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At least 793 records · Page 44
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

A Trichomonas vaginalis C2-XYPPX-repeat protein with a structured C2 domain displaying dampened flexibility upon binding calcium

C2 domains are ubiquitous membrane-binding modules of ∼130 residues in eukaryotes that are often associated with proteins involved in membrane trafficking and lipid modification. The genome of Trichomonas vaginalis, the most common, non-viral, sexually transmitted human pathogen, encodes eight genes that contain a N-terminal C2 module linked to a XYPPX-repeat domain of more than four XYPPX repeats (C2-XYPPX). While the function of the XYPPX-repeat domain remains unknown, its multiple association with C2 domains in T. vaginalis suggests it is important. Here, the C2 domain from one of these C2-XYPPX-repeat proteins, Tv-C2-1, was structurally and physically characterized using X-ray crystallography and NMR spectroscopy. The crystal structure for Tv-C2-1 shows that this domain shares a fold common to all C2 domains, a compact Greek-key motif composed of eight anti-parallel β-strands in the type-2 topology. An NMR chemical shift perturbation study with Ca 2+ showed that Tv-C2-1 bound two Ca 2+ atoms primarily via two loops (loop-1 and loop-3) on the predicted calcium binding face of the protein with K d s of 58.0 ± 0.1 μM and 232 ± 6 μM. Estimations of the overall rotational correlation time, τ c , in the apo (11.1 ns) and Ca 2+ -bound (9.2 ns) state suggests the protein becomes more compact upon Ca 2+ binding, consistent with a decrease in dynamics in loop-3 and marginally in loop-1 suggested by amide 15 N heteronuclear steady-state { 1 H}- 15 N NOEs. Showing Tv-C2-1 binds calcium and adopts a compact Greek-key motif structure, two primary features of C2 domains, suggests understanding the function of the XYPPX-repeat domain may be warranted.

NMR spectroscopy↗
DOE OSTI2024

Structural characterization of high-protein system through ultra-small and small-angle X-ray scattering

High-protein systems exhibit a hierarchical structure consisting of interconnected multiscale length assemblies. Recent investigations are directed toward identifying these structural units under various processing and environmental conditions to establish structure–function correlations. Ultra-small and small-angle X-ray scattering (USAXS/SAXS) has become a crucial tool for characterizing the structures of proteins and their clusters/aggregates, ranging from nanometers to micrometers, with minimal disruption to their original state. Here, this review first describes the facilities and principles of X-ray scattering, followed by discussions on the analysis of scattering data, including the interpretation of fitting models. It then delves into the main applications of USAXS/SAXS in plant and dairy protein-rich systems. Future strategies to enhance the utilization of scattering techniques for elucidating the structure of high-protein systems are also included.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2022

Deamidation of the human eye lens protein $\gamma$S-crystallin accelerates oxidative aging

Cataract, a clouding of the eye lens from protein precipitation, affects millions of people every year. The lens proteins, the crystallins, show extensive post-translational modifications (PTMs) in cataractous lenses. The most common PTMs, deamidation and oxidation, promote crystallin aggregation; however, it is not clear precisely how these PTMs contribute to crystallin insolubilization. Here, we report six crystal structures of the lens protein γS-crystallin (γS): one of the wild-type and five of deamidated γS variants, from three to nine deamidation sites, after sample aging. The deamidation mutations do not change the overall fold of γS; however, increasing deamidation leads to accelerated disulfide-bond formation. Addition of deamidated sites progressively destabilized protein structure, and the deamidated variants display an increased propensity for aggregation. Here these results suggest that the deamidated variants are useful as models for accelerated aging; the structural changes observed provide support for redox activity of γS-crystallin in the lens.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Engineered pH-Sensitive Protein G/IgG Interaction

While natural protein–protein interactions have evolved to be induced by complex stimuli, rational design of interactions that can be switched-on-demand still remain challenging in the protein design world. Here, we demonstrate that a computationally redesigned natural interface for improved binding affinity could further be mutated to adopt a pH switchable interaction. The redesigned interface of Protein G/human IgG Fc domain (referred to as PrG/hIgG), when incorporated with histidine and glutamic acid on PrG (PrG-EHHE), showed a switch in binding affinity by 50-fold when the pH was altered from mild acidic to mild basic. The wild-type (WT) interface showed a negligible switch. The overall binding affinity under mild acidic pH for PrG-EHHE outperformed the wild-type PrG (PrG-WT) interaction. Overall, we find that the new reagent PrG-EHHE can be revolutionary in IgG purification, since the standard method of using an extreme acidic pH for elution can be circumvented.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Correlating Protein Dynamics and Catalytic Activity of a Model Hydrogenase Using Paramagnetic and Biological Nuclear Magnetic Resonance Spectroscopy

Rational catalyst design remains a significant challenge, with electronic structure, steric, and electrostatic effects known to contribute to activity. Recently, dynamics has been recognized as another factor that impacts catalysis, though identifying and predicting these effects has remained out of reach. Nickel-substituted rubredoxin (NiRd), a protein-based mimic of a hydrogenase enzyme, serves as a model catalytic system in which dynamics can be systematically investigated with respect to activity. While over 30 secondary-sphere mutants of NiRd have been shown to be catalytically active, no significant correlation was observed between the rates and catalytic overpotential or electronic structure, prompting questions about the protein-derived factors that modulate activity. Here, in this work, NMR spectroscopy was used to investigate the roles of substrate accessibility, protein dynamics, and protein stability in controlling catalysis. Significant paramagnetic effects from the nickel center (S = 1) isolate the methylene proton resonances of the metal-coordinating cysteine residues. The sensitivity of resonance positions and linewidths to local environment offers an opportunity to study dynamical molecular changes around the metal center with high resolution. Machine learning algorithms were employed to identify correlations between the catalytic activity and the paramagnetic NMR spectra. These analyses revealed spectroscopic features of specific cysteine protons that report on catalytic overpotential and increased turnover rates, which are further supported by the results obtained using high-field NMR techniques. Collectively, these studies indicate the potential for multifrequency NMR techniques to resolve key contributors to catalytic activity and highlight the importance of local and outer-sphere dynamics.

Protein Engineering↗
DOE OSTI2020

Comparative differential cuproproteomes of Rhodobacter capsulatus reveal novel copper homeostasis related proteins

Abstract Copper (Cu) is an essential, but toxic, micronutrient for living organisms and cells have developed sophisticated response mechanisms towards both the lack and the excess of Cu in their environments. In this study, we achieved a global view of Cu-responsive changes in the prokaryotic model organism Rhodobacter capsulatus using label-free quantitative differential proteomics. Semi-aerobically grown cells under heterotrophic conditions in minimal medium (∼0.3 μM Cu) were compared with cells supplemented with either 5 μM Cu or with 5 mM of the Cu-chelator bathocuproine sulfonate. Mass spectrometry based bottom-up proteomics of unfractionated cell lysates identified 2430 of the 3632 putative proteins encoded by the genome, producing a robust proteome dataset for R. capsulatus. Use of biological and technical replicates for each growth condition yielded high reproducibility and reliable quantification for 1926 of the identified proteins. Comparison of cells grown under Cu-excess or Cu-depleted conditions to those grown under minimal Cu-sufficient conditions revealed that 75 proteins exhibited statistically significant (p < 0.05) abundance changes, ranging from 2- to 300-fold. A subset of the highly Cu-responsive proteins was orthogonally probed using molecular genetics, validating that several of them were indeed involved in cellular Cu homeostasis.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Transparent window 2D IR spectroscopy of proteins

Proteins are complex, heterogeneous macromolecules that exist as ensembles of interconverting states on a complex energy landscape. A complete, molecular-level understanding of their function requires experimental tools to characterize them with high spatial and temporal precision. Infrared (IR) spectroscopy has an inherently fast time scale that can capture all states and their dynamics with, in principle, bond-specific spatial resolution. Two-dimensional (2D) IR methods that provide richer information are becoming more routine but remain challenging to apply to proteins. Spectral congestion typically prevents selective investigation of native vibrations; however, the problem can be overcome by site-specific introduction of amino acid side chains that have vibrational groups with frequencies in the “transparent window” of protein spectra. This Perspective provides an overview of the history and recent progress in the development of transparent window 2D IR of proteins.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2025

Physical models reveal indirect reader protein interactions that facilitate epigenetic crosstalk

The spatial organization of chromatin is governed by epigenetic factors, including epigenetic marks and the reader proteins that bind them. By dictating the accessibility of genomic loci, epigenetic factors contribute to the physical regulation of gene expression, enabling diverse cellular phenotypes to be encoded by a shared genome in an individual. Epigenetic dysregulation can lead to aberrations in chromatin architecture, contributing to diseases such as neurological disorders and cancers. Despite the known importance of chromatin organization for human health, the physical mechanisms governing chromatin folding remain underspecified. In this work, we develop a physical model of chromatin organization based on contributions from multiple epigenetic factors. Using our model, we evaluate how conditions in the nuclear environment and crosstalk between epigenetic marks affect the compartmentalization of chromatin into dense heterochromatin and loose euchromatin. Our results emphasize the role of reader protein binding in chromatin compartmentalization. We show that reader proteins interact through an indirect mechanism facilitated by the shared chromatin “scaffold” to which they bind. Under a scenario where reader proteins compete for binding sites, we find that indirect interactions affect the program adopted by the chromatin fiber. By isolating indirect modes of epigenetic crosstalk, we demonstrate how the interplay between epigenetic patterning and environmental factors influences chromatin architecture.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Site 2 of the Yersinia pestis substrate-binding protein YfeA is a dynamic surface metal-binding site

The substrate-binding protein YfeA (also known as YPO2439 or y1897) is a polyspecific metal-binding protein that is crucial for nutrient acquisition and virulence in Yersinia pestis, the causative microbe of plague. YfeA folds into a monomeric c-clamp like other substrate-binding proteins and has two metal-binding sites (sites 1 and 2). Site 2 is a bidentate surface site capable of binding Zn and Mn atoms and is a unique feature of YfeA. Occasionally, the site 2 residues of two YfeA molecules will cooperate with the histidine tag of a third YfeA molecule in coordinating the same metal and lead to metal-dependent crystallographic packing. Here, three crystal structures of YfeA are presented at 1.85, 2.05 and 2.25 Å resolution. A comparison of the structures reveals that the metal can be displaced at five different locations ranging from ~4 to ~16 Å away from the canonical site 2. These observations reveal different configurations of site 2 that enable cooperative metal binding and demonstrate how site 2 is dynamic and freely available for inter-protein metal coordination.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

AlphaFold -assisted structure determination of a bacterial protein of unknown function using X-ray and electron crystallography

Macromolecular crystallography generally requires the recovery of missing phase information from diffraction data to reconstruct an electron-density map of the crystallized molecule. Most recent structures have been solved using molecular replacement as a phasing method, requiring an a priori structure that is closely related to the target protein to serve as a search model; when no such search model exists, molecular replacement is not possible. New advances in computational machine-learning methods, however, have resulted in major advances in protein structure predictions from sequence information. Methods that generate predicted structural models of sufficient accuracy provide a powerful approach to molecular replacement. Taking advantage of these advances, AlphaFold predictions were applied to enable structure determination of a bacterial protein of unknown function (UniProtKB Q63NT7, NCBI locus BPSS0212) based on diffraction data that had evaded phasing attempts using MIR and anomalous scattering methods. Using both X-ray and micro-electron (microED) diffraction data, it was possible to solve the structure of the main fragment of the protein using a predicted model of that domain as a starting point. The use of predicted structural models importantly expands the promise of electron diffraction, where structure determination relies critically on molecular replacement.

molecular replacement↗
DOE OSTI2023

CALCIUM‐DEPENDENT PROTEIN KINASE32 regulates cellulose biosynthesis through post‐translational modification of cellulose synthase

Summary Cellulose is an essential component of plant cell walls and an economically important source of food, paper, textiles, and biofuel. Despite its economic and biological significance, the regulation of cellulose biosynthesis is poorly understood. Phosphorylation and dephosphorylation of cellulose synthases (CESAs) were shown to impact the direction and velocity of cellulose synthase complexes (CSCs). However, the protein kinases that phosphorylate CESAs are largely unknown. We conducted research in Arabidopsis thaliana to reveal protein kinases that phosphorylate CESAs. In this study, we used yeast two‐hybrid, protein biochemistry, genetics, and live‐cell imaging to reveal the role of calcium‐dependent protein kinase32 (CPK32) in the regulation of cellulose biosynthesis in A. thaliana . We identified CPK32 using CESA3 as a bait in a yeast two‐hybrid assay. We showed that CPK32 phosphorylates CESA3 while it interacts with both CESA1 and CESA3. Overexpressing functionally defective CPK32 variant and phospho‐dead mutation of CESA3 led to decreased motility of CSCs and reduced crystalline cellulose content in etiolated seedlings. Deregulation of CPKs impacted the stability of CSCs. We uncovered a new function of CPKs that regulates cellulose biosynthesis and a novel mechanism by which phosphorylation regulates the stability of CSCs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Tandem repeats in giant archaeal Borg elements undergo rapid evolution and create new intrinsically disordered regions in proteins

Borgs are huge, linear extrachromosomal elements associated with anaerobic methane-oxidizing archaea. Striking features of Borg genomes are pervasive tandem direct repeat (TR) regions. Here, we present six new Borg genomes and investigate the characteristics of TRs in all ten complete Borg genomes. We find that TR regions are rapidly evolving, recently formed, arise independently, and are virtually absent in host Methanoperedens genomes. Flanking partial repeats and A-enriched character constrain the TR formation mechanism. TRs can be in intergenic regions, where they might serve as regulatory RNAs, or in open reading frames (ORFs). TRs in ORFs are under very strong selective pressure, leading to perfect amino acid TRs (aaTRs) that are commonly intrinsically disordered regions. Proteins with aaTRs are often extracellular or membrane proteins, and functionally similar or homologous proteins often have aaTRs composed of the same amino acids. We propose that Borg aaTR-proteins functionally diversify Methanoperedens and all TRs are crucial for specific Borg–host associations and possibly cospeciation.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Naegleria fowleri: Protein structures to facilitate drug discovery for the deadly, pathogenic free-living amoeba

Naegleria fowleri is a pathogenic, thermophilic, free-living amoeba which causes primary amebic meningoencephalitis (PAM). Penetrating the olfactory mucosa, the brain-eating amoeba travels along the olfactory nerves, burrowing through the cribriform plate to its destination: the brain’s frontal lobes. The amoeba thrives in warm, freshwater environments, with peak infection rates in the summer months and has a mortality rate of approximately 97%. A major contributor to the pathogen’s high mortality is the lack of sensitivity of N . fowleri to current drug therapies, even in the face of combination-drug therapy. To enable rational drug discovery and design efforts we have pursued protein production and crystallography-based structure determination efforts for likely drug targets from N . fowleri . The genes were selected if they had homology to drug targets listed in Drug Bank or were nominated by primary investigators engaged in N . fowleri research. In 2017, 178 N . fowleri protein targets were queued to the Seattle Structural Genomics Center of Infectious Disease (SSGCID) pipeline, and to date 89 soluble recombinant proteins and 19 unique target structures have been produced. Many of the new protein structures are potential drug targets and contain structural differences compared to their human homologs, which could allow for the development of pathogen-specific inhibitors. Five of the structures were analyzed in more detail, and four of five show promise that selective inhibitors of the active site could be found. The 19 solved crystal structures build a foundation for future work in combating this devastating disease by encouraging further investigation to stimulate drug discovery for this neglected pathogen.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Distinct Clinical Impact and Biological Function of Angiopoietin and Angiopoietin-like Proteins in Human Breast Cancer

Secreted angiopoietin/angiopoietin-like (ANGPT/ANGPTL) proteins are involved in many biological processes. However, the role of these proteins in human breast cancers (BCs) remains largely unclear. Here, we conducted integrated omics analyses to evaluate the clinical impact of ANGPT/ANGPTL proteins and to elucidate their biological functions. In BCs, we identified rare mutations in ANGPT/ANGPTL genes, frequent gains of ANGPT1, ANGPT4, and ANGPTL1, and frequent losses of ANGPT2, ANGPTL5, and ANGPTL7, but observed that ANGPTL1, 2, and 4 were robustly downregulated in multiple datasets. The expression levels of ANGPTL1, 5, and 8 were positively correlated with overall survival (OS), while the expression levels of ANGPTL4 were negatively correlated with OS. Additionally, the expression levels of ANGPTL1 and 7 were positively correlated with distant metastasis-free survival (DMFS), while the expression levels of ANGPT2 and ANGPTL4 were negatively correlated with DMFS. The prognostic impacts of ANGPT/ANGPTL genes depended on the molecular subtypes and on clinical factors. We discovered that various ANGPT/ANGPTL genes were co-expressed with various genes involved in different pathways. Finally, with the exception of ANGPTL3, the remaining genes showed significant correlations with cancer-associated fibroblasts, endothelial cells, and microenvironment score, whereas only ANGPTL6 was significantly correlated with immune score. Our findings provide strong evidence for the distinct clinical impact and biological function of ANGPT/ANGPTL proteins, but the question of whether some of them could be potential therapeutic targets still needs further investigation in BCs.

60 APPLIED LIFE SCIENCES↗
NASA NTRS2003

Ubiquitin-protein ligases in muscle wasting: multiple parallel pathways?

PURPOSE OF REVIEW: Studies in a wide variety of animal models of muscle wasting have led to the concept that increased protein breakdown via the ubiquitin-proteasome pathway is responsible for the loss of muscle mass seen as muscle atrophy. The complexity of the ubiquitination apparatus has hampered our understanding of how this pathway is activated in atrophying muscles and which ubiquitin-conjugating enzymes in muscle are responsible. RECENT FINDINGS: Recent experiments have shown that two newly identified ubiquitin-protein ligases (E3s), atrogin-1/MAFbx and MURF-1, are critical in the development of muscle atrophy. Other in-vitro studies also implicated E2(14k) and E3alpha, of the N-end rule pathway, as playing an important role in the process. SUMMARY: It seems likely that multiple pathways of ubiquitin conjugation are activated in parallel in atrophying muscle, perhaps to target for degradation specific classes of muscle proteins. The emerging challenge will be to define the protein targets for, as well as inhibitors of, these E3s.

Review↗
NASA NTRS2001

Antisense expression of an Arabidopsis ran binding protein renders transgenic roots hypersensitive to auxin and alters auxin-induced root growth and development by arresting mitotic progress

We cloned a cDNA encoding an Arabidopsis Ran binding protein, AtRanBP1c, and generated transgenic Arabidopsis expressing the antisense strand of the AtRanBP1c gene to understand the in vivo functions of the Ran/RanBP signal pathway. The transgenic plants showed enhanced primary root growth but suppressed growth of lateral roots. Auxin significantly increased lateral root initiation and inhibited primary root growth in the transformants at 10 pM, several orders of magnitude lower than required to induce these responses in wild-type roots. This induction was followed by a blockage of mitosis in both newly emerged lateral roots and in the primary root, ultimately resulting in the selective death of cells in the tips of both lateral and primary roots. Given the established role of Ran binding proteins in the transport of proteins into the nucleus, these findings are consistent with a model in which AtRanBP1c plays a key role in the nuclear delivery of proteins that suppress auxin action and that regulate mitotic progress in root tips.

Non-NASA Center↗
NASA NTRS1990

Partial purification and characterization of a Ca(2+)-dependent protein kinase from the green alga, Dunaliella salina

A calcium-dependent protein kinase was partially purified and characterized from the green alga Dunaliella salina. The enzyme was activated at free Ca2+ concentrations above 10(-7) molar. and half-maximal activation was at about 3 x 10(-7) molar. The optimum pH for its Ca(2+)-dependent activity was 7.5. The addition of various phospholipids and diolein had no effects on enzyme activity and did not alter the sensitivity of the enzyme toward Ca2+. The enzyme was inhibited by calmodulin antagonists, N-(6-aminohexyl)-1-naphthalene sulfonamide and N-(6-aminohexyl)-5-chloro-1-naphthalene sulfonamide in a dose-dependent manner while the protein kinase C inhibitor, sphingosine, had little effect on enzyme activity up to 800 micromolar. Immunoassay showed some calmodulin was present in the kinase preparations. However, it is unlikely the kinase was calmodulin regulated, since it still showed stimulation by Ca2+ in gel assays after being electrophoretically separated from calmodulin by two different methods. This gel method of detection of the enzyme indicated that a protein band with an apparent molecular weight of 40,000 showed protein kinase activity at each one of the several steps in the purification procedure. Gel assay analysis also showed that after native gel isoelectric focusing the partially purified kinase preparations had two bands with calcium-dependent activity, at isoelectric points 6.7 and 7.1. By molecular weight, by isoelectric point, and by a comparative immunoassay, the Dunaliella kinase appears to differ from at least some of the calcium-dependent, but calmodulin and phospholipid independent kinases described from higher plants.

Non-NASA Center↗
NASA NTRS1999

Interaction of plant chimeric calcium/calmodulin-dependent protein kinase with a homolog of eukaryotic elongation factor-1alpha

A chimeric Ca2+/calmodulin-dependent protein kinase (CCaMK) was previously cloned and characterized in this laboratory. To investigate the biological functions of CCaMK, the yeast two-hybrid system was used to isolate genes encoding proteins that interact with CCaMK. One of the cDNA clones obtained from the screening (LlEF-1alpha1) has high similarity with the eukaryotic elongation factor-1alpha (EF-1alpha). CCaMK phosphorylated LlEF-1alpha1 in a Ca2+/calmodulin-dependent manner. The phosphorylation site for CCaMK (Thr-257) was identified by site-directed mutagenesis. Interestingly, Thr-257 is located in the putative tRNA-binding region of LlEF-1alpha1. An isoform of Ca2+-dependent protein kinase (CDPK) phosphorylated multiple sites of LlEF-1alpha1 in a Ca2+-dependent but calmodulin-independent manner. Unlike CDPK, CCaMK phosphorylated only one site, and this site is different from CDPK phosphorylation sites. This suggests that the phosphorylation of EF-1alpha by these two kinases may have different functional significance. Although the phosphorylation of LlEF-1alpha1 by CCaMK is Ca2+/calmodulin-dependent, in vitro binding assays revealed that CCaMK binds to LlEF-1alpha1 in a Ca2+-independent manner. This was further substantiated by coimmunoprecipitation of CCaMK and EF-1alpha using the protein extract from lily anthers. Dissociation of CCaMK from EF-1alpha by Ca2+ and phosphorylation of EF-1alpha by CCaMK in a Ca2+/calmodulin-dependent manner suggests that these interactions may play a role in regulating the biological functions of EF-1alpha.

Non-NASA Center↗