Interaction of DWORF with SERCA and PLB as determined by EPR spectroscopy.

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.

Cell-free TXTL synthesis of infectious bacteriophage T4 in a single test tube reaction.

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.

Synthesis of Infectious Bacteriophages in an E. coli-based Cell-free Expression System.

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.

The All E. coli TX-TL Toolbox 2.0: A Platform for Cell-Free Synthetic Biology.

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.

Why and how does native topology dictate the folding speed of a protein?

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∕ß and ß proteins, (ii) two-state proteins (α∕ß, ß 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.