Search NASA⌕ Search

SEARCH · Search NASA

Results for “globular proteins”

Search indexed NASA NTRS and DOE OSTI research on propulsion, heat transfer, battery materials and energy systems. Follow report and document links to the original sources.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 19 records
All sourcesNASA NTRSDOE OSTI
NASA NTRS1996

Nucleation and Crystallization of Globular Proteins: What we Know and What is Missing

Recently. much progress has been made in understanding the nucleation and crystallization of globular proteins, including the formation of compositional and structural crystal defects, Insight into the interactions of (screened) protein macro-ions in solution, obtained from light scattering, small angle X-ray scattering and osmotic pressure studies. can guide the search for crystallization conditions. These studies show that the nucleation of globular proteins is governed by the same principles as that of small molecules. However, failure to account for direct and indirect (hydrodynamic) protein interactions in the solutions results in unrealistic aggregation scenarios. Microscopic studies of numerous proteins reveal that crystals grow by the attachment of growth units through the same layer-spreading mechanisms as inorganic crystals. Investigations of the growth kinetics of hen-egg-white lysozyme (HEWL) reveal non-steady behavior under steady external conditions. Long-term variations in growth rates are due to changes in step-originating dislocation groups. Fluctuations on a shorter timescale reflect the non-linear dynamics of layer growth that results from the interplay between interfacial kinetics and bulk transport. Systematic gel electrophoretic analyses suggest that most HEWL crystallization studies have been performed with material containing other proteins at percent levels. Yet, sub-percent levels of protein impurities impede growth step propagation and play a role in the formation of structural/compositional inhomogeneities. In crystal growth from highly purified HEWL solutions, however, such inhomogeneities are much weaker and form only in response to unusually large changes in growth conditions. Equally important for connecting growth conditions to crystal perfection and diffraction resolution are recent advances in structural characterization through high-resolution Bragg reflection profiling and X-ray topography.

Rosenberger, F.↗
NASA NTRS1983

Information contained in protein shapes

The sequence of local conformations at C-alpha atoms of a protein has been considered as an informational message string. The total self-information contents and self-information per letter have been evaluated for 83 globular proteins whose structures are known from X-ray crystallography. The derived information contents provide a method of quantitating structural specificity of proteins. This method of analysis enables repeating, intricate structural features to be recognized. Among the globular proteins whose structures have been solved, high potential iron protein stands out with the largest three-letter dependence.

Sundaram, K.↗
DOE OSTI2020

Real-time tracking of protein unfolding with time-resolved x-ray solution scattering

The correct folding of proteins is of paramount importance for their function, and protein misfolding is believed to be the primary cause of a wide range of diseases. Protein folding has been investigated with time-averaged methods and time-resolved spectroscopy, but observing the structural dynamics of the unfolding process in real-time is challenging. Here, we demonstrate an approach to directly reveal the structural changes in the unfolding reaction. We use nano- to millisecond time-resolved x-ray solution scattering to probe the unfolding of apomyoglobin. The unfolding reaction was triggered using a temperature jump, which was induced by a nanosecond laser pulse. We demonstrate a new strategy to interpret time-resolved x-ray solution scattering data, which evaluates ensembles of structures obtained from molecular dynamics simulations. We find that apomyoglobin passes three states when unfolding, which we characterize as native, molten globule, and unfolded. The molten globule dominates the population under the conditions investigated herein, whereas native and unfolded structures primarily contribute before the laser jump and 30 μs after it, respectively. The molten globule retains much of the native structure but shows a dynamic pattern of inter-residue contacts. Our study demonstrates a new strategy to directly observe structural changes over the cause of the unfolding reaction, providing time- and spatially resolved atomic details of the folding mechanism of globular proteins.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS1997

Liquid-Liquid Phase Separation in Supersaturated Lysozyme Solutions and Associated Precipitate Formation/Crystallization

Using cloud point determinations, the phase boundaries (binodals) for metastable liquid-liquid (L-L) separation in supersaturated hen egg white lysozyme solutions with 3%, 5%, and 7% (wlv) NaCl at pH= 4.5 and protein concentrations c between 40 and 400 mg/ml were determined. The critical temperature for the binodal increased approximately linearly with salt concentration. The coexisting liquid phases both remained supersaturated but differed widely in protein concentration. No salt repartitioning was observed between the initial and the two separated liquid phases. After the L-L separation, due to the presence of the high protein concentration phase, crystallization occurred much more rapidly than in the initial solution. At high initial protein concentrations, a metastable gel phase formed at temperatures above the liquid binodal. Both crystal nucleation and gel formation were accelerated in samples that had been cycled through the binodal. Solutions in the gel and L-L regions yielded various types of precipitates. Based on theoretical considerations, previous observations with other proteins, and our experimental results with lysozyme, a generic phase diagram for globular proteins is put forth. A limited region in the (T,c) plane favorable for the growth of protein single crystals is delineated.

Muschol, Martin↗
DOE OSTI2025

Intracellular Biomacromolecule Delivery by Stimuli-Responsive Protein Vesicles Loaded by Hydrophobic Ion Pairing

Proteins can perform ideal therapeutic functions. However, their large size and significant surface hydrophilicity and charge prohibit them from reaching intracellular targets. These chemical features also render them poorly encapsulated by nanoparticles used for intracellular delivery. In this work, a novel combination of protein vesicles and hydrophobic ion pairing (HIP) was used to load protein cargo and achieve cytosolic delivery to overcome the limitations of previous protein vesicle properties. Protein vesicles are thermally self-assembling nanoparticles made from elastin-like polypeptide (ELP) fused to an arginine-rich leucine zipper and a globular protein fused to a glutamate-rich leucine zipper. To impart stimuli-responsive disassembly, physiological stability, and small size, the ELP sequence was modified to include histidine and tyrosine residues. HIP was used to load and release protein cargo requiring endosomal escape for cytosolic function. HIP vesicles enabled delivery of cytochrome c, a cytosolically active protein, and a significant reduction in viability in both a traditional two-dimensional (2D) human cancer cell line culture and a biomimetic three-dimensional (3D) organoid model of acute myeloid leukemia. By examining the uptake of positively and negatively charged fluorescent protein cargos loaded by HIP, this work revealed the necessity of HIP for cytosolic cargo delivery and how HIP loading influences protein vesicle self-assembly and disassembly using microscopy, small-angle X-ray scattering, and nanoparticle tracking analysis. HIP protein vesicles have the potential to broaden the use of intracellular proteins as therapeutics for various diseases and extend protein vesicles to deliver other biomacromolecules, as the strategy developed here resulted in the first cytosolic protein cargo delivery using protein vesicles.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Temperature and salt controlled tuning of protein clusters

The formation of molecular assemblies in protein solutions is of strong interest both from a fundamental viewpoint and for biomedical applications. While ordered and desired protein assemblies are indispensable for some biological functions, undesired protein condensation can induce serious diseases. As a common cofactor, the presence of salt ions is essential for some biological processes involving proteins, and in aqueous suspensions of proteins can also give rise to complex phase diagrams including homogeneous solutions, large aggregates, and dissolution regimes. Here, we systematically study the cluster formation approaching the phase separation in aqueous solutions of the globular protein BSA as a function of temperature (T), the protein concentration (c p ) and the concentrations of the trivalent salts YCl 3 and LaCl 3 (c s ). As an important complement to structural, i.e. time-averaged, techniques we employ a dynamical technique that can detect clusters even when they are transient on the order of a few nanoseconds. By employing incoherent neutron spectroscopy, we unambiguously determine the short-time self-diffusion of the protein clusters depending on c p , c s and T. We determine the cluster size in terms of effective hydrodynamic radii as manifested by the cluster center-of-mass diffusion coefficients D. For both salts, we find a simple functional form D(c p , c s , T) in the parameter range explored. The calculated inter-particle attraction strength, determined from the microscopic and short-time diffusive properties of the samples, increases with salt concentration and temperature in the regime investigated and can be linked to the macroscopic behavior of the samples.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Membrane interactions of the globular domain and the hypervariable region of KRAS4b define its unique diffusion behavior

The RAS proteins are GTP-dependent switches that regulate signaling pathways and are frequently mutated in cancer. RAS proteins concentrate in the plasma membrane via lipid-tethers and hypervariable region side-chain interactions in distinct nano-domains. However, little is known about RAS membrane dynamics and the details of RAS activation of downstream signaling. Here, we characterize RAS in live human and mouse cells using single-molecule-tracking methods and estimate RAS mobility parameters. KRAS4b exhibits confined mobility with three diffusive states distinct from the other RAS isoforms (KRAS4a, NRAS, and HRAS); and although most of the amino acid differences between RAS isoforms lie within the hypervariable region, the additional confinement of KRAS4b is largely determined by the protein’s globular domain. To understand the altered mobility of an oncogenic KRAS4b, we used complementary experimental and molecular dynamics simulation approaches to reveal a detailed mechanism.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Protein Vesicles with pH-Responsive Disassembly

We report protein biomaterials offer several advantages over those made from other components because their amino acid sequence can be precisely controlled with genetic engineering to produce a diverse set of material building blocks. In this work, three different elastin-like polypeptide (ELP) sequences were designed to synthesize pH-responsive protein vesicles. ELPs undergo a thermally induced hydrophobic transition that enables self-assembly of different kinds of protein biomaterials. The transition can be tuned by the composition of the guest residue, X, within the ELP pentapeptide repeat unit, VPGXG. When the guest residue is substituted with an ionizable amino acid, such as histidine, the ELP undergoes a pH-dependent hydrophobic phase transition. We used pH-responsive ELPs with different levels of histidine substitution, in combination with leucine zippers and globular, functional proteins, to fabricate protein vesicles. We demonstrate pH-dependent self-assembly, diameter, and disassembly of the vesicles using a combination of turbidimetry, dynamic light scattering, microscopy, and small angle X-ray scattering. As the ELP transition is dependent on the sequence, the vesicle properties also depend on the histidine content in the ELP building blocks. These results demonstrate the tunability of protein vesicles endowed with pH responsiveness, which expands their potential in drug-delivery applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

OpenAWSEM with Open3SPN2: A fast, flexible, and accessible framework for large-scale coarse-grained biomolecular simulations

We present OpenAWSEM and Open3SPN2, new cross-compatible implementations of coarse-grained models for protein (AWSEM) and DNA (3SPN2) molecular dynamics simulations within the OpenMM framework. These new implementations retain the chemical accuracy and intrinsic efficiency of the original models while adding GPU acceleration and the ease of forcefield modification provided by OpenMM’s Custom Forces software framework. By utilizing GPUs, we achieve around a 30-fold speedup in protein and protein-DNA simulations over the existing LAMMPS-based implementations running on a single CPU core. We showcase the benefits of OpenMM’s Custom Forces framework by devising and implementing two new potentials that allow us to address important aspects of protein folding and structure prediction and by testing the ability of the combined OpenAWSEM and Open3SPN2 to model protein-DNA binding. The first potential is used to describe the changes in effective interactions that occur as a protein becomes partially buried in a membrane. We also introduced an interaction to describe proteins with multiple disulfide bonds. Using simple pairwise disulfide bonding terms results in unphysical clustering of cysteine residues, posing a problem when simulating the folding of proteins with many cysteines. We now can computationally reproduce Anfinsen’s early Nobel prize winning experiments by using OpenMM’s Custom Forces framework to introduce a multi-body disulfide bonding term that prevents unphysical clustering. Our protein-DNA simulations show that the binding landscape is funneled towards structures that are quite similar to those found using experiments. In summary, this paper provides a simulation tool for the molecular biophysics community that is both easy to use and sufficiently efficient to simulate large proteins and large protein-DNA systems that are central to many cellular processes. These codes should facilitate the interplay between molecular simulations and cellular studies, which have been hampered by the large mismatch between the time and length scales accessible to molecular simulations and those relevant to cell biology.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

A modular vaccine platform enabled by decoration of bacterial outer membrane vesicles with biotinylated antigens

Engineered outer membrane vesicles (OMVs) derived from Gram-negative bacteria are a promising technology for the creation of non-infectious, nanoparticle vaccines against diverse pathogens. However, antigen display on OMVs can be difficult to control and highly variable due to bottlenecks in protein expression and localization to the outer membrane of the host cell, especially for bulky and/or complex antigens. Here, we describe a universal approach for avidin-based vaccine antigen crosslinking (AvidVax) whereby biotinylated antigens are linked to the exterior of OMVs whose surfaces are remodeled with multiple copies of a synthetic antigen-binding protein (SNAP) comprised of an outer membrane scaffold protein fused to a biotin-binding protein. We show that SNAP-OMVs can be readily decorated with a molecularly diverse array of biotinylated subunit antigens, including globular and membrane proteins, glycans and glycoconjugates, haptens, lipids, and short peptides. When the resulting OMV formulations are injected in mice, strong antigen-specific antibody responses are observed that depend on the physical coupling between the antigen and SNAP-OMV delivery vehicle. Overall, these results demonstrate AvidVax as a modular platform that enables rapid and simplified assembly of antigen-studded OMVs for application as vaccines against pathogenic threats.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Intra- and inter-molecular regulation by intrinsically-disordered regions governs PUF protein RNA binding

PUF proteins are characterized by globular RNA-binding domains. They also interact with partner proteins that modulate their RNA-binding activities. Caenorhabditis elegans PUF protein fem-3 binding factor-2 (FBF-2) partners with intrinsically disordered Lateral Signaling Target-1 (LST-1) to regulate target mRNAs in germline stem cells. Here, we report that an intrinsically disordered region (IDR) at the C-terminus of FBF-2 autoinhibits its RNA-binding affinity by increasing the off rate for RNA binding. Moreover, the FBF-2 C-terminal region interacts with its globular RNA-binding domain at the same site where LST-1 binds. This intramolecular interaction restrains an electronegative cluster of amino acid residues near the 5' end of the bound RNA to inhibit RNA binding. LST-1 binding in place of the FBF-2 C-terminus therefore releases autoinhibition and increases RNA-binding affinity. This regulatory mechanism, driven by IDRs, provides a biochemical and biophysical explanation for the interdependence of FBF-2 and LST-1 in germline stem cell self-renewal.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Structure and dynamics of hydrodynamically interacting finite-size Brownian particles in a spherical cavity: Spheres and cylinders

The structure and dynamics of confined suspensions of particles of arbitrary shape are of interest in multiple disciplines from biology to engineering. Theoretical studies are often limited by the complexity of long-range particle-particle and particle-wall forces, including many-body fluctuating hydrodynamic interactions. Here, we report a computational study on the diffusion of spherical and cylindrical particles confined in a spherical cavity. We rely on an immersed-boundary general geometry Ewald-like method to capture lubrication and long-range hydrodynamics and include appropriate non-slip conditions at the confining walls. A Chebyshev polynomial approximation is used to satisfy the fluctuation-dissipation theorem for the Brownian suspension. We explore how lubrication, long-range hydrodynamics, particle volume fraction, and shape affect the equilibrium structure and the diffusion of the particles. It is found that once the particle volume fraction is greater than 10%, the particles start to form layered aggregates that greatly influence particle dynamics. Hydrodynamic interactions strongly influence the particle diffusion by inducing spatially dependent short-time diffusion coefficients, stronger wall effects on the particle diffusion toward the walls, and a sub-diffusive regime-caused by crowding-in the long-time particle mobility. The level of asymmetry of the cylindrical particles considered here is enough to induce an orientational order in the layered structure, decreasing the diffusion rate and facilitating a transition to the crowded mobility regime at low particle concentrations. Our results offer fundamental insights into the diffusion and distribution of globular and fibrillar proteins inside cells.

36 MATERIALS SCIENCE↗
DOE OSTI2025

Hydrogel-Immobilized Multienzyme Systems for Cell-Free Chemical Bioproduction

Cell-free gene expression systems derived from bacterial lysates enable the expression of biosynthetic pathways from inexpensive and easily prepared DNA templates. These systems hold great promise for modular and on-demand bioproduction of valuable small molecules in resource-limited settings but are constrained in their long-term stability, reusability, and deployability. In this work, we demonstrate that multiple cell-free expressed enzymes can be co-immobilized in biocompatible hydrogels made from poly(ethylene glycol) diacrylate (PEGDA) with added glycerol for enhanced gel integrity. Using small-angle X-ray scattering (SAXS), we show that the mesh size of PEGDA-glycerol hydrogels is comparable to the globular sizes of many proteins and enzymes, which could be used for protein entrapment. We found that the combination between entrapment and chemical ligation of the enzymes was effective to retain proteins. By employing a method for direct fluorescence measurement from hydrogels, we found that proteins can be retained in PEGDA-glycerol for at least a week. By separating the cell-free enzyme expression from the immobilization step, we successfully fabricated enzyme-laden hydrogels with three heterologous cell-free enzymes for the bioconversion of pyruvic acid to malic acid, an industrially valuable and versatile precursor chemical. Both heterologous and endogenous enzymes from the lysate remain functional in photo-cross-linked hydrogels and can be reused for multiple biocatalytic cycles. Moreover, we also found that the immobilized enzymes exhibit up to 1.6-fold higher activity and 2-fold longer lifetimes than free enzymes in liquid reactions. Furthermore, these results could advance the deployment of cell-free synthetic biology because they show that reusable, stable, and durable multienzyme systems can be created using readily available materials and fabrication techniques.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Miniaturized Hybrid Materials Inspired by Nature (Final Technical Report)

The objective of our research program is to develop a fundamental scientific understanding of assembly in biomolecular materials mediated by mechanisms that mimic complex events in the cellular environment. The building blocks that we study include globular, disordered, and filamentous proteins, lipids, and nucleic acids. Notably, the nanoscale assemblies may either be similar to, or highly distinct from, those occurring in vivo, owing to the complex nature of the systems. Novel assemblies may further result due to controlled shape changes in building blocks.

36 MATERIALS SCIENCE↗
NASA NTRS2022

Resolving the fine structure in the energy landscapes of repeat proteins

Ankyrin (ANK) repeat proteins are coded by tandem occurrences of patterns with around 33 amino acids. They often mediate protein–protein interactions in a diversity of biological systems. These proteins have an elongated non-globular shape and often display complex folding mechanisms. This work investigates the energy landscape of representative proteins of this class made up of 3, 4 and 6 ANK repeats using the energy-landscape visualisation method (ELViM). By combining biased and unbiased coarse-grained molecular dynamics AWSEM simulations that sample conformations along the folding trajectories with the ELViM structure-based phase space, one finds a three-dimensional representation of the globally funnelled energy surface. In this representation, it is possible to delineate distinct folding pathways. We show that ELViMs can project, in a natural way, the intricacies of the highly dimensional energy landscapes encoded by the highly symmetric ankyrin repeat proteins into useful low-dimensional representations. These projections can discriminate between multiplicities of specific parallel folding mechanisms that otherwise can be hidden in oversimplified depictions.

Murilo N. Sanches↗
NASA NTRS1999

Left-handed Z-DNA: structure and function

Z-DNA is a high energy conformer of B-DNA that forms in vivo during transcription as a result of torsional strain generated by a moving polymerase. An understanding of the biological role of Z-DNA has advanced with the discovery that the RNA editing enzyme double-stranded RNA adenosine deaminase type I (ADAR1) has motifs specific for the Z-DNA conformation. Editing by ADAR1 requires a double-stranded RNA substrate. In the cases known, the substrate is formed by folding an intron back onto the exon that is targeted for modification. The use of introns to direct processing of exons requires that editing occurs before splicing. Recognition of Z-DNA by ADAR1 may allow editing of nascent transcripts to be initiated immediately after transcription, ensuring that editing and splicing are performed in the correct sequence. Structural characterization of the Z-DNA binding domain indicates that it belongs to the winged helix-turn-helix class of proteins and is similar to the globular domain of histone-H5.

Review↗