influencing the accessibility of active and inhibitory conformations of these proteins. The results presented here support our previous suggestion of a gradient of factors that differentiate between HCM and DCM. Further characterization of HCM- and DCM-causing actin variants using in vitro and in silico methods is required for better understanding cardiomyopathy and improving clinical outcomes.

Actin is essential to eukaryotic cellular processes. Actin's C-terminus appears to play a direct role in modulating actin's structure and properties, facilitating the binding and function of actin-binding proteins (ABPs). The structural and functional characterization of filamentous actin's C-terminus has been impeded by its inherent flexibility, as well as actin's resistance to crystallization for x-ray diffraction and the historical resolution constraints associated with electron microscopy. Many biochemical studies have established that actin's C-terminus must retain its flexibility and structural integrity to modulate actin's structure and functions. For example, C-terminal structural changes are known to affect nucleotide binding and exchange, as well as propagate actin structural changes throughout extensive allosteric networks, facilitating the binding and function of ABPs. Advances in electron microscopy have resulted in high-resolution structures of filamentous actin, providing insights into subtle structural changes that are mediated by actin's C-terminus. Here, we review existing knowledge establishing the importance of actin's C-terminus within actin structural changes and functions and discuss how modern structural characterization techniques provide the tools to understand the role of actin's C-terminus in cellular processes.

Many therapeutics for cardiomyopathy treat the symptoms of the disease rather than the underlying mechanism. The mechanism of cardiomyopathy onset is believed to include two means: calcium sensitivity changes and myosin activity alteration. Trifluoperazine is a compound that binds troponin, and other components of the calcium pathway, which impacts calcium regulation of contraction. Here, the ability of TFP to shift calcium sensitivity was examined in vitro with purified proteins and the impact of TFP on heart function was assessed in vivo using embryonic zebrafish. The binding of TFP to troponin was modeled in silico and a model of zebrafish troponin was generated. TFP increased regulated cardiac actomyosin activity in vitro and elevated embryonic zebrafish heart rates at effective drug concentrations. Troponin structural changes predicted in silico suggest altered protein interactions within thin filaments that would affect the regulation of heart function.

Actin plays an essential role in fundamental life processes, interacting with more proteins than any other protein. Actin’s structure is dynamic, undergoing structural changes that facilitate interactions with actin binding proteins (ABPs) and their subsequent functions. Many of actin’s structural changes and actin:ABP interactions appear to involve actin’s C-terminus. Due to its inherent flexibility, actin’s C-terminus had not been resolved at high-resolution in actin structures until recently. The lack of structural resolution limited actin C-terminal research over the last 20 years, leaving the details of how actin’s C-terminus modulates actin structure and functions a mystery. In this thesis, I aimed to present evidence of a dynamic allosteric network within actin that are modulated by actin’s C-terminus through its inherent flexibility. In vitro motility assays suggest that removing the inherent flexibility of actin’s C-terminus inhibits the generation of cellular forces by myosin, though the specific mechanisms of inhibition remain unclear. To explore the structural impacts of crosslinking actin’s C-terminus, I developed and employed in silico methods utilizing molecular dynamics (MD) simulations of crosslinked actin and disease-causing actin variants, as such methods have been shown to supplement in vitro and in vivo studies. In silico results presented in this thesis support the suggestion that actin’s C-terminus modulates actin structural properties and changes through dynamic allosteric networks. The disruption of C-terminal allostery has negative functional effects, impacting actin’s nucleotide binding and hydrolysis as well as interactions with binding partners such as myosin. It is therefore recommended that actin’s C-terminus becomes a focus of actin research to better understand actin’s roles in essential cellular functions for life on Earth.

ICAA20, Berlin (2026)

Aluminum alloys play an integral role in modern society, with aluminum alloys allowing the tailoring of specific properties for engineering applications. Cerium is a lanthanide found in great abundance and possessing intriguing chemical properties for a range of applications, including catalysis, earning a designation as a critical raw material by many world governments. The addition of rare earth elements (REEs) such as Ce can impact the microstructure formation and refinement, improve corrosive behaviour, and improve mechanical properties. Currently, however, Ce is viewed as a by-product of extraction processes for other critical minerals, with few large-scale applications. The identification of Ce alloying applications within Al would therefore have tremendous economic and societal implications. To understand the thermodynamics driving formation of beneficial AlCe intermetallic phases, this work sought to understand the diffusion kinetics of Ce within AlCe solid solution. In this work, AlCe solid solutions were modelled using density functional theory (DFT), identifying ground state atomic positions and energies following the addition of single and double Ce or vacancy defects. Climbing image nudged elastic band (CI-NEB) calculations identified energy barriers associated with vacancy jumps within the Al matrix according to the five-frequency model, in the presence and absence of Ce. Utilizing DFT energetics, diffusion kinetics were modelled through KineCluE, extracting activation energies, prefactors, and diffusion behaviours for Ce atoms within the Al matrix.

Muscle Health Awareness Day, York University (2024)

Actin plays a central role in cellular processes through interactions with actin binding proteins (ABPs). In the sarcomere, actin’s interactions with myosin, tropomyosin, and troponin regulate contraction, with amino acid substitutions disrupting these interactions and resulting in diseases like hypertrophic (HCM) and dilated cardiomyopathy (DCM). Actin amino acid substitutions R312C/H have been found in patients with HCM and DCM, respectively. Previously, we characterized the R312C/H variants biochemically, observing altered interactions with myosin, troponin, and tropomyosin. R312H actin exhibited increased actomyosin activity under low calcium conditions, and both variants displayed decreased calcium sensitivity in vitro, contrary to the prevailing hypothesis that HCM and DCM are linked to increased and decreased calcium sensitivity, respectively. It was hypothesized that factors other than changes in calcium sensitivity differentiate between the onset of HCM and DCM. Understanding how actin variants result in different forms of cardiomyopathy is critical for guiding the development and application of treatments for heart disease. Here, in silico modeling revealed structural mechanisms behind altered actin:ABP interactions in vitro. Notable changes to structural properties and conformations occurred in the DnaseI binding loop (D-loop) and 222-230 helix in both variants. While both variants displayed decreased flexibility in D-loop residues, simulations predicted that: the D-loop of R312C moves closer to the binding sites of myosin and troponin which stabilizes those interactions, increasing actomyosin force output; the D-loop of R312H moves away from myosin and troponin binding sites, destabilizing those interactions which decreases actomyosin force output and alters troponin’s regulation of myosin binding under low calcium conditions. Both variants exhibited large changes in the position of the 222-230 helix that would interfere with tropomyosin’s ability to access the open state, decreasing activity under high calcium conditions, in line with decreased calcium sensitivity in vitro. Predictions from molecular dynamics simulations therefore support the hypothesis that factors such as actomyosin force output and the stability of interactions with regulatory proteins, rather than changes to calcium sensitivity, drive the differentiation between HCM and DCM and must be considered in the development of clinical treatments for heart disease.

Tri-University Protein Symposium, Wilfrid Laurier University (2024)

Actin facilitates essential cellular processes through interactions with actin binding proteins (ABPs). In the sarcomere, actin and myosin generate contractile forces, a process regulated in a calcium dependent manner by ABPs troponin (Tn), tropomyosin (Tm), and myosin binding protein C (cMyBP-C). The disruption of actin’s interactions by amino acid substitutions leads to diseases such as hypertrophic (HCM) and dilated (DCM) cardiomyopathy, characterized by hyper and hypoactivity of the sarcomere, respectively. The prevailing hypothesis surrounding HCM and DCM-causing actin variants is that differences in calcium sensitivity differentiate between HCM and DCM, with increased calcium sensitivity leading to sarcomere hyperactivity/HCM and the opposite resulting in DCM. The calcium sensitivity hypothesis has guided cardiomyopathy treatments, though recent characterization of actin variants suggests other factors are involved. Our previous in vitro characterization of R312C (HCM) and R312H (DCM) actin variants found that both variants displayed decreased calcium sensitivity, typically associated with DCM, as well as hyperactivity under low calcium conditions, suggestive of HCM. The onset of opposing diseases due to amino acid substitutions at the same residue that present similar biochemical profiles provides an opportunity to explore the structural mechanisms differentiating the onset of HCM vs DCM. In silico modelling reveals that actin’s DNaseI binding loop adopts distinct conformations in the R312C/H variants that can destabilize interactions with Tn, exposing myosin binding sites under low calcium conditions. Stronger interactions are formed with myosin in R312C, increasing force output, with less favourable actomyosin interactions in R312H decreasing force output. Further, the favourability of actin:TM and actin:cMyBP-C interactions is altered, resulting in variable regulation of actomyosin activity. Our results support the suggestion that factors other than calcium sensitivity, namely actomyosin force output and altered regulation of actomyosin interactions under low calcium conditions, differentiate between HCM and DCM.

Gordon Research Conference-Cytoskeletal Motors, Mount Snow Vermont (2022)

Actin, in conjunction with a wide range of binding partners, is essential for life on Earth, with mis-regulation of actin resulting in many diseases and disorders. Understanding how actin contributes to these interactions is essential for developing novel treatments and therapeutics, but complete characterization of actin’s structure has been difficult. In recent years, technological advancements in cryo-EM and computational methods have presented the opportunity to accurately model actin structural elements whose functions remain unknown. Actin’s highly flexible C-terminus appears to be a critical component of actin’s structure and functions by stabilizing actin-actin interactions during polymerization and facilitating the binding of actin binding proteins (ABPs). Past studies suggest that actin’s C-terminus might change conformation upon ABP binding, and that its structural integrity is essential for the binding and function of ABPs. Further, the C-terminus is connected allosterically to distant regions of actin’s structure, potentially propagating long-range conformational changes within actin that facilitate the functions and cooperative binding of ABPs. My research aims to identify how C-terminal flexibility contributes to actin structural changes and interactions with ABPs for life-sustaining processes. To examine the functional role of C-terminal flexibility, I have developed a novel process for the purification of actin oligomers whose C-termini have been constrained by chemical crosslinks. The impact of constraining C-terminal flexibility in actin:ABP interactions has been observed through in vitro motility assays, where the generation of cellular forces by myosin is reduced relative to unconstrained controls. The structural mechanisms behind these impacts are being modelled through computational methods, including energy minimization and molecular dynamics, which seek to identify how chemical crosslinks change C-terminal conformation, restrict its movement, and alter its ability to propagate structural changes through allosteric networks. This work will provide a fundamental understanding of how actin structural changes propagate and facilitate life-sustaining functions.

Graduate Student Symposium, University of Guelph (2022)

Actin is among the most abundant proteins on Earth, with its dynamic structure and diverse interactions with actin binding proteins (ABPs) facilitating processes that are essential for life. Mis-regulation of actin has been implicated in a wide range of diseases, including cancer and heart disease, and so the complete characterization of actin’s structure and actin-ABP interactions is essential for the development of targeted treatments and therapeutics. Despite actin’s importance, a lack of structural information has hindered attempts to characterize various structural elements, in particular actin’s dynamic and highly flexible C-terminus. Past research suggests that actin’s C-terminus plays an active role in actin-ABP interactions, for example in the generation of cellular forces with myosin during the actomyosin force generation cycle. Fluorescently labelled actin demonstrates that actin’s C-terminus undergoes conformational changes upon myosin binding, while proteolytic cleavage of actin’s C-terminus reduces myosin’s force generating ATPase activity. In addition, chemical crosslinking of actin’s C-terminus has been theorized to reduce myosin force generation proportional to the number of crosslinks present. To characterize the role of actin’s C-terminus in actin-ABP interactions, I have developed and optimized a process for the purification of crosslinked actin oligomers. Utilizing in-vitro motility (IVM) and ATPase assays, I am examining the functional impact of constraining actin’s C-terminus on actomyosin force generation. With the use of direct electron detectors and cryo-electron microscopy (cryo-EM), I will develop structural models to understand how the C-terminus facilitates actomyosin activity. Further, I am developing computational methods including energy minimization and molecular dynamics simulations to model the role of actin’s C-terminus in actomyosin interactions, as well the propagation of conformational changes within actin filaments. Characterizing how actin’s C-terminus contributes to actin-ABP interactions and actin structural changes will provide a better understanding of how actin’s dynamic structure contributes to life sustaining processes.

Graduate Student Symposium (2020)

Movement is essential for life. While an individual might move to find food or breathe, movement at the cellular level facilitates this larger scale motility. Dynamic protein-protein interactions between actin and myosin generate the forces needed for these cellular and larger scale movements. Despite the abundance and prevalence of actin, the details of its highly flexible C-terminus remain a mystery due to a lack of structural information, but there are hints of a dynamic role within actomyosin force generation. Proteolytic cleavage of actin’s two C-terminal residues reduces myosin’s force generating ATPase activity, while fluorescence studies indicate conformational changes at the C-terminus upon myosin binding. Furthermore, constraining actin’s C-terminus with chemical crosslinking restricts myosin force generation proportional to the number of crosslinks formed. To determine the role of actin’s C-terminus in actomyosin force generation, I am developing an optimized purification of crosslinked actin oligomers to produce actin filaments composed exclusively of crosslinked dimers (F-dimer) or trimers (F-trimer). The biochemical properties of these filaments are being studied through myosin ATPase and in vitro motility (IVM) assays. In addition, cryo-EM with direct electron detectors will be used to develop high resolution structures of crosslinked actomyosin, revealing the conformation of and interactions with actin’s C-terminus. Characterizing the biochemical function and dynamic structure of actin’s C-terminus will provide a deeper understanding of how actin interacts with myosin to generate forces for movement in essential life processes.

University of Guelph (2022/2023)

In 2022 and 2023, I hosted seminars to teach the basics of computational biochemistry I introduced common UNIX commands, the use of high-performance computing clusters through the Digital Research Alliance of Canada, and the use of GROMACS for MD simulations. This seminar tought the theory and individual steps behind preparing biologically relevant structures, generating solvated boxes in GROMACS, periodic boundary conditions, energy minimization, equilibration, production run MD simulations, and common analyses.

University of Guelph (2022)

As an executive member of the Centre for Cardiovascular Investigations (CCVI) at the University of Guelph, I organized and hosted a one day symposium for guest talks of featured researchers, including Dr. Anthony Cammarato from Johns Hopkins University School of Medicine, and moderated Q&As between attending students and guest speakers.