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DOE OSTI2020

Who Is Metabolizing What? Discovering Novel Biomolecules in the Microbiome and the Organisms Who Make Them

Even as the field of microbiome research has made huge strides in mapping microbial community composition in a variety of environments and organisms, explaining the phenotypic influences on the host by microbial taxa – both known and unknown – and their specific functions still remain major challenges. A pressing need is the ability to assign specific functions in terms of enzymes and small molecules to specific taxa or groups of taxa in the community. This knowledge will be crucial for advancing personalized therapies based on the targeted modulation of microbes or metabolites that have predictable outcomes to benefit the human host. This perspective article advocates for the combined use of standards-free metabolomics and activity-based protein profiling strategies to address this gap in functional knowledge in microbiome research via the identification of novel biomolecules and the attribution of their production to specific microbial taxa.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2025

Amino Acid Sequence Controls Enhanced Electron Transport in Heme-Binding Peptide Monolayers

Metal-binding proteins have the exceptional ability to facilitate long-range electron transport in nature. Despite recent progress, the sequence-structure–function relationships governing electron transport in heme-binding peptides and protein assemblies are not yet fully understood. In this work, the electronic properties of a series of heme-binding peptides inspired by cytochrome bc1 are studied using a combination of molecular electronics experiments, molecular modeling, and simulation. Self-assembled monolayers (SAMs) are prepared using sequence-defined heme-binding peptides capable of forming helical secondary structures. Following monolayer formation, the structural properties and chemical composition of assembled peptides are determined using atomic force microscopy and X-ray photoelectron spectroscopy, and the electronic properties (current density–voltage response) are characterized using a soft contact liquid metal electrode method based on eutectic gallium–indium alloys (EGaIn). Our results show a substantial 1000-fold increase in current density across SAM junctions upon addition of heme compared to identical peptide sequences in the absence of heme, while maintaining a constant junction thickness. These findings show that amino acid composition and sequence directly control enhancements in electron transport in heme-binding peptides. Overall, this study demonstrates the potential of using sequence-defined synthetic peptides inspired by nature as functional bioelectronic materials.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

Sneaking in SpyCatcher using cell penetrating peptides for in vivo imaging

Abstract In vivo imaging of protein complexes is a powerful method for understanding the underlying biological function of these key biomolecules. Though the engineering of small, high affinity nanobodies have become more prevalent, the off-rates of these tags may result in incomplete or partial labeling of proteins in live cells. The SpyCatcher003 and SpyTag split protein system allow for irreversible, covalent binding to a short target peptide unlike nanobody-affinity based probes. However, delivering these tags into a cell without disrupting its normal function is a key challenge. Cell penetrating peptides (CPPs) are short peptide sequences that facilitate the transduction of otherwise membrane-impermeable ‘cargo’ , such as proteins, into cells. Here we report on our efforts to engineer and characterize CPP-SpyCatcher003 fusions as modular imaging probes. We selected three CPPs, CUPID, Pentratin, and pVEC, to engineer fusion protein probes for superresolution microscopy, with the aim to eliminate prior permeabilization treatments that could introduce imaging artifacts. We find that fusing the CPP sequences to SpyCatcher003 resulted in dimer and multimer formation as determined by size exclusion chromatography, dynamic light scattering, and SDS resistant dimers on SDS-PAGE gels. By isolating and labeling the monomeric forms of the engineered protein, we show these constructs retained their ability to bind SpyTag and all three CPP sequences remain membrane active, as assessed by CD spectroscopy in the presence of SDS detergent. Using fluorescence and super resolution Lattice structured illumination microscopy (Lattice SIM) imaging we show that the CPPs did not enhance uptake of SpyCatcher by E. coli, however with Caulobacter crescentus cells, we show that Penetratin, and to a lesser degree CUPID, does enhance uptake. Our results demonstrate the ability of the CPP-SpyCatcher003 to label targets within living cells, providing the groundwork for using split protein systems for targeted in vivo imaging.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2021

Determination of affinities of lanthanide-binding proteins using chelator-buffered titrations

The recent discoveries of the first proteins that bind lanthanides as part of their biological function not only are relevant to the emerging field of lanthanide-dependent biology, but also hold promise to revolutionize the technologically critical rare earths industry. Although protocols to assess the thermodynamics of metal–protein interactions are well established for “traditional” metal ions in biology, the characterization of lanthanide-binding proteins presents a challenge to biochemists due to the lanthanides' Lewis acidity, propensity for hydrolysis, and high-affinity complexes with biological ligands. These properties necessitate the preparation of metal stock solutions with very low buffered “free” metal concentrations (e.g., femtomolar to nanomolar) for such determinations. Here in this paper we describe several protocols to overcome these challenges. First, we present standardization methods for the preparation of chelator-buffered solutions of lanthanide ions with easily calculated free metal concentrations. We also describe how these solutions can be used in concert with analytical methods including UV–visible spectrophotometry, circular dichroism spectroscopy, Förster resonance energy transfer (FRET), and sensitized terbium luminescence, in order to accurately determine dissociation constants (K d s) of lanthanide–protein complexes. Finally, we highlight how application of these methods to lanthanide-binding proteins, such as lanmodulin, has yielded insights into selective recognition of lanthanides in biology. We anticipate that these protocols will facilitate discovery and characterization of additional native lanthanide-binding proteins, will motivate the understanding of their biological context, and will prompt their applications in biotechnology.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2021

LDIP cooperates with SEIPIN and LDAP to facilitate lipid droplet biogenesis in Arabidopsis

Cytoplasmic lipid droplets (LDs) are evolutionarily conserved organelles that store neutral lipids and play critical roles in plant growth, development, and stress responses. However, the molecular mechanisms underlying their biogenesis at the endoplasmic reticulum (ER) remain obscure. Here we show that a recently identified protein termed LD-associated protein [LDAP]-interacting protein (LDIP) works together with both endoplasmic reticulum-localized SEIPIN and the LD-coat protein LDAP to facilitate LD formation in Arabidopsis thaliana. Heterologous expression in insect cells demonstrated that LDAP is required for the targeting of LDIP to the LD surface, and both proteins are required for the production of normal numbers and sizes of LDs in plant cells. LDIP also interacts with SEIPIN via a conserved hydrophobic helix in SEIPIN and LDIP functions together with SEIPIN to modulate LD numbers and sizes in plants. Further, the co-expression of both proteins is required to restore normal LD production in SEIPIN-deficient yeast cells. These data, combined with the analogous function of LDIP to a mammalian protein called LD Assembly Factor 1, are discussed in the context of a new model for LD biogenesis in plant cells with evolutionary connections to LD biogenesis in other eukaryotes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

An anti-PD-1–GITR-L bispecific agonist induces GITR clustering-mediated T cell activation for cancer immunotherapy

Costimulatory receptors such as glucocorticoid-induced tumor necrosis factor receptor–related protein (GITR) play key roles in regulating the effector functions of T cells. In human clinical trials, however, GITR agonist antibodies have shown limited therapeutic effect, which may be due to suboptimal receptor clustering-mediated signaling. To overcome this potential limitation, a rational protein engineering approach is needed to optimize GITR agonist-based immunotherapies. Here we show a bispecific molecule consisting of an anti-PD-1 antibody fused with a multimeric GITR ligand (GITR-L) that induces PD-1-dependent and FcγR-independent GITR clustering, resulting in enhanced activation, proliferation and memory differentiation of primed antigen-specific GITR + PD-1 + T cells. The anti-PD-1–GITR-L bispecific is a PD-1-directed GITR-L construct that demonstrated dose-dependent, immunologically driven tumor growth inhibition in syngeneic, genetically engineered and xenograft humanized mouse tumor models, with a dose-dependent correlation between target saturation and Ki67 and TIGIT upregulation on memory T cells. Anti-PD-1–GITR-L thus represents a bispecific approach to directing GITR agonism for cancer immunotherapy.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Resin Assisted Capture Coupled with Isobaric Tandem Mass Tag Labeling for Multiplexed Quantification of Protein Thiol Oxidation

Reversible oxidative modifications on protein thiols have recently emerged as important mediators of cellular function. Herein we describe the detailed procedure of a quantitative redox proteomics method that utilizes resin-assisted capture (RAC) in combination with tandem mass tag (TMT) isobaric labeling and liquid chromatography-tandem mass spectrometry (LC-MS/MS) to allow multiplexed stochiometric quantification of oxidized protein thiols at the proteome level. The site-specific quantitative information on oxidized cysteine residues provides additional insight into the functional impacts of such modifications. The workflow is adaptable across many sample types including cultured cells (e.g. mammalian, prokaryotic) and whole tissues (e.g. heart, lung, muscle) which are initially lysed/homogenized with free thiols being alkylated to prevent artificial oxidation. The oxidized protein thiols are then reduced and captured by a thiol affinity resin which streamlines and simplifies the workflow steps by allowing the proceeding digestion, labeling, and washing procedures to be performed without additional transfer of proteins/peptides. Finally, labeled peptides are eluted and analyzed by LC-MS/MS to reveal comprehensive stoichiometric changes related to thiol oxidation across the entire proteome. This method greatly improves our understanding of the role of redox dependent regulation under physiological and pathophysiological states related to protein thiol oxidation.

RAC, TMT, thiol redox proteomics, cysteine, PTM st↗
DOE OSTI2025

Characterization of Gas-Phase Native(-like) Proteins Using Structures for Lossless Ion Manipulations

High-resolution mobility-based ion separations in Structures for Lossless Ion Manipulations (SLIM) have been useful for ion mobility separations for a variety of molecular classes in the gas phase. Here, in this study, we present multipass SLIM separations for gas-phase proteins in their near-native state exhibiting charge-state-dependent arrival time distributions using carbonic anhydrase (29 kDa), alcohol dehydrogenase (148 kDa), and apo-transferrin (79 kDa). The experimental CCS values were obtained from calibration curves for the arrival times of Agilent Tune Mix ions. For multipass separations, the ATDs were converted to CCS values by deconvoluting the multipass arrival times into accurate single-pass values amenable to the single-pass calibration curves. Mass spectra of carbonic anhydrase (CA) showed three different charge states (z = 9+ to 11+). Their corresponding mobility peaks were baseline-separated by using 8-m single-pass separations. When compared to the corresponding drift tube ion mobility (DTIMS) measurements, the CCS values obtained from DTIMS and SLIM were in agreement within experimental error. Single-pass analysis of alcohol dehydrogenase (ADH) exhibits three predominant charge states (z = 23+ to 25+) with mobility overlap between adjacent charge states. The mobility peak resolution for ADH improved with multipass separations (up to 24-m path length). In addition, CCS distributions obtained for charge states z = 16+ to 18+ of apo-transferrin reveal a transition from a compact unimodal form (z = 18+ and 19+) to broader multimodal CCS distributions for z = 16+. For apo-transferrin, 40-m multipass separations were performed allowing for complete isolation of the selected mobility range corresponding to z = 17+, leading to selective isolation of a narrow arrival time window. The extended mobility separations provided minimal alterations to the structure of the proteins, and the experimentally derived CCS values showed minimal change as a function of the separation time or number of passes. Mobility-based ion separations for native-like proteins, using SLIM, open opportunities for native-IMS applications as well as other manipulations enabled by SLIM-like mobility-selective isolation and collection.

charge state distribution↗
DOE OSTI2020

Computational design of closely related proteins that adopt two well-defined but structurally divergent folds

The plasticity of naturally occurring protein structures, which can change shape considerably in response to changes in environmental conditions, is critical to biological function. While computational methods have been used for de novo design of proteins that fold to a single state with a deep free-energy minimum [P.-S. Huang, S. E. Boyken, D. Baker, Nature 537, 320–327 (2016)], and to reengineer natural proteins to alter their dynamics [J. A. Davey, A. M. Damry, N. K. Goto, R. A. Chica, Nat. Chem. Biol.13, 1280–1285 (2017)] or fold [P. A. Alexander, Y. He, Y. Chen, J. Orban, P. N. Bryan, Proc. Natl. Acad. Sci. U.S.A. 106, 21149–21154 (2009)], the de novo design of closely related sequences which adopt well-defined but structurally divergent structures remains an outstanding challenge. We designed closely related sequences (over 94% identity) that can adopt two very different homotrimeric helical bundle conformations—one short (~66 Å height) and the other long (~100 Å height)—reminiscent of the conformational transition of viral fusion proteins. Crystallographic and NMR spectroscopic characterization shows that both the short- and long-state sequences fold as designed. We sought to design bistable sequences for which both states are accessible, and obtained a single designed protein sequence that populates either the short state or the long state depending on the measurement conditions. The design of sequences which are poised to adopt two very different conformations sets the stage for creating large-scale conformational switches between structurally divergent forms.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A functional map of HIV-host interactions in primary human T cells

Human Immunodeficiency Virus (HIV) relies on host molecular machinery for replication. Systematic attempts to genetically or biochemically define these host factors have yielded hundreds of candidates, but few have been functionally validated in primary cells. Here, we target 426 genes previously implicated in the HIV lifecycle through protein interaction studies for CRISPR-Cas9-mediated knock-out in primary human CD4+ T cells in order to systematically assess their functional roles in HIV replication. We achieve efficient knockout (>50% of alleles) in 364 of the targeted genes and identify 86 candidate host factors that alter HIV infection. 47 of these factors validate by multiplex gene editing in independent donors, including 23 factors with restrictive activity. Both gene editing efficiencies and HIV-1 phenotypes are highly concordant among independent donors. Importantly, over half of these factors have not been previously described to play a functional role in HIV replication, providing numerous novel avenues for understanding HIV biology. These data further suggest that host-pathogen protein-protein interaction datasets offer an enriched source of candidates for functional host factor discovery and provide an improved understanding of the mechanics of HIV replication in primary T cells.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2023

Low complexity domains of the nucleocapsid protein of SARS-CoV-2 form amyloid fibrils

The self-assembly of the Nucleocapsid protein (NCAP) of SARS-CoV-2 is crucial for its function. Computational analysis of the amino acid sequence of NCAP reveals low-complexity domains (LCDs) akin to LCDs in other proteins known to self-assemble as phase separation droplets and amyloid fibrils. Previous reports have described NCAP’s propensity to phase-separate. Here we show that the central LCD of NCAP is capable of both, phase separation and amyloid formation. Within this central LCD we identified three adhesive segments and determined the atomic structure of the fibrils formed by each. Those structures guided the design of G12, a peptide that interferes with the self-assembly of NCAP and demonstrates antiviral activity in SARS-CoV-2 infected cells. Our work, therefore, demonstrates the amyloid form of the central LCD of NCAP and suggests that amyloidogenic segments of NCAP could be targeted for drug development.

60 APPLIED LIFE SCIENCES↗
DOE OSTI2021

Effects of pH on an IDP conformational ensemble explored by molecular dynamics simulation

The conformational ensemble of intrinsically disordered proteins, such as α-synuclein, are responsible for their function and malfunction. Misfolding of α-synuclein can lead to neurodegenerative diseases, and the ability to study their conformations and those of other intrinsically disordered proteins under varying physiological conditions can be crucial to understanding and preventing pathologies. In contrast to well-folded peptides, a consensus feature of IDPs is their low hydropathy and high charge, which makes their conformations sensitive to pH perturbation. We examine a prominent member of this subset of IDPs, α-synuclein, using a divide-and-conquer scheme that provides enhanced sampling of IDP structural ensembles. We constructed conformational ensembles of α-synuclein under neutral (pH ~ 7) and low (pH ~ 3) pH conditions and compared our results with available information obtained from smFRET, SAXS, and NMR studies. Specifically, α-synuclein has been found to in a more compact state at low pH conditions and the structural changes observed are consistent with those from experiments. We also characterize the conformational and dynamic differences between these ensembles and discussed the implication on promoting pathogenic fibril formation. We find that under low pH conditions, neutralization of negatively charged residues leads to compaction of the C-terminal portion of α-synuclein while internal reorganization allows α-synuclein to maintain its overall end-to-end distance. Here, we also observe different levels of intra-protein interaction between three regions of α-synuclein at varying pH and a shift towards more hydrophilic interactions with decreasing pH.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗
DOE OSTI2023

An expectation–maximization framework for comprehensive prediction of isoform-specific functions

Advances in RNA sequencing technologies have achieved an unprecedented accuracy in the quantification of mRNA isoforms, but our knowledge of isoform-specific functions has lagged behind. There is a need to understand the functional consequences of differential splicing, which could be supported by the generation of accurate and comprehensive isoform-specific gene ontology annotations. We present isoform interpretation, a method that uses expectation–maximization to infer isoform-specific functions based on the relationship between sequence and functional isoform similarity. We predicted isoform-specific functional annotations for 85 617 isoforms of 17 900 protein-coding human genes spanning a range of 17 430 distinct gene ontology terms. Comparison with a gold-standard corpus of manually annotated human isoform functions showed that isoform interpretation significantly outperforms state-of-the-art competing methods. We provide experimental evidence that functionally related isoforms predicted by isoform interpretation show a higher degree of domain sharing and expression correlation than functionally related genes. We also show that isoform sequence similarity correlates better with inferred isoform function than with gene-level function.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

A fusion of the Bacteroides fragilis ferrous iron import proteins reveals a role for FeoA in stabilizing GTP-bound FeoB

Iron is an essential element for nearly all organisms, and under anoxic and/or reducing conditions, Fe 2+ is the dominant form of iron available to bacteria. The ferrous iron transport (Feo) system is the primary prokaryotic Fe 2+ import machinery, and two constituent proteins (FeoA and FeoB) are conserved across most bacterial species. However, how FeoA and FeoB function relative to one another remains enigmatic. In this work, we explored the distribution of feoAB operons encoding a fusion of FeoA tethered to the N-terminal, G-protein domain of FeoB via a connecting linker region. We hypothesized that this fusion poises FeoA to interact with FeoB to affect function. To test this hypothesis, we characterized the soluble NFeoAB fusion protein from Bacteroides fragilis, a commensal organism implicated in drug-resistant infections. Using X-ray crystallography, we determined the 1.50-Å resolution structure of BfFeoA, which adopts an SH3-like fold implicated in protein–protein interactions. Using a combination of structural modeling, small-angle X-ray scattering, and hydrogen–deuterium exchange mass spectrometry, we show that FeoA and NFeoB interact in a nucleotide-dependent manner, and we mapped the protein–protein interaction interface. Finally, using guanosine triphosphate (GTP) hydrolysis assays, we demonstrate that BfNFeoAB exhibits one of the slowest known rates of Feo-mediated GTP hydrolysis that is not potassium-stimulated. Importantly, truncation of FeoA from this fusion demonstrates that FeoA–NFeoB interactions function to stabilize the GTP-bound form of FeoB. Taken together, our work reveals a role for FeoA function in the fused FeoAB system and suggests a function for FeoA among prokaryotes.

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

Spatial proteomics reveals signal sequence characteristics correlated with localization in cyanobacteria

Abstract Cyanobacteria have an inner and outer cell membrane enclosing the periplasm and cell wall and an additional set of internal membranes (called the thylakoid membranes) enclosing the thylakoid lumen. The periplasm and thylakoid lumen have unique proteomes, but the mechanisms regulating protein sorting to these locations have remained elusive. Here, proximity-based proteomics using the engineered peroxidase APEX2 was performed in the cyanobacteria Synechococcus sp. PCC 7002 to profile the proteomes of the cytoplasm, thylakoid lumen, and the periplasm and outer membrane (P-OM). Our analyses revealed specific roles for the thylakoid lumen in photosynthesis and energy generation, as well as roles for the periplasm in metabolite transport and binding, cell motility, and cell wall maintenance. Forty proteins localized to both the thylakoid lumen and the P-OM; however, their biological functions remain unclear. We also analyzed the correlation between signal sequence characteristics and differential protein localization to either the thylakoid lumen or the P-OM. In PCC 7002, as well as Synechocystis sp. PCC 6803 and Nostoc sp. PCC 7120, thylakoid lumen proteins translocated across membranes via the Secretory (Sec) system possessed more hydrophobic and alpha-helical signal sequence H-regions than P-OM proteins. The signal sequences of homologous proteins in Gloeobacter violaceus PCC 7421, a cyanobacterial species with a combined thylakoid lumen and periplasmic space, did not exhibit such differences. Therefore, the pattern of increased H-region hydrophobicity and alpha helix content is specific to cyanobacteria with a separate thylakoid lumen space and likely contributes to proper protein sorting between the thylakoid lumen and periplasm.

Plant Sciences↗
DOE OSTI2024

Dynamic Implications of Noncovalent Interactions in Amphiphilic Single-Chain Polymer Nanoparticles

Single-chain polymer nanoparticles (SCNPs) combine the chemical diversity of synthetic polymers with the intricate structure of biopolymers, generating versatile biomimetic materials. The mobility of polymer chain segments at length scales similar to secondary structural elements in proteins is critical to SCNP structure and thus function. However, the influence of noncovalent interactions used to form SCNPs (e.g., hydrogen-bonding and biomimetic secondary-like structure) on these conformational dynamics is challenging to quantitatively assess. To isolate the effects of noncovalent interactions on SCNP structure and conformational dynamics, we synthesized a series of amphiphilic copolymers containing dimethylacrylamide and monomers capable of forming these different interactions: (1) di(phenylalanine) acrylamide that forms intramolecular β-sheet-like cross-links, (2) phenylalanine acrylamide that forms hydrogen-bonds but lacks a defined local structure, and (3) benzyl acrylamide that has the lowest propensity for hydrogen-bonding. Each SCNP formed folded structures comparable to those of intrinsically disordered proteins, as observed by size exclusion chromatography and small angle neutron scattering. The dynamics of these polymers, as characterized by a combination of dynamic light scattering and neutron spin echo spectroscopy, was well described using the Zimm with internal friction (ZIF) model, highlighting the role of each noncovalent interaction to additively restrict the internal relaxations of SCNPs. These results demonstrate the utility of local scale interactions to control SCNP polymer dynamics, guiding the design of functional biomimetic materials with refined binding sites and tunable kinetics.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗