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Results for “Protein fragment complementation”

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.

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13 records
All sourcesNASA NTRSDOE OSTI
DOE OSTI2025

Engineering a new tripartite split-ccGFP system from Corynactis californica for detecting protein–protein interactions

Protein-protein interactions (PPIs) are critical to a range of biological processes and, consequently, aberrant interactions are implicated in many disorders. The study of the complex networks of PPIs promises to elucidate undiscovered roles in cellular processes and the mechanisms of disease. To accomplish this, tools to effectively sense PPIs are necessary. Effective PPI sensors must rapidly detect interactions in real-time with high sensitivity without perturbing the proteins of interest (POIs) under study. Split fluorescent proteins have previously been used to successfully monitor PPIs, in part due to the small size of the tags. Here, we developed an optimized tripartite split GFP system based on Corynactis californica GFP (ccGFP) to detect PPIs in vitro. In this sensor system, ccGFP fragments ccGFP10 and ccGFP11 are tagged to two POIs. PPIs can then be detected via fluorescence by complementation to the third fragment, ccGFP1-9, which reconstitutes functional ccGFP. The optimized ccGFP system shows improved detection kinetics and pH and temperature stability compared to a previous system. We then validated the sensor by monitoring PPIs in two model systems: attractive/repulsive coiled-coils and rapamycin-inducible FRB/FKBP heterodimerization. Finally, we developed an anti-tripartite ccGFP single-chain variable fragment (scFv), which could enable versatile detection of identified protein-protein complexes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2020

A large accessory protein interactome is rewired across environments

To characterize how protein-protein interaction (PPI) networks change, we quantified the relative PPI abundance of 1.6 million protein pairs in the yeast Saccharomyces cerevisiae across 9 growth conditions, with replication, for a total of 44 million measurements. Our multi-condition screen identified 13,764 pairwise PPIs, a 3-fold increase over PPIs identified in one condition. A few 'immutable' PPIs are present across all conditions, while most 'mutable' PPIs are rarely observed. Immutable PPIs aggregate into highly connected 'core' network modules, with most network remodeling occurring within a loosely connected 'accessory' module. Mutable PPIs are less likely to co-express, co-localize, and be explained by simple mass action kinetics, and more likely to contain proteins with intrinsically disordered regions, implying that environment-dependent association and binding is critical to cellular adaptation. Our results show that protein interactomes are larger than previously thought and contain highly dynamic regions that reorganize to drive or respond to cellular changes.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

High-throughput protein characterization by complementation using DNA barcoded fragment libraries

Abstract Our ability to predict, control, or design biological function is fundamentally limited by poorly annotated gene function. This can be particularly challenging in non-model systems. Accordingly, there is motivation for new high-throughput methods for accurate functional annotation. Here, we used co mplementation of aux otrophs and DNA barcode seq uencing (Coaux-Seq) to enable high-throughput characterization of protein function. Fragment libraries from eleven genetically diverse bacteria were tested in twenty different auxotrophic strains of Escherichia coli to identify genes that complement missing biochemical activity. We recovered 41% of expected hits, with effectiveness ranging per source genome, and observed success even with distant E. coli relatives like Bacillus subtilis and Bacteroides thetaiotaomicron . Coaux-Seq provided the first experimental validation for 53 proteins, of which 11 are less than 40% identical to an experimentally characterized protein. Among the unexpected function identified was a sulfate uptake transporter, an O-succinylhomoserine sulfhydrylase for methionine synthesis, and an aminotransferase. We also identified instances of cross-feeding wherein protein overexpression and nearby non-auxotrophic strains enabled growth. Altogether, Coaux-Seq’s utility is demonstrated, with future applications in ecology, health, and engineering.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Therapeutic efficacy of a potent anti-Venezuelan equine encephalitis virus antibody is contingent on Fc effector function

The development of specific, safe, and potent monoclonal antibodies (Abs) has led to novel therapeutic options for infectious disease. In addition to preventing viral infection through neutralization, Abs can clear infected cells and induce immunomodulatory functions through engagement of their crystallizable fragment (Fc) with complement proteins and Fc receptors on immune cells. Little is known about the role of Fc effector functions of neutralizing Abs in the context of encephalitic alphavirus infection. To determine the role of Fc effector function in therapeutic efficacy against Venezuelan equine encephalitis virus (VEEV), we compared the potently neutralizing anti-VEEV human IgG F5 (hF5) Ab with intact Fc function (hF5-WT) or containing the loss of function Fc mutations L234A and L235A (hF5-LALA) in the context of VEEV infection. We observed significantly reduced binding to complement and Fc receptors, as well as differential in vitro kinetics of Fc-mediated cytotoxicity for hF5-LALA compared to hF5-WT. The in vivo efficacy of hF5-LALA was comparable to hF5-WT at -24 and + 24 h post infection, with both Abs providing high levels of protection. However, when hF5-WT and hF5-LALA were administered + 48 h post infection, there was a significant decrease in the therapeutic efficacy of hF5-LALA. Together these results demonstrate that optimal therapeutic Ab treatment of VEEV, and possibly other encephalitic alphaviruses, requires neutralization paired with engagement of immune effectors via the Fc region.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Engineering highly stable variants of Corynactis californica green fluorescent proteins

Fluorescent proteins (FPs) are versatile biomarkers that facilitate effective detection and tracking of macromolecules of interest in real time. Engineered FPs such as superfolder green fluorescent protein (sfGFP) and superfolder Cherry (sfCherry) have exceptional refolding capability capable of delivering fluorescent readout in harsh environments where most proteins lose their native functions. Our recent work on the development of a split FP from a species of strawberry anemone, Corynactis californica, delivered pairs of fragments with up to threefold faster complementation than split GFP. We present the biophysical, biochemical, and structural characteristics of five full-length variants derived from these split C. californica GFP (ccGFP). These ccGFP variants are more tolerant under chemical denaturation with up to 8 kcal/mol lower unfolding free energy than that of the sfGFP. It is likely that some of these ccGFP variants could be suitable as biomarkers under more adverse environments where sfGFP fails to survive. A structural analysis suggests explanations of the variations in stabilities among the ccGFP variants.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2022

Engineered human antibodies for the opsonization and killing of Staphylococcus aureus

Gram-positive organisms with their thick envelope cannot be lysed by complement alone. Nonetheless, antibody-binding on the surface can recruit complement and mark these invaders for uptake and killing by phagocytes, a process known as opsonophagocytosis. The crystallizable fragment of immunoglobulins (Fcγ) is key for complement recruitment. The cell surface of S. aureus is coated with Staphylococcal protein A (SpA). SpA captures the Fcγ domain of IgG and interferes with opsonization by anti-S. aureus antibodies. In principle, the Fcγ domain of therapeutic antibodies could be engineered to avoid the inhibitory activity of SpA. However, the SpA-binding site on Fcγ overlaps with that of the neonatal Fc receptor (FcRn), an interaction that is critical for prolonging the half-life of serum IgG. This evolutionary adaptation poses a challenge for the exploration of Fcγ mutants that can both weaken SpA–IgG interactions and retain stability. Here, we use both wild-type and transgenic human FcRn mice to identify antibodies with enhanced half-life and increased opsonophagocytic killing in models of S. aureus infection and demonstrate that antibody-based immunotherapy can be improved by modifying Fcγ. Our experiments also show that by competing for FcRn-binding, staphylococci effectively reduce the half-life of antibodies during infection. These observations may have profound impact in treating cancer, autoimmune, and asthma patients colonized or infected with S. aureus and undergoing monoclonal antibody treatment.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2019

Charge Movement and Structural Changes in the Gas-Phase Unfolding of Multimeric Protein Complexes Captured by Native Top-Down Mass Spectrometry

The extent to which noncovalent protein complexes retain native structure in the gas phase is highly dependent on experimental conditions. Energetic collisions with background gas can cause structural changes ranging from unfolding to subunit dissociation. Additionally, recent studies have highlighted the role of charge in such structural changes, but the mechanism is not completely understood. In this study, native top down (native TD) mass spectrometry was used to probe gas-phase structural changes of alcohol dehydrogenase (ADH, 4mer) under varying degrees of in-source activation. Changes in covalent backbone fragments produced by electron capture dissociation (ECD) or 193 nm ultraviolet photodissociation (UVPD) were correlated to structural changes of the ADH 4mer. ECD fragments indicated unfolding started at the N-terminus, and the charge states of UVPD fragments enabled monitoring of charge migration to the unfolded regions. Interestingly, UVPD fragments also indicated that the charge at the “unfolding” N-terminus of ADH decreased at high in-source activation energies after the initial increase. We proposed a possible “refolding-after-unfolding” mechanism, as further supported by monitoring hydrogen elimination from radical a-ions produced by UVPD at the N-terminus of ADH. However, “refolding-after-unfolding” with increasing in-source activation was not observed for charge-reduced ADH, which likely adopted compact structures that are resistant to both charge migration and unfolding. Combined, these results support a charge-directed unfolding mechanism for protein complexes. Overall, an experimental framework was outlined for utilizing native TD to generate structure-informative mass spectral signatures for protein complexes that complement other structure characterization techniques, such as ion mobility spectrometry and computational modeling.

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

A bipartite bacterial virulence factor targets the complement system and neutrophil activation

Abstract The complement system and neutrophils constitute the two main pillars of the host innate immune defense against infection by bacterial pathogens. Here, we identify T-Mac, a novel virulence factor of the periodontal pathogen Treponema denticola that allows bacteria to evade both defense systems. We show that T-Mac is expressed as a pre-protein that is cleaved into two functional units. The N-terminal fragment has two immunoglobulin-like domains and binds with high affinity to the major neutrophil chemokine receptors FPR1 and CXCR1, blocking N -formyl-Met-Leu-Phe- and IL-8-induced neutrophil chemotaxis and activation. The C-terminal fragment functions as a cysteine protease with a unique proteolytic activity and structure, which degrades several components of the complement system, such as C3 and C3b. Murine infection studies further reveal a critical T-Mac role in tissue damage and inflammation caused by bacterial infection. Collectively, these results disclose a novel innate immunity-evasion strategy, and open avenues for investigating the role of cysteine proteases and immunoglobulin-like domains of gram-positive and -negative bacterial pathogens.

Kurniyati, Kurni↗
DOE OSTI2021

Two human antibodies to a meningococcal serogroup B vaccine antigen enhance binding of complement Factor H by stabilizing the Factor H binding site

Microbial pathogens bind host complement regulatory proteins to evade the immune system. The bacterial pathogen Neisseria meningitidis, or meningococcus, binds several complement regulators, including human Factor H (FH). FH binding protein (FHbp) is a component of two licensed meningococcal vaccines and in mice FHbp elicits antibodies that inhibit binding of FH to FHbp, which defeat the bacterial evasion mechanism. However, humans vaccinated with FHbp develop antibodies that enhance binding of FH to the bacteria, which could limit the effectiveness of the vaccines. In the present study, we show that two vaccine-elicited antibody fragments (Fabs) isolated from different human subjects increase binding of complement FH to meningococcal FHbp by ELISA. The two Fabs have different effects on the kinetics of FH binding to immobilized FHbp as measured by surface plasmon resonance. The 1.7- and 2.0-Å resolution X-ray crystal structures of the Fabs in complexes with FHbp illustrate that the two Fabs bind to similar epitopes on the amino-terminal domain of FHbp, adjacent to the FH binding site. Superposition models of ternary complexes of each Fab with FHbp and FH show that there is likely minimal contact between the Fabs and FH. Collectively, the structures reveal that the Fabs enhance binding of FH to FHbp by altering the conformations and mobilities of two loops adjacent to the FH binding site of FHbp. In addition, the 1.5 Å-resolution structure of one of the isolated Fabs defines the structural rearrangements associated with binding to FHbp. The FH-enhancing human Fabs, which are mirrored in the human polyclonal antibody responses, have important implications for tuning the effectiveness of FHbp-based vaccines.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2023

Amino Acids Essential for the Assembly of Cellulose Synthase Complexes

Over 40 years ago, Andrew Staehelin's group showed that plant cellulose microfibrils are synthesized by protein complexes arranged in hexagonal arrays called 'particle rosettes'. Plant cellulose synthases (CESAs) differ from their bacterial ancestral forms by insertion of three unique sequences responsible for the assembly into compact multimeric units that aggregate further into rosettes. We used site-directed mutagenesis to replace amino acids within these sequences predicted to be essential for assembly and developed an in vivo method to determine the ability of mutated CesA1 transgenes to complement an Arabidopsis temperature-sensitive root-swelling1 (rsw1) mutant. Replacement of a Cys residue in the Class-Specific Region (CSR) or Pro417 and Arg453 of the Plant-Conserved Region (P-CR) rendered an AtCesA1 transgene unable to complement the rsw1 mutation. Despite an expected role for Arg457 in the trimerization of CESA proteins, AtCesA1 transgenes with Arg457Ala mutations were able to fully restore the wild-type phenotype in rsw1. Staehelin observed a third order of assembly in Micrasterias dendiculata, where hundreds of rosettes form large hexagonal arrays during the synthesis of secondary wall cellulose macrofibrils. We found by SEM of cytosolic faces of Micrasterias cell fragments that these arrays are associated with fields of regularly spaced slime secretion pore complexes.

arabidopsis↗
DOE OSTI2023

Dissecting the structural heterogeneity of proteins by native mass spectrometry

Abstract A single gene yields many forms of proteins via combinations of posttranscriptional/posttranslational modifications. Proteins also fold into higher‐order structures and interact with other molecules. The combined molecular diversity leads to the heterogeneity of proteins that manifests as distinct phenotypes. Structural biology has generated vast amounts of data, effectively enabling accurate structural prediction by computational methods. However, structures are often obtained heterologously under homogeneous states in vitro. The lack of native heterogeneity under cellular context creates challenges in precisely connecting the structural data to phenotypes. Mass spectrometry (MS) based proteomics methods can profile proteome composition of complex biological samples. Most MS methods follow the “bottom‐up” approach, which denatures and digests proteins into short peptide fragments for ease of detection. Coupled with chemical biology approaches, higher‐order structures can be probed via incorporation of covalent labels on native proteins that are maintained at the peptide level. Alternatively, native MS follows the “top‐down” approach and directly analyzes intact proteins under nondenaturing conditions. Various tandem MS activation methods can dissect the intact proteins for in‐depth structural elucidation. Herein, we review recent native MS applications for characterizing heterogeneous samples, including proteins binding to mixtures of ligands, homo/hetero‐complexes with varying stoichiometry, intrinsically disordered proteins with dynamic conformations, glycoprotein complexes with mixed modification states, and active membrane protein complexes in near‐native membrane environments. We summarize the benefits, challenges, and ongoing developments in native MS, with the hope to demonstrate an emerging technology that complements other tools by filling the knowledge gaps in understanding the molecular heterogeneity of proteins.

59 BASIC BIOLOGICAL SCIENCES↗
DOE OSTI2024

Versatile allelic replacement and self-excising integrative vectors for plasmid genome mutation and complementation

ABSTRACT The ability to better understand the function of proteins expressed by bacteria has typically relied upon the development of genetic mutant strains. This approach has been especially challenging for plasmid-encoded genes, as most of the previously described allelic replacement vectors are inefficient for plasmid genome mutation as they either rely on plasmid-derived counterselection toxins or depend on other strategies suchsacB,tetA, andrpsLwhich have been proven to be less efficient for mutant selection. Integrative vectors lack chromophore indicators, thus requiring laborious screening or excision of the vector’s backbone relies on the introduction of a flippase (FLP)-expressing plasmid. The allelic replacement vector, designated here as pDG1, expresses an X-Gal hydrolyzing enzyme (BgaB) that can be used for blue/white screening allowing identification of colonies that integrated and successfully removed the mutagenesis plasmid without a bias for those still carrying it. pDG1 was further improved by including the rhamnose-inducible Tse2 toxin as a potent counterselection system. The efficacy of pDG1 was validated by deleting portions of the plasmid-encoded VirB4/D4 type IV secretion system and aerobactin-synthesizing operons inSalmonella enterica. The integrative vectors, which contain an ΦC31attPsite and genes encoding ΦC31 integrase (int), can seamlessly integrate to target ΦC31attBonSalmonellaplasmids or chromosome. These vectors were improved by insertingbgaBand FLP-encoding genes so that, following integration, most of the vector’s backbone encompassingint,bgaB, and FLP genes can be excised by FLP, without the need for another FLP-expressing vector, creating white colonies carrying a stably integrated target gene. As such, we were able to integrate a 9.3-kb DNA fragment toSalmonellachromosome and flipped out most of the integrated vector in one step, leaving the target fragment in the chromosome. IMPORTANCE In spite of the dissemination of multidrug-resistant plasmids among Gram-negative pathogens, including those carrying virulence genes, vector tools for studying plasmid-born genes are lacking. The allelic replacement vectors can be used to generate plasmid or chromosomal mutations including markless point mutations. This is the first report describing a self-excising integrative vector that can be used as a stable single-copy complementing tool to study medically important pathogens includingin vivostudies without the need for antibiotic selection. Overall, our newly developed vectors can be applied for the assessment of the function of plasmid-encoded genes by specifically creating mutations, moving large operons between plasmids and to/from the chromosome, and complementing phenotypes associated with gene mutation. Furthermore, the vectors express chromophores for the detection of target gene modification or colony isolation, avoiding time-consuming screening procedures.

Microbiology↗
DOE OSTI2024

Antigen‐binding fragments with improved crystal lattice packing and enhanced conformational flexibility at the elbow region as crystallization chaperones

It has been shown previously that a set of three modifications—termed S1, Crystal Kappa, and elbow—act synergistically to improve the crystallizability of an antigen-binding fragment (Fab) framework. Here, we prepared a phage-displayed library and performed crystallization screenings to identify additional substitutions—located near the heavy-chain elbow region—which cooperate with the S1, Crystal Kappa, and elbow modifications to increase expression and improve crystallizability of the Fab framework even further. One substitution (K141Q) supports the signature Crystal Kappa-mediated Fab:Fab crystal lattice packing interaction. Another substitution (E172G) improves the compatibility of the elbow modification with the Fab framework by alleviating some of the strain incurred by the shortened and bulkier elbow linker region. A third substitution (F170W) generates a split-Fab conformation, resulting in a powerful crystal lattice packing interaction comprising the biological interaction interface between the variable heavy and light chain domains. In sum, we have used K141Q, E172G, and F170W substitutions—which complement the S1, Crystal Kappa, and elbow modifications—to generate a set of highly crystallizable Fab frameworks that can be used as chaperones to enable facile elucidation of Fab:antigen complex structures by x-ray crystallography.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗