Engineering Papers⌕ Search

SEARCH · Engineering Papers

Results for “protein function”

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 1,333 records · Page 74
All sourcesNASA NTRSDOE OSTI
NASA NTRS1999

The Role of Calcium in the Response of Osteoblasts to Mechanical Stimulation

A major biomedical concern in the exploration and development of space is the rapid loss of bone associated with extended periods of spaceflight. Mineral content, bone formation, matrix protein production and total body calcium are all reduced during long-term periods of weightlessness. These effects of weightlessness appears to be due to decreases in the anabolic function of osteoblasts and osteocytes rather than changes in the resorptive activity of osteoclasts. Conversely, subjecting the skeleton to exogenous mechanical loading increases matrix protein synthesis and bone formation rate, a process which also appears mediated through osteogenic cells. Osteoblasts have been shown to respond to a number of types of mechanical stimulation. However recently we have demonstrated that osteoblasts respond to fluid shear, but not physiologic levels of mechanical strain, with increases in expression of the matrix protein, osteopontin. We have also shown similar responses in other markers for the anabolic response in bone. The expression of the early response gene, c-fos, and the inducible-isoform of the prostaglandin synthetic enzyme, cyclooygenase-2 (COX-2), both increase rapidly in response to fluid shear, but not strain. How osteoblasts and osteocytes perceive mechanical stimuli and convert this stimulus into a biochemical event within the cell is still unknown. However, examination of the cellular events following mechanical stimulation indicate that two of the earliest responses are a rapid increase in intracellular calcium ([Ca(2+)](sub i)) and a reorganization of the actin cytoskeleton. The increase in [Ca(2+)](sub i) is dependent on the presence of extracellular Ca(2+), suggesting the activation of membrane Ca(2+) channel. We have previously characterized a mechanosensitive, cation-selective channel (MSCC) in osteoblast-like clonal cells, which we postulate is important in this early response to mechanical loading. Using an antisense oligodeoxynucleotide strategy, we have tentatively identified this channel as an isoform of the alc subunit of the dihydropyridine-sensitive, voltage sensitive Ca(2+) channel (VSCC). However, a major component in this mechanically induced rise in [Ca(2+)](sub i) is the release of Ca(2+) from intracellular stores. The actin cytoskeleton also rapidly responds to fluid shear with an increase in stress fiber formation and a realignment of the cell parallel to the direction of flow. To ascertain whether these two observations are related and how they effect shear-induced gene expression, we examined the role of Ca(2+) channels and intracellular Ca(2+) release on cytoskeletal reorganization and the resultant increases in the expression and production of c-fos and COX-2 in response to fluid shear.

Duncan, R. L.↗
DOE OSTI2021

Implication of rare genetic variants of NODAL and ACVR1B in congenital heart disease patients from Indian population

Highlights: • Screening of 300 CHD patients revealed two rare missense variants (p.P51L & p.D327E) in NODAL. • A novel missense variation p.M345I was identified in ACVR1B. • Bioinformatic analyses suggested that these variants could have deleterious role. • Three-dimensional modelling suggested conformation changes in the mutated proteins compared to wild-type. • In vitro studies demonstrated that missense variants affect the function of NODAL and ACVR1B. NODAL signaling plays an essential role in vertebrate embryonic patterning and heart development. Accumulating evidences suggest that genetic mutations in TGF-β/NODAL signaling pathway can cause congenital heart disease in humans. To investigate the implication of NODAL signaling in isolated cardiovascular malformation, we have screened 300 non-syndromic CHD cases and 200 controls for NODAL and ACVR1B by Sanger sequencing and identified two rare missense (c.152C > T; p.P51L and c.981 T > A; p.D327E) variants in NODAL and a novel missense variant c.1035G > A; p.M345I in ACVR1B. All these variants are absent in 200 controls. Three-dimensional protein-modelling demonstrates that both p.P51L and p.D327E variations of NODAL and p.M345I mutation of ACVR1B, affect the tertiary structure of respective proteins. Variants of NODAL (p.P51L and p.D327E) and ACVR1B (p.M345I), significantly reduce the transactivation of AR3-Luc, (CAGA){sub 12}-Luc and (SBE){sub 4}-Luc promoters. Moreover, qRT-PCR results have also deciphered a reduction in the expression of cardiac-enriched transcription factors namely Gata4, Nkx2-5, and Tbx5 in both the mutants of NODAL. Decreased expression of, Gata4, Nkx2-5, Tbx5, and lefty is observed in p.M345I mutant of ACVR1B as well. Additionally, reduced phosphorylation of SMAD2/3 in response to these variants, suggests impaired NODAL signaling and possibly responsible for defective cell fate decision and differentiation of cardiomyocytes leading to CHD phenotype.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2025

PET-FBA: A lightweight enzyme allocation and thermodynamics-constrained flux analysis approach to explore Escherichia coli metabolic adaptation to intracellular acidification

Escherichia coli employs diverse strategies to adapt to acidic environments that disrupt enzyme activity and the thermodynamic feasibility of essential reactions. To understand the impact of pH stress on cell metabolism, we present the PET-FBA (pH-, Enzyme protein allocation-, and Thermodynamics-constrained Flux Balance Analysis) framework. PET-FBA extends genome-scale modeling by integrating enzyme protein costs and reaction Gibbs free energy changes. Additionally, by incorporating pH-dependent enzyme kinetics in response to intracellular acidification, this framework enables the simulation of E. coli's metabolic adjustments across varying external pH levels. The model's accuracy is validated by comparing in silico growth simulations with experimental measurements under both anaerobic and aerobic conditions, as well as in silico gene knockouts of essential genes. By explicitly incorporating pH effects, our model accurately replicates the metabolic shift towards lactate production as the primary fermentation product at low pH in anaerobic conditions. This shift is only predicted when enzyme kinetics are dynamically adjusted as a function of pH. Further analysis revealed that this shift can be attributed to the reduced protein efficiency of the acetyl-CoA branch compared to lactate dehydrogenase under acidic stress, which then becomes crucial for maintaining NAD regeneration and cell growth at low pH. Furthermore, we identified strategies for enhancing cell growth under acidic anaerobic conditions by improving the enzyme activity of lactate dehydrogenase and pyruvate formate lyase, which increases NAD production efficiency and reduces enzyme protein allocation costs. Designed as a lightweight yet versatile framework, PET-FBA enables efficient genome-scale metabolic analysis. Using E. coli as a model system, our framework provides a systematic approach to understanding metabolic responses to environmental stress, pinpointing key metabolic bottlenecks, and identifying potential targets for strain optimization.

42 ENGINEERING↗
DOE OSTI2025

Experimental and Computational Insights into the Structural Dynamics of the Fc Fragment of IgG1 Subtype from Biosimilar VEGF‐Trap

The constant fragment (Fc) of the immunoglobulin G1 (IgG1) subtype is increasingly recognized as a crucial scaffold in the development of advanced therapeutics due to its enhanced specificity, efficacy, and extended half‐life. A prime example is VEGF‐Trap (Aflibercept), a recombinant fusion protein that merges the Fc region of the IgG1 subtype with the binding domains of vascular endothelial growth factor receptors (VEGFR)‐1 and VEGFR‐2. The Fc region's role in N‐glycosylation is particularly important, as it significantly influences protein stability. Herein, the first near‐physiological temperature structures of the N‐glycan‐bound Fc fragment of IgG1 subtype from a biosimilar VEGF‐Trap are presented, determined using the SPring‐8 Angstrom Compact free electron LAser (SACLA) and the Turkish Light Source (Turkish DeLight). Comparative analysis with cryogenic structures, including existing data, reveals alternate conformations within the glycan‐binding pocket. Furthermore, molecular dynamics simulations indicate the presence of a high degree of structural plasticity, explaining how the protein adapts its structure through conformational changes. The observed structural fluctuations/conformational changes demonstrate the effect of N‐glycans on protein stability. These findings offer new insights into the molecular basis of Fc‐mediated functions and provide valuable information for the design of next‐generation therapeutics.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Dissecting components of the Campylobacter jejuni fetMP-fetABCDEF gene cluster under iron limitation

Campylobacter jejuni is a leading cause of bacterial gastroenteritis worldwide. Acute infection can be an antecedent to highly debilitating long-term sequelae. The expression of iron acquisition systems is vital for C. jejuni to survive the low iron availability within the human gut. The C. jejuni fetMP-fetABCDEF gene cluster is known to be upregulated during human infection and under iron limitation. While FetM and FetP have been functionally linked to iron transport in prior work, here we assess the contribution of each of the downstream genes (fetABCDEF) to C. jejuni growth during both iron-depleted and iron-replete conditions. Significant growth impairment was observed upon disruption of fetA, fetB, fetC, and fetD, suggesting a role in FetMP-mediated iron acquisition for each encoded protein. FetA expression was not dependent on the presence of FetB, FetC, FetD, FetE, or FetF. The functions of the putative thioredoxins FetE and FetF were redundant under iron-limited growth, requiring a double deletion (ΔfetEF) to exhibit a growth defect. C. jejuni FetE was expressed, and the structure was solved to 1.50 Å, revealing structural similarity to thiol-disulfide oxidases. Functional characterization in biochemical assays showed that FetE reduced insulin at a slower rate than Escherichia coli Trx and that together, FetEF promoted substrate oxidation in cell extracts, suggesting that FetE (and presumably FetF) are oxidoreductases that can mediate oxidation in vivo. This study advances our understanding of the contributions of the fetMP-fetABCDEF gene cluster to virulence at a genetic and functional level, providing foundational knowledge toward mitigating C. jejuni-related morbidity and mortality.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2001

Use of abnormal preprophase bands to decipher division plane determination

Many premitotic plant cells possess a cortical preprophase band of microtubules and actin filaments that encircles the nucleus. In vacuolated cells, the preprophase band is visibly connected to the nucleus by a cytoplasmic raft of actin filaments and microtubules termed the phragmosome. Typically, the location of the preprophase band and phragmosome corresponds to, and thus is thought to influence, the location of the cell division plane. To better understand the function of the preprophase band and phragmosome in orienting division, we used a green fluorescent protein-based microtubule reporter protein to observe mitosis in living tobacco bright yellow 2 cells possessing unusual preprophase bands. Observations of mitosis in these unusual cells support the involvement of the preprophase band/phragmosome in properly positioning the preprophase nucleus, influencing spindle orientation such that the cytokinetic phragmoplast initially grows in an appropriate direction, and delineating a region in the cell cortex that attracts microtubules and directs later stages of phragmoplast growth. Thus, the preprophase band/phragmosome appears to perform several interrelated functions to orient the division plane. However, functional information associated with the preprophase band is not always used or needed and there appears to be an age or distance-dependent character to the information. Cells treated with the anti-actin drug, latrunculin B, are still able to position the preprophase nucleus suggesting that microtubules may play a dominant role in premitotic positioning. Furthermore, in treated cells, spindle location and phragmoplast insertion are frequently abnormal suggesting that actin plays a significant role in nuclear anchoring and phragmoplast guidance. Thus, the microtubule and actin components of the preprophase band/phragmosome execute complementary activities to ensure proper orientation of the division plane.

NASA Discipline Plant Biology↗
DOE OSTI2022

Induced nanoscale membrane curvature bypasses the essential endocytic function of clathrin

During clathrin-mediated endocytosis (CME), flat plasma membrane is remodeled to produce nanometer-scale vesicles. The mechanisms underlying this remodeling are not completely understood. The ability of clathrin to bind membranes of distinct geometries casts uncertainty on its specific role in curvature generation/stabilization. Here, we used nanopatterning to produce substrates for live-cell imaging, with U-shaped features that bend the ventral plasma membrane of a cell into shapes resembling energetically unfavorable CME intermediates. This induced membrane curvature recruits CME proteins, promoting endocytosis. Upon AP2, FCHo1/2, or clathrin knockdown, CME on flat substrates is severely diminished. However, induced membrane curvature recruits CME proteins in the absence of FCHo1/2 or clathrin and rescues CME dynamics/cargo uptake after clathrin (but not AP2 or FCHo1/2) knockdown. Induced membrane curvature enhances CME protein recruitment upon branched actin assembly inhibition under elevated membrane tension. These data establish that membrane curvature assists in CME nucleation and that the essential function of clathrin during CME is to facilitate curvature evolution, rather than scaffold protein recruitment.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2026

Transcriptomic and functional analyses uncover a conserved effector driving genotype-dependent virulence in the Sphaerulina musiva-Populus trichocarpa interaction

The introduction of invasive microbes compromises the structure, biodiversity, and function of naïve ecosystems. Sphaerulina musiva, a hemibiotrophic pathogen that causes leaf spot and stem cankers in Populus species, exemplifies an invasive fungal pathogen spread by human activities. However, the genetic mechanisms of pathogenicity and virulence are poorly understood, impeding mitigation strategies. We utilized RNA sequencing to identify fungal effectors linked to stem canker formation, informing the development of future strategies for effective disease management. Our analysis revealed 70 genes differentially expressed at 2 weeks and 110 genes at 3 weeks between inoculated trees and controls. Notably, the gene with the highest expression at 2 weeks and the second highest at 3 weeks was homologous to Extracellular protein 2 (Ecp2). Complementary genome-wide association studies linked sequence polymorphisms in this locus to phenotypic variation in disease severity. Infiltration of S. musiva Ecp2 into Populus trichocarpa leaves induced necrosis in susceptible genotypes. Gene disruption using a CRISPR-Cas9 RNP system resulted in a genotype-dependent reduction of stem canker and disease severity. Tracing the evolutionary history of this effector across the fungal kingdom, we uncovered clade-specific gene-family expansions and orthologs in new species. These findings raise questions about the function and adaptive significance of these gene families in fungal lifestyles. Our study provides the first tractable target for breeding resistant poplar genotypes, addressing the challenges of managing S. musiva and uncovering mechanisms that drive its virulence, and provides deeper insights into the evolutionary dynamics of a conserved small-secreted protein with a diversity of functions.

Sondreli, Kelsey L [Oregon State University]↗
DOE OSTI2024

Structural and biochemical analyses of the nuclear IκBζ protein in complex with the NF-κB p50 homodimer

As part of the efforts to understand nuclear IκB function in NF-κB-dependent gene expression, we report an X-ray crystal structure of the IκBζ ankyrin repeat domain in complex with the dimerization domain of the NF-κB p50 homodimer. IκBζ possesses an N-terminal α helix that conveys domain folding stability. Affinity and specificity of the complex depend on a small portion of p50 at the nuclear localization signal. The model suggests that only one p50 subunit supports binding with IκBζ, and biochemical experiments confirm that IκBζ associates with DNA-bound NF-κB p50:RelA heterodimers. Comparisons of IκBζ:p50 and p50:κB DNA complex crystallographic models indicate that structural rearrangement is necessary for ternary complex formation of IκBζ and p50 with DNA.

Cell Biology↗
DOE OSTI2021

Recombinant production of a functional SARS-CoV-2 spike receptor binding domain in the green algae Chlamydomonas reinhardtii

Recombinant production of viral proteins can be used to produce vaccine antigens or reagents to identify antibodies in patient serum. Minimally, these proteins must be correctly folded and have appropriate post-translation modifications. Here we report the production of the SARS-CoV-2 spike protein Receptor Binding Domain (RBD) in the green algae Chlamydomonas . RBD fused to a fluorescent reporter protein accumulates as an intact protein when targeted for ER-Golgi retention or secreted from the cell, while a chloroplast localized version is truncated. The ER-retained RBD fusion protein was able to bind the human ACE2 receptor, the host target of SARS-CoV-2, and was specifically out-competed by mammalian cell-produced recombinant RBD, suggesting that the algae produced proteins are sufficiently post-translationally modified to act as authentic SARS-CoV-2 antigens. Because algae can be grown at large scale very inexpensively, this recombinant protein may be a low cost alternative to other expression platforms.

59 BASIC BIOLOGICAL SCIENCES↗
NASA NTRS2000

The Effects of Thermal History on Nucleation of Tetragonal Lysozyme Crystals, or Hot Protein and Cold Nucleation

Chicken egg white lysozyme has a well characterized thermally driven phase transition. Between pH 4.2 and 5.2, the transition temperature, as defined by the point where the tetragonal and orthorhombic solubilities are equal, is a function of the pH, salt (precipitant) type and concentration, and most likely of the buffer concentration as well. This phase transition can be carried out with protein solution alone, prior to addition of precipitant solution. Warming a lysozyme solution above the phase transition point, then cooling it back below this point, has been shown to affect the subsequent nucleation rate, as determined by the numbers and size of crystals formed, but not the growth rate for the tetragonal crystal form . We have now measured the kinetics of this process and investigated its reversibility. The transition effects are progressive with temperature, having a half time of about 1 hour at 37C at pH 4.8. After holding a lysozyme solution at 37C (prior to addition of precipitant) for 16 hours, then cooling it back to 4C no return to the pre-warmed nucleation kinetics are observed after at least 4 weeks. Orthorhombic lysozyme crystals apparently do not undergo the flow-induced growth cessation of tetragonal lysozyme crystals. Putting the protein in the orthorhombic form does not affect the averaged face growth kinetics, only nucleation, for tetragonal crystals. This differential behaviour may be exploited to elucidate how and where flow affects the lysozyme crystal growth process. The presentation will focus on the results of these and ongoing studies in this area.

Burke, Michael↗
DOE OSTI2022

Proton transfer reactions in donor site mutants of ESR, a retinal protein from Exiguobacterium sibiricum

Light-driven proton transport by microbial retinal proteins such as archaeal bacteriorhodopsin involves carboxylic residues as internal proton donors to the catalytic center which is a retinal Schiff base (SB). The proton donor, Asp96 in bacteriorhodopsin, supplies a proton to the transiently deprotonated Schiff base during the photochemical cycle. Subsequent proton uptake resets the protonated state of the donor. Here this two step process became a distinctive signature of retinal based proton pumps. Similar steps are observed also in many natural variants of bacterial proteorhodopsins and xanthorhodopsins where glutamic acid residues serve as a proton donor. Recently, however, an exception to this rule was found. A retinal protein from Exiguobacterium sibiricum, ESR, contains a Lys residue in place of Asp or Glu, which facilitates proton transfer from the bulk to the SB. Lys96 can be functionally replaced with the more common donor residues, Asp or Glu. Proton transfer to the SB in the mutants containing these replacements (K96E and K96D/A47T) is much faster than in the proteins lacking the proton donor (K96A and similar mutants), and in the case of K96D/A47T, comparable with that in the wild type, indicating that carboxylic residues can replace Lys96 as proton donors in ESR. We show here that there are important differences in the functioning of these residues in ESR from the way Asp96 functions in bacteriorhodopsin. Reprotonation of the SB and proton uptake from the bulk occur almost simultaneously during the M to N transition (as in the wild type ESR at neutral pH), whereas in bacteriorhodopsin these two steps are well separated in time and occur during the M to N and N to O transitions, respectively.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
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↗
NASA NTRS1979

Influence of hypokinesis on physiological functions in fowl

The effects of hypokinesis and postincubation stress (which are characteristic for modern techniques of poultry cage keeping) on the endocrine functions, metabolic reactions, body weight growth and proteosynthesis in the muscle of cocks was investigated. The stress due to hypokinesis was observed in growing cocks housed in metallic cages in which they could hardly turn around. The findings obtained indicate that a 35-day hypokinesis did not exert any more significant influence both on physiological functions and body weight growth as well as on proteosynthesis in the muscle of cocks under study; however, it speeded up the protein metabolism in the muscle. The postincubation stress modified significantly the hypokinesis effect. Findings recorded in birds differed considerably from findings obtained in laboratory mammals, in which the hypokinesis induced significant changes in endocrine functions, body weight decrease and proteosynthesis disorders. A good tolerance of hypokinesis by fowl can be interpreted not only by the phylogenetic remoteness of the compared species but also by the domestication.

Nvota, J.↗
NASA NTRS2010

Multifunctional and biologically active matrices from multicomponent polymeric solutions

The present invention relates to a biologically active functionalized electrospun matrix to permit immobilization and long-term delivery of biologically active agents. In particular the invention relates to a functionalized polymer matrix comprising a matrix polymer, a compatibilizing polymer and a biomolecule or other small functioning molecule. In certain aspects the electrospun polymer fibers comprise at least one biologically active molecule functionalized with low molecular weight heparin. Examples of active molecules that may be used with the multicomponent polymer of the invention include, for example, a drug, a biopolymer, for example a growth factor, a protein, a peptide, a nucleotide, a polysaccharide, a biological macromolecule or the like. The invention is further directed to the formation of functionalized crosslinked matrices, such as hydrogels, that include at least one functionalized compatibilizing polymer capable of assembly.

Kiick, Kristi L.↗
DOE OSTI2022

Allosteric control of electron transfer in nitrogenase and nitrogenase-like enzymes (Final Report)

More than a third of all known proteins to date are either oxidoreductases or metalloproteins. The diversity of metal cofactors and redox centers in these proteins make them attractive candidates for engineering protein-based biocatalysts, biosensors, and biofuel cells, among other necessary tools for the bioeconomy. Many of these enzymes function as large multi-subunit complexes arranged as symmetric or asymmetric assemblies, and the purpose of this architecture is unclear. The goal of the project was to address this fundamental question by investigating the mechanism of action of two oxidoreductases: Nitrogenase and DPOR (Dark-Operative Protochlorophyllide Oxidoreductase, a nitrogenase-like oxidoreductase). Nitrogenase and DPOR play key roles in nitrogen fixation and photosynthesis, processes essential to sustaining life. Both proteins are arranged as two symmetrical halves with each half capable of substrate reduction. For more than half a century, substrate reduction by nitrogenase has been presumed to occur independently within the two identical halves. Funded by this DOE grant we have now shown that both nitrogenase and DPOR function asymmetrically. Electron transfer in one half suppresses activity in the other. Thus, we have demonstrated that the two halves do not function in an independent manner; rather, catalytic events in one-half allosterically suppress activity and electron transfer (ET) in the other. This alternating-site mechanism has significant implications on our current understanding of substrate reduction by these enzymes and offers a new mechanistic paradigm for the reactions they catalyze. Our work now provides a mechanistic map of the how the two symmetric catalytic halves work together to establish and maintain the sequential-site mechanism through multiple ET cycles to achieve substrate reduction. There is substantive need for bio-inspired tools that enable reduction of nitrogen and other substrates, as well as photosynthesis, for energy and food production. Understanding the workings of enzymes like nitrogenase and DPOR that mediate multi-electron redox chemistry is vital for designing such synthetic tools. The timing of our research coincides with the biotechnological breakthrough of light-driven artificial N2 reduction by nitrogenase, achieved using the native enzyme with both halves arranged in a mirror-like architecture as catalyst. Together with such discoveries, our research now reveal unknown facets of the reaction mechanism and hold promise for designing more effective catalysts.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Selected humanization of yeast U1 snRNP leads to global suppression of pre-mRNA splicing and mitochondrial dysfunction in the budding yeast

The recognition of the 5′ splice site (5′ ss) is one of the earliest steps of pre-mRNA splicing. To better understand, the mechanism and regulation of 5′ ss recognition, we selectively humanized components of the yeast U1 (yU1) snRNP to reveal the function of these components in 5′ ss recognition and splicing. We targeted U1C and Luc7, two proteins that interact with and stabilize the yU1 snRNA and the 5′ ss RNA duplex. We replaced the zinc-finger (ZnF) domain of yeast U1C (yU1C) with its human counterpart, which resulted in a cold-sensitive growth phenotype and moderate splicing defects. We next added an auxin-inducible degron to yeast Luc7 (yLuc7) protein (to mimic the lack of Luc7Ls in human U1 snRNP). We found that Luc7-depleted yU1 snRNP resulted in the concomitant loss of Prp40 and Snu71 (two other essential yU1 snRNP proteins), and further biochemical analyses suggest a model of how these three proteins interact with each other in the U1 snRNP. The loss of these proteins resulted in a significant growth retardation accompanied by a global suppression of pre-mRNA splicing. The splicing suppression led to mitochondrial dysfunction as revealed by a release of Fe 2+ into the growth medium and an induction of mitochondrial reactive oxygen species. Together, these observations indicate that the human U1C ZnF can substitute that of yeast, Luc7 is essential for the incorporation of the Luc7–Prp40–Snu71 trimer into yU1 snRNP, and splicing plays a major role in the regulation of mitochondrial function in yeast.

Biochemistry & Molecular Biology↗
DOE OSTI2023

A single amino acid change led to structural and functional differentiation of PvHd1 to control flowering in switchgrass

Abstract Switchgrass, a forage and bioenergy crop, occurs as two main ecotypes with different but overlapping ranges of adaptation. The two ecotypes differ in a range of characteristics, including flowering time. Flowering time determines the duration of vegetative development and therefore biomass accumulation, a key trait in bioenergy crops. No causal variants for flowering time differences between switchgrass ecotypes have, as yet, been identified. In this study, we mapped a robust flowering time quantitative trait locus (QTL) on chromosome 4K in a biparental F2 population and characterized the flowering-associated transcription factor gene PvHd1, an ortholog of CONSTANS in Arabidopsis and Heading date 1 in rice, as the underlying causal gene. Protein modeling predicted that a serine to glycine substitution at position 35 (p.S35G) in B-Box domain 1 greatly altered the global structure of the PvHd1 protein. The predicted variation in protein compactness was supported in vitro by a 4 °C shift in denaturation temperature. Overexpressing the PvHd1-p.35S allele in a late-flowering CONSTANS-null Arabidopsis mutant rescued earlier flowering, whereas PvHd1-p.35G had a reduced ability to promote flowering, demonstrating that the structural variation led to functional divergence. Our findings provide us with a tool to manipulate the timing of floral transition in switchgrass cultivars and, potentially, expand their cultivation range.

59 BASIC BIOLOGICAL SCIENCES↗