Several recent reports have implicated somatic mosaic variants as phenocopies of germline defects of inborn errors of immunity (IEI). Somatic variants can also result from clonal hematopoiesis (CH), which has been associated with increased risk of hematological malignancies and cardiovascular disorders. The extent of clinical relevance of somatic variants remain uncertain.

Insufficient sarco/endoplasmic reticulum calcium ATPase (SERCA) activity significantly contributes to heart failure, which is a leading cause of death worldwide. A characteristic pathology of cardiac disease is the slow and incomplete Ca2+ removal from the myocyte cytoplasm in diastole, which is primarily driven by SERCA, the integral transmembrane Ca2+ pump. Phospholamban (PLB) allosterically inhibits SERCA by reducing its apparent Ca2+ affinity. Recently, the 34-codon novel dwarf open reading frame (DWORF) micropeptide has been identified as a muscle-specific SERCA effector, capable of reversing the inhibitory effects of PLB and independently activating SERCA in the absence of PLB. However, the structural basis for these functions has not yet been determined in a system of defined molecular components. We have used electron paramagnetic resonance (EPR) spectroscopy to investigate the protein-protein interactions of DWORF, co-reconstituted in proteoliposomes with SERCA and spin-labeled PLB. We analyzed the change of PLB rotational mobility in response to varying DWORF concentration, to quantify competitive binding of DWORF and PLB. We determined that DWORF competes with PLB for binding to SERCA at low [Ca2+], although the measured affinity of DWORF for SERCA is an order of magnitude weaker than that of PLB for SERCA, indicating cooperativity. The sensitivity of EPR to structural dynamics, using stereospecifically attached spin labels, allows us to obtain new information needed to refine the molecular model for regulation of SERCA activity, as needed for development of novel therapeutic remedies against cardiac pathologies.

Exome and genome sequencing (ES and GS) allow detection of somatic mosaic variants that are present at a lower variant allele fraction (VAF) than is observed for a typical heterozygous germline variant, which is around 50%. Recent guidelines from the American College of Medical Genetics and Genomics highlight 21 genes in which mosaic variants are commonly identified as a part of routine ES/GS for germline testing in patients with personal and/or family history inconsistent with disease associated with pathogenic germline variants these genes.

My thesis work is divided into two periods: research in the School of Physics and Astronomy, advised by Dr. Vincent Noireaux (Ch. 1-3), followed by research in the Department of Biochemistry, Molecular Biology and Biophysics, advised by Dr. David Thomas (Ch. 4). In Chapter 1, I cover the functional scope and optimizations of the cell-free transcription and translation (TX-TL) platform developed in the Noireaux lab, as described by my 2016 ACS Synthetic Biology publication. This work discusses the breadth of synthetic biological applications available with a cell-free TX-TL system, such as the programming and execution of biological circuits, the bottom-up synthesis of functional biological systems, and the development of a minimal cell system. My work was focused on the cell-free synthesis of infectious bacteriophage particles. I performed parameter space optimization of biochemical reaction components for multiple phage systems. Chapter 2 describes the cell-free synthesis of bacteriophage T4, one of the most complex systems to be fully recapitulated within a cell-free platform, reproducing my 2018 Synthetic Biology publication. In Chapter 3, I share my 2017 peer-reviewed scientific video journal for The Journal of Visual Experiments where I describe the protocol for general cell-free bacteriophage synthesis. Chapter 4 presents my work in the Thomas Lab, where I utilized site-directed spin labeling (SDSL) and electron paramagnetic resonance (EPR) to investigate the structural model by which SERCA, PLB, and DWORF regulate intracellular calcium transport in cardiac muscle. I detail the methodology behind purifying and co-reconstituting these membrane proteins in vesicles, and I present the dynamical information of these proteins obtained by EPR spectroscopy. My experimental background in cell-free synthetic biology, protein dynamics, and magnetic resonance has revealed to me the exciting nature of biophysical investigations. The societal impact and importance of progress in this field has never been more apparent than it is now, in the face of a novel pandemic. After my PhD work at the University of Minnesota, I will continue research in biophysics on the computational side, working as a Data Scientist for the National Institute of Allergy and Infectious Disease, where my new position begins in September.

We have used electron paramagnetic resonance (EPR) spectroscopy to examine the protein-protein interaction of a recently discovered 34-codon micropeptide, dwarf open reading frame (DWORF), with the sarco/endoplasmic reticulum calcium ATPase (SERCA) in the presence and absence of phospholamban (PLB) in lipid vesicles. Heart failure is one of the leading causes of death in developed countries. A characteristic pathology of cardiac disease is inadequate activity of SERCA, the integral transmembrane Ca2+ pump, so SERCA activation is an important goal in developing treatments for heart failure. PLB is known to decrease SERCA's Ca2+ affinity through allosteric interactions. However, it has been shown that co-expression of DWORF with PLB and SERCA, in HEK293 cells, restores SERCA activity (Nelson et al., 2016). It has been proposed that this relief of SERCA inhibition is due to direct competition by DWORF for PLB binding to SERCA. To test this hypothesis in a membrane of defined protein and lipid composition, we co-reconstituted purified SERCA and spin-labeled PLB, with and without DWORF, in lipid vesicles. We then performed NADH-coupled Ca-ATPase assays to determine SERCA activity, and EPR experiments to determine the binding of PLB to SERCA. The sensitivity of EPR to structural dynamics, using stereospecifically attached spin labels, made the experiments extremely sensitive to PLB immobilization by SERCA. The results indicate that DWORF's restoration of SERCA activity correlates with some direct displacement of PLB from SERCA, but the fractional relief of inhibition exceeds the fractional PLB displacement, suggesting a more complex mechanism. These results provide direct insight of the equilibrium dynamics of SERCA and its regulators PLB and DWORF, providing valuable information for the development of novel therapeutic remedies for cardiac pathologies.

We have used electron paramagnetic resonance (EPR) spectroscopy to investigate the protein-protein interaction of a recently discovered micropeptide, dwarf open reading frame (DWORF), with the sarco/endoplasmic reticulum calcium ATPase (SERCA) in the presence and absence of phospholamban (PLB) in lipid bilayer vesicles. Heart failure is one of the leading causes of death in developed countries. A characteristic pathology of cardiac disease is inadequate activity of SERCA, the integral transmembrane Ca2+ pump. For every ATP hydrolyzed, SERCA transports two Ca2+ ions from the cytosol to the sarcoplasmic reticulum (SR), enabling muscle relaxation. A primary regulator of SERCA is PLB, an additional integral transmembrane protein of SR. PLB allosterically affects SERCA by reducing its apparent Ca2+ affinity. Recently, the 34-codon micropeptide DWORF was discovered, and when co-expressed with PLB and SERCA, it was found to restore the activity of PLB-inhibited SERCA to that of the uninhibited values. However, the structural basis for this observation has not yet been determined. We have investigated the protein-protein interactions of co-reconstituted SERCA, DWORF and PLB in lipid vesicles using electron paramagnetic resonance (EPR) spectroscopy and associated kinetics assays. The sensitivity of EPR to structural dynamics, using stereospecifically attached spin labels, allows us to develop a model explaining the observed effects on SERCA activity, which will aid in the development of novel therapeutic remedies for cardiac pathologies.

The bottom-up construction of biological entities from genetic information provides a broad range of opportunities to better understand fundamental processes within living cells, as well as holding great promise for the development of novel biomedical applications. Cell-free transcription-translation (TXTL) systems have become suitable platforms to tackle such topics because they recapitulate the process of gene expression. TXTL systems have advanced to where the in vitro construction of viable, complex, self-assembling deoxyribonucleic acid-programmed biological entities is now possible. Previously, we demonstrated the cell-free synthesis of three bacteriophages from their genomes: MS2, ΦX174, T7. In this work, we present the complete synthesis of the phage T4 from its 169-kbp genome in one-pot TXTL reactions. This achievement, for one of the largest coliphages, demonstrates the integration of complex gene regulation, metabolism and self-assembly, and brings the bottom-up synthesis of biological systems to a new level.

A new generation of cell-free transcription-translation (TXTL) systems, engineered to have a greater versatility and modularity, provide novel capabilities to perform basic and applied sciences in test tube reactions. Over the past decade, cell-free TXTL has become a powerful technique for a broad range of novel multidisciplinary research areas related to quantitative and synthetic biology. The new TXTL platforms are particularly useful to construct and interrogate biochemical systems through the execution of synthetic or natural gene circuits. In vitro TXTL has proven convenient to rapidly prototype regulatory elements and biological networks as well as to recapitulate molecular self-assembly mechanisms found in living systems. In this article, we describe how infectious bacteriophages, such as MS2 (RNA), ΦΧ174 (ssDNA), and T7 (dsDNA), are entirely synthesized from their genome in one-pot reactions using an all Escherichia coli, cell-free TXTL system. Synthesis of the three coliphages is quantified using the plaque assay. We show how the yield of synthesized phage depends on the biochemical settings of the reactions. Molecular crowding, emulated through a controlled concentration of PEG 8000, affects the amount of synthesized phages by orders of magnitudes. We also describe how to amplify the phages and how to purify their genomes. The set of protocols and results presented in this work should be of interest to multidisciplinary researchers involved in cell-free synthetic biology and bioengineering.

We report on and provide a detailed characterization of the performance and properties of a recently developed, all Escherichia coli, cell-free transcription and translation system. Gene expression is entirely based on the endogenous translation components and transcription machinery provided by an E. coli cytoplasmic extract, thus expanding the repertoire of regulatory parts to hundreds of elements. We use a powerful metabolism for ATP regeneration to achieve more than 2 mg/mL of protein synthesis in batch mode reactions, and more than 6 mg/mL in semicontinuous mode. While the strength of cell-free expression is increased by a factor of 3 on average, the output signal of simple gene circuits and the synthesis of entire bacteriophages are increased by orders of magnitude compared to previous results. Messenger RNAs and protein degradation, respectively tuned using E. coli MazF interferase and ClpXP AAA+ proteases, are characterized over a much wider range of rates than the first version of the cell-free toolbox. This system is a highly versatile cell-free platform to construct complex biological systems through the execution of DNA programs composed of synthetic and natural bacterial regulatory parts.

Cell-free transcription-translation (TX-TL) systems are becoming powerful platforms to construct complex biological systems in vitro through the expression of DNA programs. Considerable efforts have been made to improve those systems in the past decade. Our laboratory has developed the most efficient and versatile in vitro E. coli TX-TL system to express synthetic or natural DNA programs encoding for self-assembly processes. Either in test tube reactions, in emulsion droplets or in liposomes, cell-free TX-TL reactions are now used for research studies ranging from elementary gene circuits to minimal cells. I will present our custom-made cell-free TX-TL system, its current capabilities and limitations. Recently we have shown that entire phages can be synthesized in cell-free TX-TL reactions from their genomes. The bacteriophage T7, composed of about 60 genes, is entirely synthesized in a single test tube reaction from its 40 kbp genomic DNA. Replication of the T7 DNA instructions occurs concurrently with phage expression and self-assembly. The phage phiX174 composed of a dozen of genes can also be synthesized from its genome. Encapsulated inside cell-sized phospholipid liposomes, the TX-TL system is used to construct a prototype of minimal cell. I will present this cell-free synthetic biology platform, our last experiments and how this system can be used to study the relationship between information and self-organization.

Since the pioneering work of Plaxco, Simons, and Baker, it is now well known that the rates of protein folding strongly correlate with the average sequence separation (absolute contact order (ACO)) of native contacts. In spite of multitude of papers, our understanding to the basis of the relation between folding speed and ACO is still lacking. We model the transition state as a gaussian polymer chain decorated with weak springs between native contacts while the unfolded state is modeled as a gaussian chain only. Using these hamiltonians, our perturbative calculation explicitly shows folding speed and ACO are linearly related when only the first order term in the series is considered. However, to the second order, we notice the existence of two new topological metrics, termed COC(1) and COC(2) (COC stands for contact order correction). These additional correction terms are needed to properly account for the entropy loss due to overlapping (nested or linked) loops that are not well described by simple addition of entropies in ACO. COC(1) and COC(2) are related to fluctuations and correlations among different sequence separations. The new metric combining ACO, COC(1), and COC(2) improves folding speed dependence on native topology when applied to three different databases: (i) two-state proteins with only α∕β and β proteins, (ii) two-state proteins (α∕β, β and purely helical proteins all combined), and (iii) master set (multi-state and two-state) folding proteins. Furthermore, the first principle calculation provides us direct physical insights to the meaning of the fit parameters. The coefficient of ACO, for example, is related to the average strength of the contacts, while the constant term is related to the protein folding speed limit. With the new scaling law, our estimate of the folding speed limit is in close agreement with the widely accepted value of 1 μs observed in proteins and RNA. Analyzing an exhaustive set (7367) of monomeric proteins from protein data bank, we find our new topology based metric (combining ACO, COC(1), and COC(2)) scales as N(0.54), N being the number of amino acids in a protein. This is in remarkable agreement with a previous argument based on random systems that predict protein folding speed depends on exp (-N(0.5)). The first principle calculation presented here provides deeper insights to the role of topology in protein folding and unifies many parallel arguments, seemingly disconnected, demonstrating the existence of universal mechanism in protein folding kinetics that can be understood from simple polymer physics based principles.