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Mark Rustad, Graduate Student, Physics

Mark RustadCurrent Research

The sarco(endo)plasmic reticulum calcium ATPase (SERCA) transports calcium from the cytosol into the lumen of the sarcoplasmic reticulum after muscle contraction.  It is known that physiological problems arise in living systems if calcium concentrations in cells are improperly regulated; various types of heart disease are linked to improper SERCA activity.  A newly discovered peptide named dwarf open reading frame (DWORF) has been found to play a role in the regulation of calcium ion concentration in muscle cells in both vertebrates and invertebrates.  Specifically, DWORF enhances the activity levels of SERCA by interacting with SERCA-inhibitors: phospholamban (PLB), sarcolipin (SLN), and myoregulin (MLN) in vertebrates; and sarcolamban A and B (sclA and sclB) in invertebrates.  It is important to understand the mechanism of interaction between DWORF, SERCA, and inhibitors of SERCA due to the correlation of heart disease and insufficient DWORF production in people and animals.  Ischemic failing human hearts showed severe down-regulation of DWORF, and similarly, mice with hypertrophic heart disease exhibit down-regulation of DWORF as well.

I use electron paramagnetic resonance (EPR) spectroscopy to investigate the mechanism of interaction of DWORF with SERCA, and inhibitors of SERCA.  EPR is an experimental technique that utilizes the absorbance of magnetic fields by paramagnetic molecular probes attached to proteins to provide orientationally, structural and dynamical information of proteins and protein complexes.  Continuous wave electron paramagnetic resonance (CWEPR) can probe protein interactions that occur on time scales ranging from picoseconds to hundreds of nanoseconds (10-12 to 10-7 s) while saturation transfer electron paramagnetic resonance (STEPR) can investigate protein dynamics on slower time scales on the order of milliseconds (10-3 s). 


Past Research Projects

Noireaux Lab, University of Minnesota, Department of Physics (2013-2016):

I optimized and expanded the scope of synthetic biological systems that can be executed by the cell-free transcription and translation platform developed at the Noireaux lab.  Cell-free transcription and translation (TX-TL) platforms are invaluable in the fields of synthetic biology and bioengineering because of their versatility with respect to experiment design and capability.  During my time at the Noireaux lab, I helped improve the protein yield of the cell-free TX-TL platform to over 2 mg/mL, making this cell-free system the most powerful of its kind.

I utilized this system to study and reconstitute entire biological systems, such as bacteriophages.  I investigated the mechanisms of self-assembly of bacteriophages and the sensitivity of these phage systems to environmental conditions (e.g. molecular crowding effects on phage synthesis).


Ghosh Group, University of Denver, Department of Physics & Astronomy (2009-2011):

Folding speed of proteins based on native topology

From first principles, I helped develop a model describing the dependence of protein folding speed with the native topology of a protein.  I improved the correlation of folding speed with absolute contact order (characterized by a protein’s topology) of three databases of protein: i) two-state proteins without helical proteins, ii) all two-state proteins, and iii) multi-state proteins.  This model gave the first direct proof of a well-known empirical expression relating protein folding speed to native topology.  Moreover, the developed model estimated a folding speed limit that is close to the well-established observed value of 1 μs.  Finally, our novel topological model correlates with protein chain length over a large set of monomeric proteins from the Protein Data Bank (PDB).


Intrinsically Disordered Proteins in Organisms

I examined the prevalence and purpose of intrinsically disordered proteins (IDPs) in organisms across the three domains of life: Eukaryotes, Bacteria and Archaea.   The central dogma of biology describes how a protein’s amino acid sequence encodes a specific, three-dimensional structure, and that structure was a necessary condition for protein functionality.  Contrary to this paradigm are the existence of proteins that do not fold into a specific 3D structure but still possess biological relevance.  These are the proteins that are classified as intrinsically disordered.

IDPs characteristically possess high net charge (at pH 7) and low average hydrophobicity of amino acids.  From this information, a protein can be classified (with high accuracy) as intrinsically disordered or ordered based upon amino acid sequence alone.  I applied this sequence based prediction method to a set of 96 organisms and analyzed what fraction of an organism’s proteome are intrinsically disordered.  I found that thermophilic organisms (those that thrive in high temperature environments) have a relatively low content of IDPs in their proteome while halophilic organisms contain a large fraction of IDPs in their proteome.



Rustad, M., & Ghosh, K. (2012). Why and how does native topology dictate the folding speed of a protein? Journal of Chemical Physics, 137(20).

Garamella, J., Marshall, R., Rustad, M., & Noireaux, V. (2016). The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology. ACS Synthetic Biology, 5(4), 344–355.

Rustad, M., Eastlund, A., Marshall, R., Jardine, P., & Noireaux, V. (In-Press). Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System. J. Vis. Exp.