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At least 1,135 records · Page 63
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DOE OSTI2020

Resolving individual atoms of protein complex by cryo-electron microscopy

Breakthroughs in single-particle cryo-electron microscopy (cryo-EM) technology have made near-atomic resolution structure determination possible. Cryo-EM has resolved over four thousand structures at near-atomic resolutions (2–4 Å). It is rapidly becoming the method of choice for structure determination of membrane proteins, large assemblies, and multi-protein complexes partly because it does not require a crystal and partly because it can accommodate specimens with heterogeneous composition and/or conformation. This powerful technique is now capable of resolving protein complexes to better than 2 Å resolution3 and has been used to solve 3.7 Å resolution structure of RNA as small as ~40 kilodaltons.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Minimally disruptive optical control of protein tyrosine phosphatase 1B

Protein tyrosine phosphatases regulate a myriad of essential subcellular signaling events, yet they remain difficult to study in their native biophysical context. Here we develop a minimally disruptive optical approach to control protein tyrosine phosphatase 1B (PTP1B)—an important regulator of receptor tyrosine kinases and a therapeutic target for the treatment of diabetes, obesity, and cancer—and we use that approach to probe the intracellular function of this enzyme. Our conservative architecture for photocontrol, which consists of a protein-based light switch fused to an allosteric regulatory element, preserves the native structure, activity, and subcellular localization of PTP1B, affords changes in activity that match those elicited by post-translational modifications inside the cell, and permits experimental analyses of the molecular basis of optical modulation. Findings indicate, most strikingly, that small changes in the activity of PTP1B can cause large shifts in the phosphorylation states of its regulatory targets.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

Synthetic antibodies against BRIL as universal fiducial marks for single-particle cryoEM structure determination of membrane proteins

We propose the concept of universal fiducials based on a set of pre-made semi-synthetic antibodies (sABs) generated by customized phage display selections against the fusion protein BRIL, an engineered variant of apocytochrome b562a. These sABs can bind to BRIL fused either into the loops or termini of different GPCRs, ion channels, receptors and transporters without disrupting their structure. A crystal structure of BRIL in complex with an affinity-matured sAB (BAG2) that bound to all systems tested delineates the footprint of interaction. Negative stain and cryoEM data of several examples of BRIL-membrane protein chimera highlight the effectiveness of the sABs as universal fiducial marks. Taken together with a cryoEM structure of sAB bound human nicotinic acetylcholine receptor, this work demonstrates that these anti-BRIL sABs can greatly enhance the particle properties leading to improved cryoEM outcomes, especially for challenging membrane proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Single-cell spatial metabolomics with cell-type specific protein profiling for tissue systems biology

Metabolic reprogramming in cancer and immune cells occurs to support their increasing energy needs in biological tissues. Here we propose Single Cell Spatially resolved Metabolic (scSpaMet) framework for joint protein-metabolite profiling of single immune and cancer cells in male human tissues by incorporating untargeted spatial metabolomics and targeted multiplexed protein imaging in a single pipeline. We utilized the scSpaMet to profile cell types and spatial metabolomic maps of 19507, 31156, and 8215 single cells in human lung cancer, tonsil, and endometrium tissues, respectively. The scSpaMet analysis revealed cell type-dependent metabolite profiles and local metabolite competition of neighboring single cells in human tissues. Deep learning-based joint embedding revealed unique metabolite states within cell types. Trajectory inference showed metabolic patterns along cell differentiation paths. Here we show scSpaMet’s ability to quantify and visualize the cell-type specific and spatially resolved metabolic-protein mapping as an emerging tool for systems-level understanding of tissue biology.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Assembly of respiratory syncytial virus matrix protein lattice and its coordination with fusion glycoprotein trimers

Respiratory syncytial virus (RSV) is an enveloped, filamentous, negative-strand RNA virus that causes significant respiratory illness worldwide. RSV vaccines are available, however there is still significant need for research to support the development of vaccines and therapeutics against RSV and related Mononegavirales viruses. Individual virions vary in size, with an average diameter of ~130 nm and ranging from ~500 nm to over 10 µm in length. Though the general arrangement of structural proteins in virions is known, we use cryo-electron tomography and sub-tomogram averaging to determine the molecular organization of RSV structural proteins. We show that the peripheral membrane-associated RSV matrix (M) protein is arranged in a packed helical-like lattice of M-dimers. We report that RSV F glycoprotein is frequently observed as pairs of trimers oriented in an anti-parallel conformation to support potential interactions between trimers. Our sub-tomogram averages indicate the positioning of F-trimer pairs is correlated with the underlying M lattice. These results provide insight into RSV virion organization and may aid in the development of RSV vaccines and anti-viral targets.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Principles of paralog-specific targeted protein degradation engaging the C-degron E3 KLHDC2

Abstract PROTAC® (proteolysis-targeting chimera) molecules induce proximity between an E3 ligase and protein-of-interest (POI) to target the POI for ubiquitin-mediated degradation. Cooperative E3-PROTAC-POI complexes have potential to achieve neo-substrate selectivity beyond that established by POI binding to the ligand alone. Here, we extend the collection of ubiquitin ligases employable for cooperative ternary complex formation to include the C-degron E3 KLHDC2. Ligands were identified that engage the C-degron binding site in KLHDC2, subjected to structure-based improvement, and linked to JQ1 for BET-family neo-substrate recruitment. Consideration of the exit vector emanating from the ligand engaged in KLHDC2’s U-shaped degron-binding pocket enabled generation of SJ46421, which drives formation of a remarkably cooperative, paralog-selective ternary complex with BRD3 BD2 . Meanwhile, screening pro-drug variants enabled surmounting cell permeability limitations imposed by acidic moieties resembling the KLHDC2-binding C-degron. Selectivity for BRD3 compared to other BET-family members is further manifested in ubiquitylation in vitro, and prodrug version SJ46420-mediated degradation in cells. Selectivity is also achieved for the ubiquitin ligase, overcoming E3 auto-inhibition to engage KLHDC2, but not the related KLHDC1, KLHDC3, or KLHDC10 E3s. In sum, our study establishes neo-substrate-specific targeted protein degradation via KLHDC2, and provides a framework for developing selective PROTAC protein degraders employing C-degron E3 ligases.

Science & Technology - Other Topics↗
DOE OSTI2025

Structural studies of the IFNλ4 receptor complex using cryoEM enabled by protein engineering

Abstract IFNλ4 has posed a conundrum in human immunology since its discovery in 2013, with its expression linked to complications with viral clearance. While genetic and cellular studies revealed the detrimental effects of IFNλ4 expression, extensive structural and functional characterization has been limited by the inability to express and purify the protein, complicating explanations of its paradoxical behavior. In this work, we report a method for robust production of IFNλ4. We then use yeast surface display to affinity-mature IL10Rβ and solve the 72 kilodalton structures of IFNλ4 (3.26 Å) and IFNλ3 (3.00 Å) in complex with their receptors IFNλR1 and IL10Rβ using cryogenic electron microscopy. Comparison of the structures highlights differences in receptor engagement and reveals a distinct 12-degree rotation in overall receptor geometry, providing a potential mechanistic explanation for differences in cell signaling, downstream gene induction, and antiviral activities. Further, we perform a structural analysis using molecular modeling and simulation to identify a unique region of IFNλ4 that, when replaced, enables secretion of the protein from cells. These findings provide a structural and functional understanding of the IFNλ4 protein and enable future comprehensive studies towards correcting IFNλ4 dysfunction in large populations of affected patients.

Science & Technology - Other Topics↗
DOE OSTI2022

Predicting stress response and improved protein overproduction in Bacillus subtilis

Abstract Bacillus subtilis is a well-characterized microorganism and a model for the study of Gram-positive bacteria. The bacterium can produce proteins at high densities and yields, which has made it valuable for industrial bioproduction. Like other cell factories, metabolic modeling of B. subtilis has discovered ways to optimize its metabolism toward various applications. The first genome-scale metabolic model (M-model) of B. subtilis was published more than a decade ago and has been applied extensively to understand metabolism, to predict growth phenotypes, and served as a template to reconstruct models for other Gram-positive bacteria. However, M-models are ill-suited to simulate the production and secretion of proteins as well as their proteomic response to stress. Thus, a new generation of metabolic models, known as metabolism and gene expression models (ME-models), has been initiated. Here, we describe the reconstruction and validation of a ME model of B. subtilis , i JT964-ME. This model achieved higher performance scores on the prediction of gene essentiality as compared to the M-model. We successfully validated the model by integrating physiological and omics data associated with gene expression responses to ethanol and salt stress. The model further identified the mechanism by which tryptophan synthesis is upregulated under ethanol stress. Further, we employed i JT964-ME to predict amylase production rates under two different growth conditions. We analyzed these flux distributions and identified key metabolic pathways that permitted the increase in amylase production. Models like i JT964-ME enable the study of proteomic response to stress and the illustrate the potential for optimizing protein production in bacteria.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Preservation of corneous β-proteins in Mesozoic feathers

Fossil proteins are valuable tools in evolutionary biology. Recent technological advances and better integration of experimental methods have confirmed the feasibility of biomolecular preservation in deep time, yielding new insights into the timing of key evolutionary transitions. Keratins (formerly α-keratins) and corneous β-proteins (CBPs, formerly β-keratins) are of particular interest as they define tissue structures that underpin fundamental physiological and ecological strategies and have the potential to inform on the molecular evolution of the vertebrate integument. Reports of CBPs in Mesozoic fossils, however, appear to conflict with experimental evidence for CBP degradation during fossilization. Further, the recent model for molecular modification of feather chemistry during the dinosaur–bird transition does not consider the relative preservation potential of different feather proteins. Here we use controlled taphonomic experiments coupled with infrared and sulfur X-ray spectroscopy to show that the dominant β-sheet structure of CBPs is progressively altered to α-helices with increasing temperature, suggesting that (α-)keratins and α-helices in fossil feathers are most likely artefacts of fossilization. Our analyses of fossil feathers shows that this process is independent of geological age, as even Cenozoic feathers can comprise primarily α-helices and disordered structures. Critically, our experiments show that feather CBPs can survive moderate thermal maturation. Importantly, as predicted by our experiments, analyses of Mesozoic feathers confirm that evidence of feather CBPs can persist through deep time.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

The Mycobacterium tuberculosis protein O -phosphorylation landscape

Bacterial phosphosignaling has long been synonymous with the histidine kinases of the two component systems (TCSs), but many bacteria, including Mycobacterium tuberculosis (Mtb), also code for Ser/Thr protein kinases (STPKs). STPKs are the main phosphosignaling enzymes in eukaryotes, but the full extent of phosphorylation on protein Ser/Thr and Tyr (O-phosphorylation) in bacteria is unclear. Here, we explored the global signaling capacity of the STPKs in Mtb. We generated a panel of STPK loss- and gain-of-function strains and measured the resulting O-phosphorylation and transcriptional changes. This deep phosphoproteome shows that O-phosphorylation in Mtb is a vastly underexplored protein modification that affects >70% of the proteome. The substrate-kinase interactions of STPKs show an extensive interface with the transcriptional machinery, resulting in STPK regulation of gene expression of over 30% of Mtb genes. Finally, Mtb O-phosphorylation gives rise to an expansive, distributed, and cooperative network of a complexity that has previously only been associated with eukaryotic phosphosignaling networks.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

De novo design of pH-responsive self-assembling helical protein filaments

Abstract Biological evolution has led to precise and dynamic nanostructures that reconfigure in response to pH and other environmental conditions. However, designing micrometre-scale protein nanostructures that are environmentally responsive remains a challenge. Here we describe the de novo design of pH-responsive protein filaments built from subunits containing six or nine buried histidine residues that assemble into micrometre-scale, well-ordered fibres at neutral pH. The cryogenic electron microscopy structure of an optimized design is nearly identical to the computational design model for both the subunit internal geometry and the subunit packing into the fibre. Electron, fluorescent and atomic force microscopy characterization reveal a sharp and reversible transition from assembled to disassembled fibres over 0.3 pH units, and rapid fibre disassembly in less than 1 s following a drop in pH. The midpoint of the transition can be tuned by modulating buried histidine-containing hydrogen bond networks. Computational protein design thus provides a route to creating unbound nanomaterials that rapidly respond to small pH changes.

77 NANOSCIENCE AND NANOTECHNOLOGY↗
DOE OSTI2026

Programmed synthesis of mesoporous protein crystals in cellular reactors

Protein crystals are naturally derived mesoporous materials with versatile structures and physicochemical properties. Here we introduce an intracellular synthesis platform that enables controllable and programmable protein crystallization. In live cells, we show that, after initial nucleation, steady protein expression governs crystal growth, yielding predictable, tunable dynamics in live cells. Exploiting this feature, we combined HaloTag and click chemistries to achieve modular, programmable immobilization of diverse guest materials with spatial patterning down to ~100 nm resolution. We further demonstrated the sequential release of immobilized materials in physiologically relevant fluids. As a proof of concept, we programmed particles to carry human fibroblast growth factors in distinct layers, which elicited designed oscillatory Akt signalling patterns in cell culture. Finally, this work outlines a programmable method for producing mesoporous materials, with possible applications in catalysis and biomedicine.

Yang, Hongru [Johns Hopkins Univ., Baltimore, MD (↗
DOE OSTI2021

Reply to: Insufficient evidence for ageing in protein dynamics

Reference 1 states that we claimed that internal motions in single protein molecules exhibit ageing over 13 decades of time. In contrast, our primary conclusion, as expressed in the title, was that over these timescales the dynamics of single protein molecules is non-equilibrium and self-similar. Notwithstanding, we demonstrate in this reply that protein dynamics does indeed exhibit ageing according to the criteria of ref. 1.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Development of spirulina for the manufacture and oral delivery of protein therapeutics

The use of the edible photosynthetic cyanobacterium Arthrospira platensis (spirulina) as a biomanufacturing platform has been limited by a lack of genetic tools. Here we report genetic engineering methods for stable, high-level expression of bioactive proteins in spirulina, including large-scale, indoor cultivation and downstream processing methods. Following targeted integration of exogenous genes into the spirulina chromosome (chr), encoded protein biopharmaceuticals can represent as much as 15% of total biomass, require no purification before oral delivery and are stable without refrigeration and protected during gastric transit when encapsulated within dry spirulina. Oral delivery of a spirulina-expressed antibody targeting campylobacter—a major cause of infant mortality in the developing world—prevents disease in mice, and a phase 1 clinical trial demonstrated safety for human administration. Spirulina provides an advantageous system for the manufacture of orally delivered therapeutic proteins by combining the safety of a food-based production host with the accessible genetic manipulation and high productivity of microbial platforms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

The effect of protein mutations on drug binding suggests ensuing personalised drug selection

The advent of personalised medicine promises a deeper understanding of mechanisms and therefore therapies. However, the connection between genomic sequences and clinical treatments is often unclear. We studied 50 breast cancer patients belonging to a population-cohort in the state of Qatar. From Sanger sequencing, we identified several new deleterious mutations in the estrogen receptor 1 gene (ESR1). The effect of these mutations on drug treatment in the protein target encoded by ESR1, namely the estrogen receptor, was achieved via rapid and accurate protein–ligand binding affinity interaction studies which were performed for the selected drugs and the natural ligand estrogen. Four nonsynonymous mutations in the ligand-binding domain were subjected to molecular dynamics simulation using absolute and relative binding free energy methods, leading to the ranking of the efficacy of six selected drugs for patients with the mutations. Our study shows that a personalised clinical decision system can be created by integrating an individual patient’s genomic data at the molecular level within a computational pipeline which ranks the efficacy of binding of particular drugs to variant proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Mapping periplasmic binding protein oligosaccharide recognition with neutron crystallography

Numerous studies have shown how periplasmic binding proteins (PBPs) bind substrates with exquisite specificity, even distinguishing between sugar epimers and anomers, or structurally similar ions. Yet, marked substrate promiscuity is also a feature encoded in some PBPs. Except for three sub-Ångström crystal structures, there are no reports of hydrogen atom positions in the remaining (> 1000) PBP structures. The previous X-ray crystal structure of the maltodextrin periplasmic-binding protein from Thermotoga maritima (tmMBP) complexed with oligosaccharide showed a large network of interconnected water molecules stretching from one end of the substrate binding pocket to the other. These water molecules are positioned to form multiple hydrogen bonds, as well as forming interactions between the protein and substrate. Here we present the neutron crystal structure of tmMBP to a resolution of 2.1 Å. This is the first neutron crystal structure from the PBP superfamily and here we unambiguously identify the nature and orientation of the hydrogen bonding and water-mediated interactions involved in stabilizing a tetrasaccharide in the binding site. More broadly, these results demonstrate the conserved intricate mechanisms that underlie substrate-specificity and affinity in PBPs.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Induced protein expression in Leptospira spp. and its application to CRISPR/Cas9 mutant generation

Abstract Expanding the genetic toolkit for Leptospira spp. is a crucial step toward advancing our understanding of the biology and virulence of these atypical bacteria. Pathogenic Leptospiraare responsible for over 1 million human leptospirosis cases annually and significantly impact domestic animals. Bovine leptospirosis causes substantial financial losses due to abortion, stillbirths, and suboptimal reproductive performance. The advent of the CRISPR/Cas9 system has marked a turning point in genetic manipulation, with applications across multiple Leptospira species. However, incorporating controlled protein expression into existing genetic tools could further expand their utility. We developed and demonstrated the functionality of IPTG-inducible heterologous protein expression in Leptospira spp. This system was applied for regulated expression of dead Cas9 (dCas9) to generate knockdown mutants, and Cas9 to produce knockout mutants by inducing double-strand breaks (DSB) into desired targets. IPTG-induced dCas9 expression enabled validation of essential genes and non-coding RNAs. Additionally, IPTG-controlled Cas9 expression combined with a constitutive non-homologous end-joining (NHEJ) system allowed for successful recovery of knockout mutants, even in the absence of IPTG. These newly controlled protein expression systems will advance studies on the basic biology and virulence ofLeptospira, as well as facilitate knockout mutant generation for improved veterinary vaccines.

Science & Technology - Other Topics↗
DOE OSTI2020

Sequence specificity analysis of the SETD2 protein lysine methyltransferase and discovery of a SETD2 super-substrate

SETD2 catalyzes methylation at lysine 36 of histone H3 and it has many disease connections. We investigated the substrate sequence specificity of SETD2 and identified nine additional peptide and one protein (FBN1) substrates. Our data showed that SETD2 strongly prefers amino acids different from those in the H3K36 sequence at several positions of its specificity profile. Based on this, we designed an optimized super-substrate containing four amino acid exchanges and show by quantitative methylation assays with SETD2 that the super-substrate peptide is methylated about 290-fold more efficiently than the H3K36 peptide. Protein methylation studies confirmed very strong SETD2 methylation of the super-substrate in vitro and in cells. We solved the structure of SETD2 with bound super-substrate peptide containing a target lysine to methionine mutation, which revealed better interactions involving three of the substituted residues. Our data illustrate that substrate sequence design can strongly increase the activity of protein lysine methyltransferases.

36 MATERIALS SCIENCE↗