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Baker, David

Publications and source records attributed to Baker, David.

At least 469 records · Page 26
All sourcesNASA NTRSDOE OSTI
DOE OSTI2016

Determining crystal structures through crowdsourcing and coursework

We show here that computer game players can build high-quality crystal structures. Introduction of a new feature into the computer game Foldit allows players to build and real-space refine structures into electron density maps. To assess the usefulness of this feature, we held a crystallographic model-building competition between trained crystallographers, undergraduate students, Foldit players and automatic model-building algorithms. After removal of disordered residues, a team of Foldit players achieved the most accurate structure. Analysing the target protein of the competition, YPL067C, uncovered a new family of histidine triad proteins apparently involved in the prevention of amyloid toxicity. From this study, we conclude that crystallographers can utilize crowdsourcing to interpret electron density information and to produce structure solutions of the highest quality.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Accurate de novo design of hyperstable constrained peptides

Covalently-crosslinked peptides present attractive opportunities for developing new therapeutics. Lying between small molecule and protein therapeutics in size, natural crosslinked peptides play critical roles in signaling, virulence and immunity. Engineering novel peptides with precise control over their three-dimensional structures is a significant challenge. Here we describe the development of computational methods for de novo design of conformationally-restricted peptides, and the use of these methods to design hyperstable disulfide-stabilized miniproteins, heterochiral peptides, and N-C cyclic peptides. Experimentally-determined X-ray and NMR structures for 12 of the designs are nearly identical to the computational models. The computational design methods and stable scaffolds provide the basis for a new generation of peptide-based drugs.

drug-design↗
DOE OSTI2016

Accurate design of megadalton-scale two-component icosahedral protein complexes

Nature provides many examples of self- and co-assembling protein-based molecular machines, including icosahedral protein cages that serve as scaffolds, enzymes, and compartments for essential biochemical reactions and icosahedral virus capsids, which encapsidate and protect viral genomes and mediate entry into host cells. Inspired by these natural materials, we report the computational design and experimental characterization of co-assembling, two-component, 120-subunit icosahedral protein nanostructures with molecular weights (1.8 to 2.8 megadaltons) and dimensions (24 to 40 nanometers in diameter) comparable to those of small viral capsids. Electron microscopy, small-angle x-ray scattering, and x-ray crystallography show that 10 designs spanning three distinct icosahedral architectures form materials closely matching the design models. In vitro assembly of icosahedral complexes from independently purified components occurs rapidly, at rates comparable to those of viral capsids, and enables controlled packaging of molecular cargo through charge complementarity. The ability to design megadalton-scale materials with atomic-level accuracy and controllable assembly opens the door to a new generation of genetically programmable protein-based molecular machines.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

A general strategy to construct small molecule biosensors in eukaryotes

Biosensors for small molecules can be used in applications that range from metabolic engineering to orthogonal control of transcription. Here, we produce biosensors based on a ligand-binding domain (LBD) by using a method that, in principle, can be applied to any target molecule. The LBD is fused to either a fluorescent protein or a transcriptional activator and is destabilized by mutation such that the fusion accumulates only in cells containing the target ligand. We illustrate the power of this method by developing biosensors for digoxin and progesterone. Addition of ligand to yeast, mammalian, or plant cells expressing a biosensor activates transcription with a dynamic range of up to ~100-fold. We use the biosensors to improve the biotransformation of pregnenolone to progesterone in yeast and to regulate CRISPR activity in mammalian cells. This work provides a general methodology to develop biosensors for a broad range of molecules in eukaryotes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Engineering an allosteric transcription factor to respond to new ligands

Genetic regulatory proteins inducible by small molecules are useful synthetic biology tools as sensors and switches. Bacterial allosteric transcription factors (aTFs) are a major class of regulatory proteins, but few aTFs have been redesigned to respond to new effectors beyond natural aTF-inducer pairs. Altering inducer specificity in these proteins is difficult because substitutions that affect inducer binding may also disrupt allostery. In this paper, we engineered an aTF, the Escherichia coli lac repressor, LacI, to respond to one of four new inducer molecules: fucose, gentiobiose, lactitol and sucralose. Using computational protein design, single-residue saturation mutagenesis or random mutagenesis, along with multiplex assembly, we identified new variants comparable in specificity and induction to wild-type LacI with its inducer, isopropyl β-D-1-thiogalactopyranoside (IPTG). Finally, the ability to create designer aTFs will enable applications including dynamic control of cell metabolism, cell biology and synthetic gene circuits.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Exploring the repeat protein universe through computational protein design

A central question in protein evolution is the extent to which naturally occurring proteins sample the space of folded structures accessible to the polypeptide chain. Repeat proteins composed of multiple tandem copies of a modular structure unit are widespread in nature and have critical roles in molecular recognition, signalling, and other essential biological processes. Naturally occurring repeat proteins have been re-engineered for molecular recognition and modular scaffolding applications. In this paper, we use computational protein design to investigate the space of folded structures that can be generated by tandem repeating a simple helix–loop–helix–loop structural motif. Eighty-three designs with sequences unrelated to known repeat proteins were experimentally characterized. Of these, 53 are monomeric and stable at 95 °C, and 43 have solution X-ray scattering spectra consistent with the design models. Crystal structures of 15 designs spanning a broad range of curvatures are in close agreement with the design models with root mean square deviations ranging from 0.7 to 2.5 Å. Finally, our results show that existing repeat proteins occupy only a small fraction of the possible repeat protein sequence and structure space and that it is possible to design novel repeat proteins with precisely specified geometries, opening up a wide array of new possibilities for biomolecular engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Computational Design of Self-Assembling Protein Nanomaterials with Atomic Level Accuracy

We describe a general computational method for designing proteins that self-assemble to a desired symmetric architecture. Protein building blocks are docked together symmetrically to identify complementary packing arrangements, and low-energy protein-protein interfaces are then designed between the building blocks in order to drive self-assembly. We used trimeric protein building blocks to design a 24-subunit, 13-nm diameter complex with octahedral symmetry and a 12-subunit, 11-nm diameter complex with tetrahedral symmetry. The designed proteins assembled to the desired oligomeric states in solution, and the crystal structures of the complexes revealed that the resulting materials closely match the design models. The method can be used to design a wide variety of self-assembling protein nanomaterials.

King, Neil P.↗
DOE OSTI2014

High thermodynamic stability of parametrically designed helical bundles

Here we describe a procedure for designing proteins with backbones produced by varying the parameters in the Crick coiled coil–generating equations. Combinatorial design calculations identify low-energy sequences for alternative helix supercoil arrangements, and the helices in the lowest-energy arrangements are connected by loop building. We design an antiparallel monomeric untwisted three-helix bundle with 80-residue helices, an antiparallel monomeric right-handed four-helix bundle, and a pentameric parallel left-handed five-helix bundle. The designed proteins are extremely stable (extrapolated ΔG fold > 60 kilocalories per mole), and their crystal structures are close to those of the design models with nearly identical core packing between the helices. The approach enables the custom design of hyperstable proteins with fine-tuned geometries for a wide range of applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Computational design of a red fluorophore ligase for site-specific protein labeling in living cells

In this study, chemical fluorophores offer tremendous size and photophysical advantages over fluorescent proteins but are much more challenging to target to specific cellular proteins. Here, we used Rosetta-based computation to design a fluorophore ligase that accepts the red dye resorufin, starting from Escherichia coli lipoic acid ligase. X-ray crystallography showed that the design closely matched the experimental structure. Resorufin ligase catalyzed the site-specific and covalent attachment of resorufin to various cellular proteins genetically fused to a 13-aa recognition peptide in multiple mammalian cell lines and in primary cultured neurons. We used resorufin ligase to perform superresolution imaging of the intermediate filament protein vimentin by stimulated emission depletion and electron microscopies. This work illustrates the power of Rosetta for major redesign of enzyme specificity and introduces a tool for minimally invasive, highly specific imaging of cellular proteins by both conventional and superresolution microscopies.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2014

Protein Scaffolding for Small Molecule Catalysts

We aim to design hybrid catalysts for energy production and storage that combine the high specificity, affinity, and tunability of proteins with the potent chemical reactivities of small organometallic molecules. The widely used Rosetta and RosettaDesign methodologies will be extended to model novel protein / small molecule catalysts in which one or many small molecule active centers are supported and coordinated by protein scaffolding. The promise of such hybrid molecular systems will be demonstrated with the nickel-phosphine hydrogenase of DuBois et. al.We will enhance the hydrogenase activity of the catalyst by designing protein scaffolds that incorporate proton relays and systematically modulate the local environment of the catalyticcenter. In collaboration with DuBois and Shaw, the designs will be experimentally synthesized and characterized.

25 ENERGY STORAGE↗
NASA NTRS2006

Hypergol Systems: Design, Buildup, and Operation

This course was developed by personnel at the NASA JSC White Sands Test Facility in conjunction with the NASA Safety Training Center (NSTC). The NSTC was established in May 1991 by the NASA Headquarters Safety Directorate to provide up-to-date, high-quality, NASA specific safety training on location at NASA centers, or simultaneously to multiple centers over the Video Teleconferencing System (ViTS). Our desire is to establish and maintain a strong, long-lasting relationship with all NASA centers in order to fulfill your safety training needs on a cost-effective basis. Our ultimate goal is to provide a positive contribution to safe operations at NASA. NSTC Course 055 is a 2-day course discussing the safe usage of hypergols (hydrazine fuels and nitrogen tetroxide). During the course we will identify the hazards associated with hypergols including toxicity, reactivity, fire, and explosion. Management of risk is discussed in terms of the primary engineering controls design, buildup, and operation; and secondary controls personal protective equipment and detectors/monitors. The emphasis is on the design and buildup of compatible systems and the safe operation of these systems by technicians and engineers.

Baker, David↗
NASA NTRS2004

Fuel Oxidizer Reaction Products (FORP) Contamination of Service Module (SM) and Release of N-nitrosodimethylamine(NDMA)in a Humid Environment from Crew EVA Suits Contaminated with FORP

The Service Module (SM) is an element of the Russian Segment of the International Space Station (ISS). One of the functions of the SM is to provide attitude control for the ISS using thrusters when the U.S. Control Moment Gyros (CMG's) must be desaturated. Prior to an Extravehicular Activity (EVA) on the Russian Segment, the Docking Compartment (DC1) is depressurized, as it is used as an airlock. When the DC1 is depressurized, the CMG's margin of momentum is insufficient and the SM attitude control thrusters need to fire to desaturate the CMG's. SM roll thruster firings induce contamination onto adjacent surfaces with Fuel Oxidizer Reaction Products (FORP). FORP is composed of both volatile and non-volatile components. One of the components of FORP is the potent carcinogen N-nitrosdimethylamine (NDMA). Since the EVA crewmembers often enter the area surrounding the thrusters for tasks on the aft end of the SM and when translating to other areas of the Russian Segment, the presence of FORP is a concern. This paper will discuss FORP contamination of the SM surfaces, the release of NDMA in a humid environment from crew EVA suits, if they happen to be contaminated with FORP, and the toxicological risk associated with the NDMA release.

Schmidl, William↗