Dr. Robert Harkness

As an undergraduate student I became interested in how biological molecules can change their shape to perform different functions in the cell. This is now widely recognized as an important property that regulates many aspects of human heath, and when these dynamics are perturbed serious diseases can occur. I wanted to learn how to study the structural dynamics of biomolecules and this led me toward graduate and post-doctoral studies using techniques that can capture these motions and how they regulate activity with a high level of detail. It was fascinating to me that even very well studied molecules like the DNA double helix were continually being found to shift into different conformations that influence interactions with polymerases and transcription factors.

In my lab, we study what biological molecules do and how they do it, with the ultimate aim of better understanding their function in human health and diseases contexts. Broadly, our areas of interest are:

1. The formation and activation of protein molecular "machines" that perform reactions as required by the cell, notably proteases and nucleases that recycle proteins and nucleic acids respectively
2. The structural dynamics of nucleic acids, their protein partners, and how these relate to gene expression

We use suite of biophysical tools to understand how biomolecules work with a high level of detail. These include isothermal titration calorimetry (ITC) and fluorescence for ligand binding and activity assays, confromational dynamics measurements via nuclear magnetic resonance (NMR) spectroscopy, hydrodynamics methodologies for characterizing molecular assembly, and computer modeling of the resultant data with mechanisms that explain biomolecular activity.

By working in the group, trainees will establish a well-rounded skillset featuring experimental and computational methods for biophysical research. This will facilitate employment in industry, academic, and government positions. Most importantly, students will develop a framework for thinking about and quantitatively modeling the behavior of biomolecules and the world around us.

Post-doctoral fellowship, the University of Toronto and the Hospital for Sick Children Research Institute, Supervisor: Dr. Lewis Kay

PhD Chemistry, McGill University, Supervisor: Dr. Anthony Mittermaier

BSc Biochemistry Honours, Queen’s University

Proteases in cellular homeostasis

The recycling of proteins within the cell is critical to health and development. To achieve this, cells deploy a host of oligomeric protease “machines”. These proteases capture and digest substrate proteins that are no longer required or are misfolded and pose a hazard if they aggregate. Their assembly into oligomers enables allosteric communication between subunits and multiple interfaces for effector protein binding, which provides layers of regulation in substrate consumption. Together, the self-assembly and activation mechanisms of these complexes enables stringent control of proteolysis reactions. The dysregulation of these molecular machines has been linked to the development of cancers and neurodegenerative disorders and, therefore, it is critically important to understand how they work as a first step in developing treatments for illnesses. We study how these oligomers form, how they interact with substrate molecules in their assembled state, and how their activity is modulated by disease mutations and post-translational modifications, toward illuminating the molecular basis for their function.

Nucleases in the RNA life cycle

We are interested in how the mRNA life cycle is regulated. The mRNA within eukaryotic cells is appended with a 3ʹ polyadenosine (polyA) tail that provides protection from non-specific degradation and promotes translation. When an mRNA is no longer required, the polyA tail must then be removed in a process termed deadenylation, by enzymes known as deadenylases, so that the mRNA can be recycled. Deadenylation is the rate-limiting step in mRNA decay and consequently is a key determinant of the mRNA life cycle which, in turn, controls many other events in the cell. With its central role in cellular homeostasis, deadenylation must be tightly controlled to ensure mRNAs are appropriately modified. It has been suggested that the structural dynamics of deadenylases and substrate RNAs are critical to regulating deadenylation, though detailed insights into these conformational excursions and the role that they play in activity are lacking. We study the dynamics of these systems toward a better understanding of the mRNA homeostasis network.

Structural dynamics of nucleic acids

For many years, it was thought that DNA and RNA were simply static carriers of genetic information. We now understand that alternate base pairing modes in equilibrium with the traditional Watson-Crick form regulate protein partner binding to DNA and RNA, and that the folding of “non-canonical” nucleic acid structures such as G-quadruplexes, can play important roles in (mis)function. Moreover, epigenetic marks in the form of methylation, and damage such as oxidative lesions, can lead to the formation of alternate conformations that influence gene expression and the damage repair response, respectively. We study the structural landscapes of nucleic acids in healthy and disease backgrounds, and how these shift in response to the binding and action of enzymes or other protein partners, leading to (dys)functional outputs.

Proteases in the maintenance of cellular homeostasis

1. Harkness, R. W. et al. Exploring host-guest interactions in a 600 kDa DegP protease cage complex by hydrodynamics measurements and methyl-TROSY NMR. 2024, Journal of the American Chemical Society.

2. Harkness, R. W.; Ripstein, Z. A.; Di Trani, J. M; Kay, L. E. Flexible client-dependent cages in the assembly landscape of the periplasmic protease-chaperone DegP. 2023, Journal of the American Chemical Society.

3. Harkness, R. W. et al. Competing stress-dependent oligomerization pathways regulate self-assembly of the periplasmic protease-chaperone DegP. 2021, Proceedings of the National Academy of Sciences, USA.

Nucleases and nucleic acid structures in the regulation of gene expression

4. Irwin, R.; Harkness, R. W.; Forman-Kay, J. D. 2023, Methods in Molecular Biology, Deadenylation.

5. Harkness, R. W. et al. Parallel reaction pathways accelerate folding of a guanine quadruplex. 2021, Nucleic Acids Research.

6. Harkness, R. W.; Avakyan, N.; Sleiman, H. F.; Mittermaier, A. K. Mapping the energy landscapes of supramolecular assembly by thermal hysteresis. 2018, Nature Communications.