A self-assembling protein template for constrained synthesis and patterning of nanoparticle arrays
No abstract available
Alloys/chemistry↗
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Tissue-engineered human bioartificial muscles expressing a foreign recombinant protein for gene therapy
Murine skeletal muscle cells transduced with foreign genes and tissue engineered in vitro into bioartificial muscles (BAMs) are capable of long-term delivery of soluble growth factors when implanted into syngeneic mice (Vandenburgh et al., 1996b). With the goal of developing a therapeutic cell-based protein delivery system for humans, similar genetic tissue-engineering techniques were designed for human skeletal muscle stem cells. Stem cell myoblasts were isolated, cloned, and expanded in vitro from biopsied healthy adult (mean age, 42 +/- 2 years), and elderly congestive heart failure patient (mean age, 76 +/- 1 years) skeletal muscle. Total cell yield varied widely between biopsies (50 to 672 per 100 mg of tissue, N = 10), but was not significantly different between the two patient groups. Percent myoblasts per biopsy (73 +/- 6%), number of myoblast doublings prior to senescence in vitro (37 +/- 2), and myoblast doubling time (27 +/- 1 hr) were also not significantly different between the two patient groups. Fusion kinetics of the myoblasts were similar for the two groups after 20-22 doublings (74 +/- 2% myoblast fusion) when the biopsy samples had been expanded to 1 to 2 billion muscle cells, a number acceptable for human gene therapy use. The myoblasts from the two groups could be equally transduced ex vivo with replication-deficient retroviral expression vectors to secrete 0.5 to 2 microg of a foreign protein (recombinant human growth hormone, rhGH)/10(6) cells/day, and tissue engineered into human BAMs containing parallel arrays of differentiated, postmitotic myofibers. This work suggests that autologous human skeletal myoblasts from a potential patient population can be isolated, genetically modified to secrete foreign proteins, and tissue engineered into implantable living protein secretory devices for therapeutic use.
Non-NASA Center↗
Water, Solute, and Ion Transport in De Novo-Designed Membrane Protein Channels
Biological organisms engineer peptide sequences to fold into membrane pore proteins capable of performing a wide variety of transport functions. Synthetic de novo-designed membrane pores can mimic this approach to achieve a potentially even larger set of functions. Here, in this work, we explore water, solute, and ion transport in three de novo designed β-barrel membrane channels in the 5–10 Å pore size range. We show that these proteins form passive membrane pores with high water transport efficiencies and size rejection characteristics consistent with the pore size encoded in the protein structure. Ion conductance and ion selectivity measurements also show trends consistent with the pore size, with the two larger pores showing weak cation selectivity. MD simulations of water and ion transport and solute size exclusion are consistent with the experimental trends and provide further insights into structure–function correlations in these membrane pores.
59 BASIC BIOLOGICAL SCIENCES↗
Bio-Organic Nanotechnology: Using Proteins and Synthetic Polymers for Nanoscale Devices
While the ability of proteins to self-assemble makes them powerful tools in nanotechnology, in biological systems protein-based structures ultimately depend on the context in which they form. We combine the self-assembling properties of synthetic diblock copolymers and proteins to construct intricately ordered, three-dimensional polymer protein structures with the ultimate goal of forming nano-scale devices. This hybrid approach takes advantage of the capabilities of organic polymer chemistry to build ordered structures and the capabilities of genetic engineering to create proteins that are selective for inorganic or organic substrates. Here, microphase-separated block copolymers coupled with genetically engineered heat shock proteins are used to produce nano-scale patterning that maximizes the potential for both increased structural complexity and integrity.
Molnar, Linda K.↗
Model for determining vapor equilibrium rates in the hanging drop method for protein crystal growth
An engineering analysis of the rate of evaporation of solvent in the hanging drop method of protein crystal growth is presented. Results are applied to 18 drop and well arrangements commonly encountered in the laboratory. The chemical nature of the salt, drop size and shape, drop concentration, well size, well concentration, and temperature are taken into account. The rate of evaporation increases with temperature, drop size, and the salt concentration difference between the drop and the well. The evaporation in this model possesses no unique half-life. Once the salt in the drop achieves 80 percent of its final concentration, further evaporation suffers from the law of diminishing returns.
Baird, James K.↗
Evaporation kinetics in the hanging drop method of protein crystal growth
An engineering analysis of the rate of evaporation of solvent in the hanging drop method of protein crystal growth is presented; these results are applied to 18 different drop and well arrangements commonly encountered in the laboratory, taking into account the chemical nature of the salt, the drop size and shape, the drop concentration, the well size, the well concentration, and the temperature. It is found that the rate of evaporation increases with temperature, drop size, and with the salt concentration difference between the drop and the well. The evaporation possesses no unique half-life. Once the salt in the drop achieves about 80 percent of its final concentration, further evaporation suffers from the law of diminishing returns.
Baird, James K.↗
Controlled Enzyme Cargo Loading in Engineered Bacterial Microcompartment Shells
Bacterial microcompartments (BMCs) are nanometer-scale organelles with a protein-based shell that serve to colocalize and encapsulate metabolic enzymes. They may provide a range of benefits to improve pathway catalysis, including substrate channeling and selective permeability. Several groups are working toward using BMC shells as a platform for enhancing engineered metabolic pathways. The microcompartment shell of Haliangium ochraceum (HO) has emerged as a versatile and modular shell system that can be expressed and assembled outside its native host and with non-native cargo. Further, the HO shell has been modified to use the engineered protein conjugation system SpyCatcher–SpyTag for non-native cargo loading. Here, we used a model enzyme, triose phosphate isomerase (Tpi), to study non-native cargo loading into four HO shell variants and begin to understand maximal shell loading levels. We also measured activity of Tpi encapsulated in the HO shell variants and found that activity was determined by the amount of cargo loaded and was not strongly impacted by the predicted permeability of the shell variant to large molecules. All shell variants tested could be used to generate active, Tpi-loaded versions, but the simplest variants assembled most robustly. We propose that the simple variant is the most promising for continued development as a metabolic engineering platform.
59 BASIC BIOLOGICAL SCIENCES↗
Lighting up yeast: overview of optogenetics in yeast and their applications to yeast biotechnology
Abstract Optogenetics is an empowering technology that uses light-responsive proteins to control biological processes. Because of its genetic tractability, abundance of genetic tools, and robust culturing conditions, Saccharomyces cerevisiae has served for many years as an ideal platform in which to study, develop, and apply a wide range of optogenetic systems. In many instances, yeast has been used as a steppingstone in which to characterize and optimize optogenetic tools to later be deployed in higher eukaryotes. More recently, however, optogenetic tools have been developed and deployed in yeast specifically for biotechnological applications, including in nonconventional yeasts. In this review, we summarize various optogenetic systems responding to different wavelengths of light that have been demonstrated in diverse yeast species. We then describe various applications of these optogenetic tools in yeast, particularly in metabolic engineering and recombinant protein production. Finally, we discuss emerging applications in yeast cybergenetics—the interfacing of yeast and computers for closed-loop controls of yeast bioprocesses—and the potential impact of optogenetics in other future biotechnological applications.
Biotechnology & Applied Microbiology↗
Ca X ML: Chemistry‐informed machine learning explains mutual changes between protein conformations and calcium ions in calcium‐binding proteins using structural and topological features
Proteins' flexibility is a feature in communicating changes in cell signaling instigated by binding with secondary messengers, such as calcium ions, associated with the coordination of muscle contraction, neurotransmitter release, and gene expression. When binding with the disordered parts of a protein, calcium ions must balance their charge states with the shape of calcium-binding proteins and their versatile pool of partners depending on the circumstances they transmit. Accurately determining the ionic charges of those ions is essential for understanding their role in such processes. However, it is unclear whether the limited experimental data available can be effectively used to train models to accurately predict the charges of calcium-binding protein variants. Here, we developed a chemistry-informed, machine-learning algorithm that implements a game theoretic approach to explain the output of a machine-learning model without the prerequisite of an excessively large database for high-performance prediction of atomic charges. We used the ab initio electronic structure data representing calcium ions and the structures of the disordered segments of calcium-binding peptides with surrounding water molecules to train several explainable models. Network theory was used to extract the topological features of atomic interactions in the structurally complex data dictated by the coordination chemistry of a calcium ion, a potent indicator of its charge state in protein. Our design created a computational tool of Ca X ML, which provided a framework of explainable machine learning model to annotate ionic charges of calcium ions in calcium-binding proteins in response to the chemical changes in an environment. Our framework will provide new insights into protein design for engineering functionality based on the limited size of scientific data in a genome space.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
Recombinant Spidroins Fully Replicate Primary Mechanical Properties of Natural Spider Silk
Dragline spider silk is among the strongest and toughest bio-based materials, capable of outperforming most synthetic polymers and even some metal alloys.1,2,3,4 These properties have gained spider silk a growing list of potential applications that, coupled with the impracticalities of spider farming, have driven a decades-long effort to produce recombinant spider silk proteins (spidroins) in engineered heterologous hosts.2 However, these efforts have so far been unable to yield synthetic silk fibers with mechanical properties equivalent to natural spider silk, largely due to an inability to stably produce highly repetitive, high molecular weight (MW) spidroins in heterologous hosts.1,5 Here we address these issues by combining synthetic biology techniques with split intein (SI)- mediated ligation for the bioproduction of spidroins with unprecedented MW (556 kDa), containing 192 repeat motifs of the Nephila clavipes MaSp1 dragline spidroin. Fibers spun from these synthetic spidroins display ultimate tensile strength (σ), modulus (E), extensibility (ε), and toughness (UT) of 1.03 +/- 0.11 GPa, 13.7 +/- 3.0 GPa, 18 +/- 6%, and 114 +/- 51 MJ/m3, respectively-equivalent to the performance of natural N. clavipes dragline silk.6 This work demonstrates for the first time that microbially produced synthetic silk fibers can match the performance of natural silk fibers by all common metrics (σ, E, ε, UT), providing a more dependable source of high-strength fibers to replace natural spider silks for mechanically demanding applications. Furthermore, our biosynthetic platform can be potentially expanded for the assembly and production of other protein-based materials with high MW and repetitive sequences that have so far been impossible to synthesize by genetic means alone.
spider silk↗
Characterization of lignin-degrading enzyme PmdC, which catalyzes a key step in the synthesis of polymer precursor 2-pyrone-4,6-dicarboxylic acid
Pyrone-2,4-dicarboxylic acid (PDC) is a valuable polymer precursor that can be derived from the microbial degradation of lignin. The key enzyme in the microbial production of PDC is 4-carboxy-2-hydroxymuconate-6-semialdehyde (CHMS) dehydrogenase, which acts on the substrate CHMS. We present the crystal structure of CHMS dehydrogenase (PmdC from Comamonas testosteroni) bound to the cofactor NADP, shedding light on its three-dimensional architecture, and revealing residues responsible for binding NADP. Using a combination of structural homology, molecular docking, and quantum chemistry calculations, we have predicted the binding site of CHMS. Key histidine residues in a conserved sequence are identified as crucial for binding the hydroxyl group of CHMS and facilitating dehydrogenation with NADP. Mutating these histidine residues results in a loss of enzyme activity, leading to a proposed model for the enzyme's mechanism. These findings are expected to help guide efforts in protein and metabolic engineering to enhance PDC yields in biological routes to polymer feedstock synthesis.
37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
U.S.-Japan Workshop on Smart/Intelligent Materials and Systems, Honolulu, HI, Mar. 19-23, 1990, Proceedings
Topics presented include the concept of intelligent materials, the molecular engineering of channel proteins, the prospects, limitations, and requirements of intelligent micro motion systems, the modeling of a shape memory integrated actuator for vibration control of large space structures, and intelligent materials for future electronics. Also presented are the slewing of an active structure, the health monitoring of control system components, the numerical modeling of the microstructure of crystals with symmetry-related variants, and the intelligent material system concept.
Ahmad, Iqbal↗
Electrophoresis experiments in microgravity
The use of the microgravity environment to separate and purify biological cells and proteins has been a major activity since the beginning of the NASA Microgravity Science and Applications program. Purified populations of cells are needed for research, transplantation and analysis of specific cell constituents. Protein purification is a necessary step in research areas such as genetic engineering where the new protein has to be separated from the variety of other proteins synthesized from the microorganism. Sufficient data are available from the results of past electrophoresis experiments in space to show that these experiments were designed with incomplete knowledge of the fluid dynamics of the process including electrohydrodynamics. However, electrophoresis is still an important separation tool in the laboratory and thermal convection does limit its performance. Thus, there is a justification for electrophoresis but the emphasis of future space experiments must be directed toward basic research with model experiments to understand the microgravity environment and fluid analysis to test the basic principles of the process.
Snyder, Robert S.↗
Life in the Clouds of Venus? An Experimental Synthetic Biology Approach
The surface of Venus constitutes the most hellish and biologically inhospitable planetary surface in our solar system, boasting a pH of 0, blistering winds that can melt lead, and pressures of 60 atm. However, during the earlier years of the solar system, without the runaway greenhouse effect that has plagued the planet, Venus potentially housed oceans and perhaps even life. There is a possibility that microbes could have retreated into hospitable niches in the atmosphere, as suggested by Carl Sagan as early as 1967 [1]. For example, 50 km above the raging hell of the Venusian surface, exists a relatively temperate environment that might serve as reservoir for life. This astrobiology project seeks to explore life at the extremes and to theorize whether microbial communities could not only survive but also reproduce in the Venusian atmosphere. Specifically, we ask: are aerosols viable microbial environments? But before we can test for life in the clouds, we have to develop a proper reporter to visualize cell growth in situ. For this purpose, we aimed to develop cell-growth dependent reporters to serve as remote biosensors for cell growth. We developed two using the polA promoter, a DNA-replication dependent promoter, and nrd operon promoter, a cell-cycle dependent promoter. Using these cell-growth reporters, the next step is to aerosolize microbes expressing these reporters in a suspension chamber adapted from a Millikan Drop Apparatus to assay reproduction in an aerosolized environment. Better yet is to test the reproduction of microbes in a microgravity regime such as on ISS.Approach: We engineered two cell-cycle dependent genetic reporters. One was the polA promoter which codes for DNA Polymerase I, a gene active in DNA replication [2]. The other was the nrdP. The activation of ribonucleotide reductase reduces ribonucleotides into deoxyribonucleotides and is involved in the bacterial cell cycle [3]. This promoter began activation during the initiation of DNA replication and is cell-cycle dependent [4]. These promoters were fused to a GFP reporter, transformed into E. coli. The constructs were deposited in the iGEM registry as K847210: Escherichia coli DNA-replication dependent polA promoter K847211: Escherichia coli cell-division dependent nrd promoter Results: Our constructs displayed fluorescence when transformed into NEB-5alpha competent cells. While nrdP-E0840 displayed sufficient fluorescence as verified by fluorescent microscopy, the original polAP-E0840 construct (which uses mut3b GFP) exhibited low expression; while fluorescence was visible under the microscope, the signal was too weak for the camera to recognize. The polA promoter was therefore digest-ed with EcoRI and SpeI then ligated into plasmid pNCS containing a RBS, Clover, and a terminator. Clover is a highly engineered green fluorescent protein that exhibits extreme brightness [5] Fluoresence time course data demonsrated that the genes were induced in a cell cycle dependant manner [6]. Our assays via microscopy and the bulk assay shows that our promoters are functional as cell cycle reporters.Conclusions: The application of such tools are widespread and not limited to astrobiology; nrdP could be used to determine doubling times empirically and could possibly extrapolate DNA content from intensity of signals expressed by polAP. However, we are pri-marily interested in its use in astrobiology.
Escherichia↗
Engineering the green algae Chlamydomonas incerta for recombinant protein production
Chlamydomonas incerta , a genetically close relative of the model green alga Chlamydomonas reinhardtii , shows significant potential as a host for recombinant protein expression. Because of the close genetic relationship between C. incerta and C. reinhardtii , this species offers an additional reference point for advancing our understanding of photosynthetic organisms, and also provides a potential new candidate for biotechnological applications. This study investigates C. incerta ’s capacity to express three recombinant proteins: the fluorescent protein mCherry, the hemicellulose-degrading enzyme xylanase, and the plastic-degrading enzyme PHL7. We have also examined the capacity to target protein expression to various cellular compartments in this alga, including the cytosol, secretory pathway, cytoplasmic membrane, and cell wall. When compared directly with C. reinhardtii , C. incerta exhibited a distinct but notable capacity for recombinant protein production. Cellular transformation with a vector encoding mCherry revealed that C. incerta produced approximately 3.5 times higher fluorescence levels and a 3.7-fold increase in immunoblot intensity compared to C. reinhardtii . For xylanase expression and secretion, both C. incerta and C. reinhardtii showed similar secretion capacities and enzymatic activities, with comparable xylan degradation rates, highlighting the industrial applicability of xylanase expression in microalgae. Finally, C. incerta showed comparable PHL7 activity levels to C. reinhardtii , as demonstrated by the in vitro degradation of a polyester polyurethane suspension, Impranil® DLN. Finally, we also explored the potential of cellular fusion for the generation of genetic hybrids between C. incerta and C. reinhardtii as a means to enhance phenotypic diversity and augment genetic variation. We were able to generate genetic fusion that could exchange both the recombinant protein genes, as well as associated selectable marker genes into recombinant offspring. These findings emphasize C. incerta ’s potential as a robust platform for recombinant protein production, and as a powerful tool for gaining a better understanding of microalgal biology.
cell membranes↗
Peripheral positions encode transport specificity in the small multidrug resistance exporters
In secondary active transporters, a relatively limited set of protein folds have evolved diverse solute transport functions. Because of the conformational changes inherent to transport, altering substrate specificity typically involves remodeling the entire structural landscape, limiting our understanding of how novel substrate specificities evolve. In the current work, we examine a structurally minimalist family of model transport proteins, the small multidrug resistance (SMR) transporters, to understand the molecular basis for the emergence of a novel substrate specificity. We engineer a selective SMR protein to promiscuously export quaternary ammonium antiseptics, similar to the activity of a clade of multidrug exporters in this family. Using combinatorial mutagenesis and deep sequencing, we identify the necessary and sufficient molecular determinants of this engineered activity. Using X-ray crystallography, solid-supported membrane electrophysiology, binding assays, and a proteoliposome-based quaternary ammonium antiseptic transport assay that we developed, we dissect the mechanistic contributions of these residues to substrate polyspecificity. We find that substrate preference changes not through modification of the residues that directly interact with the substrate but through mutations peripheral to the binding pocket. Our work provides molecular insight into substrate promiscuity among the SMRs and can be applied to understand multidrug export and the evolution of novel transport functions more generally.
Science & Technology - Other Topics↗
Protocol for Engineered Compositional Asymmetry Within Nanodiscs
Membrane proteins remain the most challenging targets for structural characterization, yet their elucidation provides valuable insights into protein function, disease mechanisms, and drug specificity. Structural biology platforms have advanced rapidly in recent years, notably through the development and implementation of nanodiscs—discoidal lipid–protein complexes that encapsulate and solubilize membrane proteins within a controlled, native-like environment. While nanodiscs have become powerful tools for studying membrane proteins, faithfully reconstituting the compositional asymmetry intrinsic to nearly all biological membranes has not yet been achieved. Proper membrane leaflet lipid distribution is critical for accurate protein folding, stability, and insertion. Here, we share a protocol for reconstituting tailored compositional asymmetry within nanodiscs through membrane extraction from giant unilamellar vesicles (GUVs) treated with a leaflet-specific methyl-β-cyclodextrin (mβCD) lipid exchange. Nanodisc asymmetry is verified through a geometric approach: biotin-DPPE-preloaded mβCD engages in lipid exchange with the outer leaflet of POPC GUVs solubilized by the lipid-free membrane scaffold protein (MSP) Δ49ApoA-I to form nanodisc structures. Once isolated, nanodiscs are introduced to the biotin-binding bacterial protein streptavidin. High-speed atomic force microscopy imaging depicts nanodisc–dimer complexes, indicating that biotin-DPPE was successfully reconstituted into a single leaflet of the nanodiscs. This finding outlines the first step toward engineering tailored nanodisc asymmetry and mimicking the native environment of integral proteins—a potentially powerful tool for accurately reconstituting and structurally analyzing integral membrane proteins whose functions are modulated by lipid asymmetry.
Biological and medical sciences↗
Exploring the binding properties and activities of ancestral expansins
Bacterial expansins are non-lytic proteins capable of loosening cellulose networks, offering promising applications in agriculture, biotechnology, and material science. Their ability to disrupt noncovalent interactions in biopolymer matrices such as cellulose and chitin positions them as valuable tools for upgrading abundant natural materials. However, their industrial use remains limited due to their relatively low wall-loosening activity compared to plant expansins. To address this limitation, we applied Ancestral Sequence Resurrection (ASR) to reconstruct and characterize ancient variants of the Bacillus subtilis expansin BsEXLX1. ASR is a powerful evolutionary tool that enables the inference and synthesis of ancestral proteins, allowing researchers to explore functional traits that may have been lost over time. This approach not only provides insights into protein evolution but also facilitates the design of proteins with enhanced properties, such as improved substrate affinity or structural stability. In this study, we combined biochemical and biophysical assays to evaluate the activity and binding behavior of ancestral expansins. Our results reveal that ancestral variants exhibit increased cellulose affinity, reduced binding to acidic polysaccharides, and greater salt resistance. Furthermore, these traits enhance their wall-loosening activity and demonstrate the utility of ASR in engineering surface-active proteins for industrial applications, particularly in biomass processing and cellulose modification.
09 BIOMASS FUELS↗