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All sourcesNASA NTRSDOE OSTI
DOE OSTI2024

African Swine Fever Virus Protein–Protein Interaction Prediction

The African swine fever virus (ASFV) is an often deadly disease in swine and poses a threat to swine livestock and swine producers. With its complex genome containing more than 150 coding regions, developing effective vaccines for this virus remains a challenge due to a lack of basic knowledge about viral protein function and protein–protein interactions between viral proteins and between viral and host proteins. In this work, we identified ASFV-ASFV protein–protein interactions (PPIs) using artificial intelligence-powered protein structure prediction tools. We benchmarked our PPI identification workflow on the Vaccinia virus, a widely studied nucleocytoplasmic large DNA virus, and found that it could identify gold-standard PPIs that have been validated in vitro in a genome-wide computational screening. We applied this workflow to more than 18,000 pairwise combinations of ASFV proteins and were able to identify seventeen novel PPIs, many of which have corroborating experimental or bioinformatic evidence for their protein–protein interactions, further validating their relevance. Two protein–protein interactions, I267L and I8L, I267L__I8L, and B175L and DP79L, B175L__DP79L, are novel PPIs involving viral proteins known to modulate host immune response.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Sulfated glycopeptide nanostructures for multipotent protein activation

Biological systems have evolved to utilize numerous proteins with capacity to bind polysaccharides for the purpose of optimizing their function. A well-known subset of these proteins with binding domains for the highly diverse sulfated polysaccharides are important growth factors involved in biological development and tissue repair. Here, we report on supramolecular sulfated glycopeptide nanostructures, which display a trisulfated monosaccharide on their surfaces and bind five critical proteins with very different polysaccharide binding domains. Binding does not disrupt the filamentous shape of the nanostructures or their internal β-sheet backbone, but must involve accessible adaptive configurations to interact with such different proteins. The glycopeptide nanostructures amplified signaling of bone morphogenetic protein 2 significantly more than the natural sulfated polysaccharide heparin, and promoted regeneration of bone in the spine with a protein dose that is 100-fold lower than expected. These super-bioactive nanostructures may enable many therapies in the horizon involving proteins.

36 MATERIALS SCIENCE↗
DOE OSTI2019

Structures of single-layer {beta}-sheet proteins evolved from {beta}-hairpin repeats

Free-standing single-layer beta-sheets are extremely rare in naturally occurring proteins, even though beta-sheet motifs are ubiquitous. Here we report the crystal structures of three homologous, single-layer, anti-parallel beta-sheet proteins, comprised of three or four twisted beta-hairpin repeats. The structures reveal that, in addition to the hydrogen bond network characteristic of beta-sheets, additional hydrophobic interactions mediated by small clusters of residues adjacent to the turns likely play a significant role in the structural stability and compensate for the lack of a compact hydrophobic core. These structures enabled identification of a family of secreted proteins that are broadly distributed in bacteria from the human gut microbiome and are putatively involved in the metabolism of complex carbohydrates. A conserved surface patch, rich in solvent-exposed tyrosine residues, was identified on the concave surface of the beta-sheet. These new modular single-layer beta-sheet proteins may serve as a new model system for studying folding and design of beta-rich proteins.

beta-hairpin repeats↗
DOE OSTI2018

Application of multiplexed ion mobility spectrometry towards the identification of host protein signatures of treatment effect in pulmonary tuberculosis

Rationale: The monitoring of TB treatments in clinical practice and clinical trials relies on traditional sputum-based culture status indicators at specific time points. Accurate, predictive, blood-based protein markers would provide a simpler and more informative view of patient health and response to treatment. Objective: We utilized sensitive, high throughput multiplexed ion mobility-mass spectrometry (IM-MS) to characterize the serum proteome of TB patients at the start of and at 8 weeks of rifamycin-based treatment. We sought to identify treatment specific signatures within patients as well as correlate the proteome signatures to various clinical markers of treatment efficacy. Methods: Serum samples were collected from 289 subjects enrolled in CDC TB Trials Consortium Study 29 at time of enrollment and at the end of the intensive phase (after 40 doses of TB treatment). Serum proteins were immunoafinity-depleted of high abundant components, digested to peptides and analyzed for data acquisition utilizing a unique liquid chromatography IM-MS platform (LC-IM-MS). Linear mixed models were utilized to identify serum protein changes in the host response to antibiotic treatment as well as correlations with culture status end points. Results: A total of 10,137 peptides corresponding to 872 proteins were identified, quantified, and used for statistical analysis across the longitudinal patient cohort. In response to TB treatment, 244 proteins were sig- ni?cantly altered. Pathway/network comparisons helped visualize the interconnected proteins, identifying up regulated (lipid transport, coagulation cascade, endopeptidase activity) and down regulated (acute phase) processes and pathways in addition to other cross regulated networks (inflammation, cell adhesion, extracellular matrix). Detection of possible lung injury serum proteins such as HPSE, significantly downregulated upon treatment. Analyses of microbiologic data over time identified a core set of serum proteins (TTHY, AFAM, CRP, RET4, SAA1, PGRP2) which change in response to treatment and also strongly correlate with culture status. A similar set of proteins at baseline were found to be predictive of week 6 and 8 culture status. Conclusion: A comprehensive host serum protein dataset reflective of TB treatment effect is defined. A repeating set of serum proteins (TTHY, AFAM, CRP, RET4, SAA1, PGRP2, among others) were found to change significantly in response to treatment, to strongly correlate with culture status, and at baseline to be predictive of future culture conversion. If validated in cohorts with long term follow-up to capture failure and relapse of TB, these protein markers could be developed for monitoring of treatment in clinical trials and in patient care.

Kedia, Komal↗
DOE OSTI2017

Structural Characterization of Native Proteins and Protein Complexes by Electron Ionization Dissociation-Mass Spectrometry

Mass spectrometry (MS) has played an increasingly important role in the identification and structural and functional characterization of proteins. In particular, the use of tandem mass spectrometry has afforded one of the most versatile methods to acquire structural information for proteins and protein complexes. The unique nature of electron capture dissociation (ECD) for cleaving protein backbone bonds while preserving non-covalent interactions has made it especially suitable for the study of native protein structures. However, the intra- and inter-molecular interactions stabilized by hydrogen bonds and salt bridges can hinder the separation of fragments even with pre-activation, which has become particularly problematic for the study of large macromolecular proteins and protein complexes. Here, we describe the capabilities of another activation method, 30 eV electron ionization dissociation (EID), for the top-down MS characterization of native protein-ligand and protein-protein complexes. Rich structural information that cannot be delivered by ECD can be generated by EID. EID allowed for the comparison of the gas-phase and the solution-phase structural stability and unfolding process of human carbonic anhydrase I (HCA-I). In addition, the EID fragmentation patterns reflect the structural similarities and differences among apo-, Zn-, and Cu,Zn-superoxide dismutase (SOD1) dimers. In particular, the structural changes due to Cu-binding and a point mutation (G41D) were revealed by EID-MS. The performance of EID was also compared to that of 193 nm ultraviolet photodissociation (UVPD), which allowed us to explore their qualitative similarities and differences as potential valuable tools for the MS study of native proteins and protein complexes.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2019

Predicting Protein–Polymer Block Copolymer Self-Assembly from Protein Properties

Protein–polymer bioconjugate self-assembly has attracted a great deal of attention as a method to fabricate protein nanomaterials in solution and the solid state. To identify protein properties that affect phase behavior in protein–polymer block copolymers, a library of 15 unique protein- b -poly(N-isopropylacrylamide) (PNIPAM) copolymers comprising 11 different proteins was compiled and analyzed. Many attributes of phase behavior are found to be similar among all studied bioconjugates regardless of protein properties, such as formation of micellar phases at high temperature and low concentration, lamellar ordering with increasing temperature, and disordering at high concentration, but several key protein-dependent trends are also observed. In particular, hexagonal phases are only observed for proteins within the molar mass range 20–36 kDa, where ordering quality is also significantly enhanced. While ordering is generally found to improve with increasing molecular weight outside of this range, most large bioconjugates exhibited weaker than predicted assembly, which is attributed to chain entanglement with increasing polymer molecular weight. Additionally, order–disorder transition boundaries are found to be largely uncorrelated to protein size and quality of ordering. However, the primary finding is that bioconjugate ordering can be accurately predicted using only protein molecular weight and percentage of residues contained within β sheets. This model provides a basis for designing protein–PNIPAM bioconjugates that exhibit well-defined self-assembly and a modeling framework that can generalize to other bioconjugate chemistries.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Serial femtosecond crystallography of soluble proteins in lipidic cubic phase

Serial femtosecond crystallography (SFX) at X-ray free-electron lasers (XFELs) enables high-resolution protein structure determination using micrometre-sized crystals at room temperature with minimal effects from radiation damage. SFX requires a steady supply of microcrystals intersecting the XFEL beam at random orientations. An LCP–SFX method has recently been introduced in which microcrystals of membrane proteins are grown and delivered for SFX data collection inside a gel-like membrane-mimetic matrix, known as lipidic cubic phase (LCP), using a special LCP microextrusion injector. Here, it is demonstrated that LCP can also be used as a suitable carrier medium for microcrystals of soluble proteins, enabling a dramatic reduction in the amount of crystallized protein required for data collection compared with crystals delivered by liquid injectors. High-quality LCP–SFX data sets were collected for two soluble proteins, lysozyme and phycocyanin, using less than 0.1 mg of each protein.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2015

Protein structure prediction guided by crosslinking restraints – A systematic evaluation of the impact of the crosslinking spacer length

Recent development of high-resolution mass spectrometry (MS) instruments enables chemical crosslinking (XL) to become a high-throughput method for obtaining structural information about proteins. Restraints derived from XL-MS experiments have been used successfully for structure refinement and protein–protein docking. However, one formidable question is under which circumstances XL-MS data might be sufficient to determine a protein’s tertiary structure de novo? Answering this question will not only include understanding the impact of XL-MS data on sampling and scoring within a de novo protein structure prediction algorithm, it must also determine an optimal crosslinker type and length for protein structure determination. While a longer crosslinker will yield more restraints, the value of each restraint for protein structure prediction decreases as the restraint is consistent with a larger conformational space. In this study, the number of crosslinks and their discriminative power was systematically analyzed in silico on a set of 2055 non-redundant protein folds considering Lys–Lys, Lys–Asp, Lys–Glu, Cys–Cys, and Arg–Arg reactive crosslinkers between 1 and 60 Å. Depending on the protein size a heuristic was developed that determines the optimal crosslinker length. Next, simulated restraints of variable length were used to de novo predict the tertiary structure of fifteen proteins using the BCL::Fold algorithm. The results demonstrate that a distinct crosslinker length exists for which information content for de novo protein structure prediction is maximized. The sampling accuracy improves on average by 1.0 Å and up to 2.2 Å in the most prominent example. XL-MS restraints enable consistently an improved selection of native-like models with an average enrichment of 2.1.

Biochemistry & Molecular Biology↗
DOE OSTI2017

Coupling neutron reflectivity with cell-free protein synthesis to probe membrane protein structure in supported bilayers

Here, the structure of the p7 viroporin, an oligomeric membrane protein ion channel involved in the assembly and release of the hepatitis C virus, was determined from proteins expressed and inserted directly into supported model lipid membranes using cell-free protein expression. Cell-free protein expression allowed (i) high protein concentration in the membrane, (ii) control of the protein’s isotopic constitution, and (iii) control over the lipid environment available to the protein. Here, we used cell-free protein synthesis to directly incorporate the hepatitis C virus (HCV) p7 protein into supported lipid bilayers formed from physiologically relevant lipids (POPC or asolectin) for both direct structural measurements using neutron reflectivity (NR) and conductance measurements using electrical impedance spectroscopy (EIS). We report that HCV p7 from genotype 1a strain H77 adopts a conical shape within lipid bilayers and forms a viroporin upon oligomerization, confirmed by EIS conductance measurements. This combination of techniques represents a novel approach to the study of membrane proteins and, through the use of selective deuteration of particular amino acids to enhance neutron scattering contrast, has the promise to become a powerful tool for characterizing the protein conformation in physiologically relevant environments and for the development of biosensor applications.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2016

Comparative Community Proteomics Demonstrates the Unexpected Importance of Actinobacterial Glycoside Hydrolase Family 12 Protein for Crystalline Cellulose Hydrolysis

Glycoside hydrolases (GHs) are key enzymes in the depolymerization of plant-derived cellulose, a process central to the global carbon cycle and the conversion of plant biomass to fuels and chemicals. A limited number of GH families hydrolyze crystalline cellulose, often by a processive mechanism along the cellulose chain. During cultivation of thermophilic cellulolytic microbial communities, substantial differences were observed in the crystalline cellulose saccharification activities of supernatants recovered from divergent lineages. Comparative community proteomics identified a set of cellulases from a population closely related to actinobacterium Thermobispora bispora that were highly abundant in the most active consortium. Among the cellulases from T. bispora , the abundance of a GH family 12 (GH12) protein correlated most closely with the changes in crystalline cellulose hydrolysis activity. This result was surprising since GH12 proteins have been predominantly characterized as enzymes active on soluble polysaccharide substrates. Heterologous expression and biochemical characterization of the suite of T. bispora hydrolytic cellulases confirmed that the GH12 protein possessed the highest activity on multiple crystalline cellulose substrates and demonstrated that it hydrolyzes cellulose chains by a predominantly random mechanism. This work suggests that the role of GH12 proteins in crystalline cellulose hydrolysis by cellulolytic microbes should be reconsidered.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2018

Fourier Transform-Ion Cyclotron Resonance Mass Spectrometry as a Platform for Characterizing Multimeric Membrane Protein Complexes

Membrane protein characterization is consistently hampered by challenges with expression, purification, and solubilization. Among several biophysical techniques employed for their characterization, native-mass spectrometry (MS) has emerged as a powerful tool for the analysis of membrane proteins and complexes. Here, two MS platforms, the FT-ICR and Q-ToF, have been explored to analyze the homotetrameric water channel protein, AquaporinZ (AqpZ), under non-denaturing conditions. This 97 kDa membrane protein complex can be readily liberated from the octylglucoside (OG) detergent micelle under a range of instrument conditions on both MS platforms. Increasing the applied collision energy of the FT-ICR collision cell yielded varying degrees of tetramer (97 kDa) liberation from the OG micelles, as well as dissociation into the trimeric (72 kDa) and monomeric (24 kDa) substituents. Tandem-MS on the Q-ToF yielded higher intensity tetramer signal and, depending on the m/z region selected, the observed monomer signal varied in intensity. Precursor ion selection of an m/z range above the expected protein signal distribution, followed by mild collisional activation, is able to efficiently liberate AqpZ with a high S/N ratio. The tetrameric charge state distribution obtained on both instruments demonstrated superpositioning of multiple proteoforms due to varying degrees of N-terminal formylation. .

46 INSTRUMENTATION RELATED TO NUCLEAR SCIENCE AND ↗
DOE OSTI2018

Mechanism of the G-protein mimetic nanobody binding to a muscarinic G-protein-coupled receptor

Protein–protein binding is key in cellular signaling processes. Molecular dynamics (MD) simulations of protein–protein binding, however, are challenging due to limited timescales. In particular, binding of the medically important G-protein-coupled receptors (GPCRs) with intracellular signaling proteins has not been simulated with MD to date. Here, we report a successful simulation of the binding of a G-protein mimetic nanobody to the M 2 muscarinic GPCR using the robust Gaussian accelerated MD (GaMD) method. Through long-timescale GaMD simulations over 4,500 ns, the nanobody was observed to bind the receptor intracellular G-protein-coupling site, with a minimum rmsd of 2.48 Å in the nanobody core domain compared with the X-ray structure. Binding of the nanobody allosterically closed the orthosteric ligand-binding pocket, being consistent with the recent experimental finding. In the absence of nanobody binding, the receptor orthosteric pocket sampled open and fully open conformations. The GaMD simulations revealed two low-energy intermediate states during nanobody binding to the M 2 receptor. The flexible receptor intracellular loops contribute remarkable electrostatic, polar, and hydrophobic residue interactions in recognition and binding of the nanobody. As a result, these simulations provided important insights into the mechanism of GPCR–nanobody binding and demonstrated the applicability of GaMD in modeling dynamic protein–protein interactions.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2016

Structural and Functional Studies of H. seropedicae RecA Protein – Insights into the Polymerization of RecA Protein as Nucleoprotein Filament

The bacterial RecA protein plays a role in the complex system of DNA damage repair. Here, we report the functional and structural characterization of the Herbaspirillum seropedicae RecA protein (HsRecA). HsRecA protein is more efficient at displacing SSB protein from ssDNA than Escherichia coli RecA protein. HsRecA also promotes DNA strand exchange more efficiently. The three dimensional structure of HsRecA-ADP/ATP complex has been solved to 1.7 Å resolution. HsRecA protein contains a small N-terminal domain, a central core ATPase domain and a large C-terminal domain, that are similar to homologous bacterial RecA proteins. Comparative structural analysis showed that the N-terminal polymerization motif of archaeal and eukaryotic RecA family proteins are also present in bacterial RecAs. Reconstruction of electrostatic potential from the hexameric structure of HsRecA-ADP/ATP revealed a high positive charge along the inner side, where ssDNA is bound inside the filament. The properties of this surface may explain the greater capacity of HsRecA protein to bind ssDNA, forming a contiguous nucleoprotein filament, displace SSB and promote DNA exchange relative to EcRecA. In conclusion, our functional and structural analyses provide insight into the molecular mechanisms of polymerization of bacterial RecA as a helical nucleoprotein filament.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Quantitative proteomics identifies altered O-GlcNAcylation of structural, synaptic and memory-associated proteins in Alzheimer's disease: Brain protein O-GlcNAcylation in Alzheimer's disease

Protein modification by O-linked beta-N-acetylglucosamine (O-GlcNAc) is emerging as an important factor in the pathogenesis of sporadic Alzheimer’s disease. Herein we report the most comprehensive, quantitative proteomics analysis for protein O-GlcNAcylation in post-mortem human brains with and without Alzheimer’s using isobaric tandem mass tags labeling, chemoenzymatic photocleavage enrichment and liquid chromatography coupled to mass spectrometry. A total of 1,850 O-GlcNAc peptides covering 1,094 O-GlcNAcylation sites were identified from 530 proteins in the human brain. 128 O-GlcNAc peptides covering 78 proteins were altered significantly in Alzheimer’s brain as compared to controls (q<0.05). Moreover, alteration of the O-GlcNAc peptide abundance could be attributed more to O-GlcNAcylation level than to protein level changes. The altered O-GlcNAcylated proteins belong to several structural and functional categories, including synaptic proteins, cytoskeleton proteins, and memory-associated proteins. These findings suggest that dysregulation of O-GlcNAcylation of multiple brain proteins may be involved in the development of sporadic Alzheimer’s disease.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2017

Cytochrome b561, copper, β-cleaved amyloid precursor protein and niemann-pick C1 protein are involved in ascorbate-induced release and membrane penetration of heparan sulfate from endosomal S-nitrosylated glypican-1

Ascorbate-induced release of heparan sulfate from S-nitrosylated heparan sulfate proteoglycan glypican-1 takes place in endosomes. Heparan sulfate penetrates the membrane and is transported to the nucleus. This process is dependent on copper and on expression and processing of the amyloid precursor protein. It remains unclear how exogenously supplied ascorbate can generate HS-anMan in endosomes and how passage through the membrane is facilitated. Here we have examined wild-type, Alzheimer Tg2576 and amyloid precursor protein (-/-) mouse fibroblasts and human fetal and Niemann-Pick C1 fibroblasts by using deconvolution immunofluorescence microscopy, siRNA technology and [S{sup 35}]sulfate-labeling, vesicle isolation and gel chromatography. We found that ascorbate-induced release of heparan sulfate was dependent on expression of endosomal cytochrome b561. Formation and nuclear transport of heparan sulfate was suppressed by inhibition of β-processing of the amyloid precursor protein and formation was restored by copper (I) ions. Membrane penetration was not dependent on amyloid beta channel formation. Inhibition of endosomal exit resulted in accumulation of heparan sulfate in vesicles that exposed the C-terminal of the amyloid precursor protein externally. Endosome-to-nucleus transport was also dependent on expression of the Niemann-Pick C1 protein. We propose that ascorbate is taken up from the medium and is oxidized by cytochrome b561 which, in turn, reduces copper (II) to copper (I) present in the N-terminal, β-cleaved domain of the amyloid precursor protein. Re-oxidation of copper (I) is coupled to reductive, deaminative release of heparan sulfate from glypican-1. Passage through the membrane may be facilitated by the C-terminal, β-cleaved fragment of the amyloid precursor protein and the Niemann-Pick C1 protein. - Highlights: • Cytochrome b561 is a mediator of ascorbate-induced release of heparan sulfate from glypican-1 in endosomes. • Beta-cleavage of the amyloid precursor protein is required for release and membrane penetration of heparan sulfate. • The Niemann-Pick C1 protein is involved in membrane penetration.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2016

Fast Photochemical Oxidation of Proteins Maps the Topology of Intrinsic Membrane Proteins: Light-Harvesting Complex 2 in a Nanodisc

Although membrane proteins are crucial participants in photosynthesis and other biological processes, many lack high-resolution structures. Prior to achieving a high-resolution structure, we are investigating whether MS-based footprinting can provide coarse-grained protein structure by following structural changes that occur upon ligand binding, pH change, and membrane binding. Our platform probes topology and conformation of membrane proteins by combining MS-based footprinting, specifically fast photochemical oxidation of proteins (FPOP), and lipid Nanodiscs, which are more similar to the native membrane environment than are the widely used detergent micelles. We describe here results that show a protein’s outer membrane regions are more heavily footprinted by OH radicals whereas the regions spanning the lipid bilayer remain inert to the labeling. Nanodiscs generally exhibit more protection of membrane proteins compared to detergent micelles and less shielding to those protein residues that exist outside the membrane. The combination of immobilizing the protein in Nanodiscs and footprinting with FPOP is a feasible approach to map extra-membrane protein surfaces, even at the amino-acid level, and to illuminate intrinsic membrane protein topology.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗