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Reagent to label proteins via lysine isopeptide bonds
Covalently cross-linked pilus polymers displayed on the cell surface of Gram-positive bacteria are assembled by class C sortase enzymes. These pilus-specific transpeptidases located on the bacterial membrane catalyze a two-step protein ligation reaction—first, cleaving the LPXTG motif of one pilin protomer to form an acyl-enzyme intermediate, and second, joining the terminal threonine to the nucleophilic lysine residue residing within the pilin motif of another pilin protomer. Informed by the high-resolution crystal structures of corynebacterial pilus-specific sortase (SrtA) and by developing structural variants of the sortase enzyme whose catalytic pocket has been unmasked by activating mutations, we have developed new reagents capable of forming isopeptide bonds in vitro. The reagents disclosed herein can catalyze ligation of isolated SpaA domains in vitro provide a facile and versatile new platform for protein engineering and bio-conjugation that has major implications for biotechnology.
Proximity-based proteomics reveals the thylakoid lumen proteome in the cyanobacterium Synechococcus sp. PCC 7002
Abstract Cyanobacteria possess unique intracellular organization. Many proteomic studies have examined different features of cyanobacteria to learn about the intracellular structures and their respective functions. While these studies have made great progress in understanding cyanobacterial physiology, the conventional fractionation methods used to purify cellular structures have limitations; specifically, certain regions of cells cannot be purified with existing fractionation methods. Proximity-based proteomics techniques were developed to overcome the limitations of biochemical fractionation for proteomics. Proximity-based proteomics relies on spatiotemporal protein labeling followed by mass spectrometry of the labeled proteins to determine the proteome of the region of interest. We performed proximity-based proteomics in the cyanobacterium Synechococcus sp. PCC 7002 with the APEX2 enzyme, an engineered ascorbate peroxidase. We determined the proteome of the thylakoid lumen, a region of the cell that has remained challenging to study with existing methods, using a translational fusion between APEX2 and PsbU, a lumenal subunit of photosystem II. Our results demonstrate the power of APEX2 as a tool to study the cell biology of intracellular features and processes, including photosystem II assembly in cyanobacteria, with enhanced spatiotemporal resolution.
Proximity-based proteomics reveals the thylakoid lumen proteome in the cyanobacterium Synechococcus sp. PCC 7002
Cyanobacteria possess unique intracellular organization. Many proteomic studies have examined different features of cyanobacteria to learn about the intracellular structures and their respective functions. While these studies have made great progress in understanding cyanobacterial physiology, the conventional fractionation methods used to purify cellular structures have limitations; specifically, certain regions of cells cannot be purified with existing fractionation methods. Proximity-based proteomics techniques were developed to overcome the limitations of biochemical fractionation for proteomics. Proximity-based proteomics relies on spatiotemporal protein labeling followed by mass spectrometry of the labeled proteins to determine the proteome of the region of interest. We performed proximity-based proteomics in the cyanobacterium Synechococcus sp. PCC 7002 with the APEX2 enzyme, an engineered ascorbate peroxidase. We determined the proteome of the thylakoid lumen, a region of the cell that has remained challenging to study with existing methods, using a translational fusion between APEX2 and PsbU, a lumenal subunit of photosystem II. Our results demonstrate the power of APEX2 as a tool to study the cell biology of intracellular features and processes, including photosystem II assembly in cyanobacteria, with enhanced spatiotemporal resolution.
Protein specific fluorescent microspheres for labelling a protein
Highly fluorescent, stable and biocompatible microspheres are obtained by copolymerizing an acrylic monomer containing a covalent bonding group such as hydroxyl, amine or carboxyl, for example, hydroxyethylmethacrylate, with an addition polymerizable fluorescent comonomer such as dansyl allyl amine. A lectin or antibody is bound to the covalent site to provide cell specificity. When the microspheres are added to a cell suspension the marked microspheres will specifically label a cell membrane by binding to a specific receptor site thereon. The labeled membrane can then be detected by fluorescence of the fluorescent monomer.
An automated liquid jet for fluorescence dosimetry and microsecond radiolytic labeling of proteins
X-ray radiolytic labeling uses broadband X-rays for in situ hydroxyl radical labeling to map protein interactions and conformation. High flux density beams are essential to overcome radical scavengers. However, conventional sample delivery environments, such as capillary flow, limit the use of a fully unattenuated focused broadband beam. An alternative is to use a liquid jet, and we have previously demonstrated that use of this form of sample delivery can increase labeling by tenfold at an unfocused X-ray source. Here we report the first use of a liquid jet for automated inline quantitative fluorescence dosage characterization and sample exposure at a high flux density microfocused synchrotron beamline. Our approach enables exposure times in single-digit microseconds while retaining a high level of side-chain labeling. This development significantly boosts the method’s overall effectiveness and efficiency, generates high-quality data, and opens up the arena for high throughput and ultrafast time-resolved in situ hydroxyl radical labeling.
Activity-targeted metaproteomics uncovers rare syntrophic bacteria central to anaerobic community metabolism
Syntrophic microbial consortia can contribute significantly to the activity and function of anoxic ecosystems, yet are often too rare to study their in situ physiologies using traditional molecular methods. Here, in this study, we describe a technical innovation combining bioorthogonal non-canonical amino acid tagging (BONCAT), stable isotope probing, and metaproteomics to improve the recovery of proteins from active community members and track isotope incorporation. Both click chemistry-enabled cell-sorting and direct protein pulldown coupled to metaproteomics improved recovery of isotopically labeled proteins during acetate oxidation within a full-scale anaerobic digester. Resulting labeled protein expression profiles revealed elevated activity of a rare and uncharacterized syntrophic bacterium belonging to the family Natronincolaceae. BONCAT-based capture of newly translated proteins provided direct molecular evidence for the expression of a previously hypothesized oxidative glycine pathway for syntrophic acetate oxidation by this microorganism, showcasing the potential of targeted metaproteomics to characterize rare and active cells central to community metabolism in natural and engineered ecosystems.
De Novo Design of Proteins That Bind Naphthalenediimides, Powerful Photooxidants with Tunable Photophysical Properties
De novo protein design provides a framework to test our understanding of protein function and build proteins with cofactors and functions not found in nature. Here, we report the design of proteins designed to bind powerful photooxidants and the evaluation of the use of these proteins to generate diffusible small-molecule reactive species. Because excited-state dynamics are influenced by the dynamics and hydration of a photooxidant’s environment, it was important to not only design a binding site but also to evaluate its dynamic properties. Thus, we used computational design in conjunction with molecular dynamics (MD) simulations to design a protein, designated NBP (NDI Binding Protein), that held a naphthalenediimide (NDI), a powerful photooxidant, in a programmable molecular environment. Solution NMR confirmed the structure of the complex. We evaluated two NDI cofactors in this de novo protein using ultrafast pump–probe spectroscopy to evaluate light-triggered intra- and intermolecular electron transfer function. Moreover, we demonstrated the utility of this platform to activate multiple molecular probes for protein labeling.
Light controlled reversible Michael addition of cysteine: a new tool for dynamic site-specific labeling of proteins
Cysteine-based Michael addition is a widely employed strategy for covalent conjugation of proteins, peptides, and drugs. The covalent reaction is irreversible in most cases, leading to a lack of control over the process. Utilizing spectroscopic analyses along with X-ray crystallographic studies, we demonstrate Michael addition of an engineered cysteine residue in human Cellular Retinol Binding Protein II (hCRBPII) with a coumarin analog that creates a non-fluorescent complex. UV-illumination reverses the conjugation, yielding a fluorescent species, presumably through a retro-Michael process. Furthermore, this series of events can be repeated between a bound and non-bound form of the cysteine reversibly, resulting in the ON–OFF control of fluorescence. The details of the mechanism of photoswitching was illuminated by recapitulation of the process in light irradiated single crystals, confirming the mechanism at atomic resolution.
Characterizing intracellular proteomes for microbes: An experimental approach using label-free protein quantitation
Genomic and transcriptomic studies generate a rich source of molecular information; however, neither a static genome nor the presence of a messenger RNA provides the most direct insight into the functional state of a microbial cell at any given time. Rather, it is the ensemble of proteins (i.e., proteome) that is the primary workhorse for most biological processes that are concurrent and coordinately active in a living cell. Currently, mass-spectrometry-based (MS) technologies provide unprecedented information into the composition of the proteome, shedding light on the numerous complex biological processes actively shaping observed phenotypes. Herein, we detail a protocol for the exploration of intracellular proteomes through the large-scale, unbiased identification of proteins and their relative abundances using liquid chromatography (LC) coupled to tandem mass spectrometry (MS/MS). In general, this protocol is applicable to both microbial and eukaryotic systems.
SoDA-PAL Protein Profiling Proteomics of Sorgoleone Catabolizing Microbial Isolate Acinetobacter pittii SO1
PerCon SFA project mass spectrometry data analysis of SoDA-PAL photoaffinity probe labeled proteins and global proteome profiling of microbial isolate A. pittii SO1 sorgoleone interactome grown under three different culture conditions using acetate and sorgoleone.
Correlative nano-imaging of metals and proteins in primary neurons by synchrotron X-ray fluorescence and STED super resolution microscopy: Experimental validation
It is becoming increasingly clear that biological metals such as iron, copper or zinc are involved in synaptic functions, and in particular in the mechanisms of synaptogenesis and subsequent plasticity. Understanding the role of metals on synaptic functions is a difficult challenge due to the very low concentration of these elements in neurons and to the submicrometer size of synaptic compartments. To address this challenge we have developed a correlative nano-imaging approach combining metal and protein detection. First, stimulated emission depletion (STED) microscopy, a super resolution optical microscopy technique, is applied to locate fluorescently labeled proteins. Then, synchrotron radiation induced X-ray fluorescence (SXRF) is performed on the same regions of interest, e.g. synaptic compartments. Here, we present here the principle scheme that allows this correlative nano-imaging and its experimental validation. We applied this correlative nano-imaging to the study of the physiological distribution of metals in synaptic compartments of primary rat hippocampal neurons. We thus compared the nanometric distribution of metals with that of synaptic proteins, such as PSD95 or cytoskeleton proteins. Compared to correlative imaging approaches currently used to characterize synaptic structures, such as electron microscopy correlated with optical fluorescence, our approach allows for ultra-sensitive detection of trace metals using highly focused synchrotron radiation beams. We provide proof-of-principle for correlative imaging of metals and proteins at the synaptic scale and discuss the present limitations and future developments in this area.
Fed-batch production of deuterated protein in Escherichia coli for neutron scattering experimentation
Neutron scattering is a powerful technique for determining the structure and dynamics of biological materials in a variety of environmental conditions. A distinguishing property of the neutron is its sensitivity to detecting hydrogen and distinguishing it from its isotope deuterium. This enables unique types of experiments that take advantage of this differential sensitivity called isotopic contrast variation. Using this approach, the chemistry of the system is not changed, but the visibility of individual sample components can be tuned by varying the deuterium content of the system under investigation. Deuterated proteins are commonly produced in bacterial systems that are adapted to growth in D2O minimal media. To maximize the yield of deuterium-labeled protein and efficiently utilize D2O and occasionally the deuterated substrate, fed-batch processes are routinely used to maximize biomass production without compromising cell viability. A step-by-step procedure will be described along with a case study of the production of deuterated green fluorescent protein. Limitations of the process will also be discussed.
Fabrication of poly(ethylene glycol) hydrogel microstructures using photolithography
The fabrication of hydrogel microstructures based upon poly(ethylene glycol) diacrylates, dimethacrylates, and tetraacrylates patterned photolithographically on silicon or glass substrates is described. A silicon/silicon dioxide surface was treated with 3-(trichlorosilyl)propyl methacrylate to form a self-assembled monolayer (SAM) with pendant acrylate groups. The SAM presence on the surface was verified using ellipsometry and time-of-flight secondary ion mass spectrometry. A solution containing an acrylated or methacrylated poly(ethylene glycol) derivative and a photoinitiator (2,2-dimethoxy-2-phenylacetophenone) was spin-coated onto the treated substrate, exposed to 365 nm ultraviolet light through a photomask, and developed with either toluene, water, or supercritical CO2. As a result of this process, three-dimensional, cross-linked PEG hydrogel microstructures were immobilized on the surface. Diameters of cylindrical array members were varied from 600 to 7 micrometers by the use of different photomasks, while height varied from 3 to 12 micrometers, depending on the molecular weight of the PEG macromer. In the case of 7 micrometers diameter elements, as many as 400 elements were reproducibly generated in a 1 mm2 square pattern. The resultant hydrogel patterns were hydrated for as long as 3 weeks without delamination from the substrate. In addition, micropatterning of different molecular weights of PEG was demonstrated. Arrays of hydrogel disks containing an immobilized protein conjugated to a pH sensitive fluorophore were also prepared. The pH sensitivity of the gel-immobilized dye was similar to that in an aqueous buffer, and no leaching of the dye-labeled protein from the hydrogel microstructure was observed over a 1 week period. Changes in fluorescence were also observed for immobilized fluorophore labeled acetylcholine esterase upon the addition of acetyl acholine.
Casein kinase II protein kinase is bound to lamina-matrix and phosphorylates lamin-like protein in isolated pea nuclei
A casein kinase II (CK II)-like protein kinase was identified and partially isolated from a purified envelope-matrix fraction of pea (Pisum sativum L.) nuclei. When [gamma-32P]ATP was directly added to the envelope-matrix preparation, the three most heavily labeled protein bands had molecular masses near 71, 48, and 46 kDa. Protein kinases were removed from the preparation by sequential extraction with Triton X-100, EGTA, 0.3 M NaCl, and a pH 10.5 buffer, but an active kinase still remained bound to the remaining lamina-matrix fraction after these treatments. This kinase had properties resembling CK II kinases previously characterized from animal and plant sources: it preferred casein as an artificial substrate, could use GTP as efficiently as ATP as the phosphoryl donor, was stimulated by spermine, was calcium independent, and had a catalytic subunit of 36 kDa. Some animal and plant CK II kinases have regulatory subunits near 29 kDa, and a lamina-matrix-bound protein of this molecular mass was recognized on immunoblot by anti-Drosophila CK II polyclonal antibodies. Also found associated with the envelope-matrix fraction of pea nuclei were p34cdc2-like and Ca(2+)-dependent protein kinases, but their properties could not account for the protein kinase activity bound to the lamina. The 71-kDa substrate of the CK II-like kinase was lamin A-like, both in its molecular mass and in its cross-reactivity with anti-intermediate filament antibodies. Lamin phosphorylation is considered a crucial early step in the entry of cells into mitosis, so lamina-bound CK II kinases may be important control points for cellular proliferation.
Preparation and Characterization of Fluorescent Derivatives of Chicken Egg White Lysozyme
Fluorescence is one of the most versatile and powerful tools for the study of macromolecules. While most proteins are intrinsically fluorescent, working at crystallization concentrations require the use of covalently prepared derivatives added as tracers. This approach requires derivatives that do not markedly affect the crystal packing. We have prepared a number of fluorescent derivatives of chicken egg white lysozyme with probes bound to one of two different sites on the protein molecule. Lucifer yellow, cascade blue, and 5-(2-aminoethyl)aminonapthalene-l-sulfonic acid (EDANS) have been attached to the side chain carboxyl of asp101 using a carbodiimide coupling procedure. asp101 lies within the active site cleft, and it is believed that the probes are at least partially "buried" within that cleft. Lucifer yellow and MANS probes with iodoacetamide reactive groups have been bound to hisl5, located on the "back side" of the molecule relative to the active site. The fluorescently labeled protein is readily purified from the starting material by cation exchange chromatography. All the derivatives fluoresce in both the solution and the crystalline states. Fluorescence characterization has focused on determining the bound probe quantum yields, lifetimes, absorption and emission spectra, and quenching by added solutes in comparison to the free probe. No appreciable changes are found in the lifetimes of any of the probes except for cascade blue, where Tau(sub free) = 3.52 ns vrs Tau(sub bound) = 2.8 ns. Spectral shifts are found in most cases. Particularly strong quenching upon binding is found in the case of the cascade blue derivative, likely due to probe interactions with the active site cleft. While none of the asp101 bound probes are well quenched by commonly employed solutes, such as potassium and sodium iodide, acrylamide, primuline, the chloride salts of manganese, cesium, and cobalt, trifluoroacetamide, trichloroethanol, and thallium iodide, in those cases where quenching is observed the bound probe is less efficiently quenched relative to the free probe. This indicates that the bound probes are less accessible to the bulk solution, an expected finding for attachment within the active site cleft. Attempts have been made to bind other molecules to these sites, with varying success. Interestingly, all three probes contain one or more sulfonate ((Ar-S03)-) groups. We have not been successful in binding analogous probes without sulfate groups such as pyrene, or with derivatized sulfonate groups such as dansyl type probes, analogous to MANS but where the sulfonate group is derivatized, Ar-S02-N2C2H7. None of the probes is rigidly bound to the protein, i.e., they all have a probe motion superimposed on that of the protein.
Subdomain cryo-EM structure of nodaviral replication protein A crown complex provides mechanistic insights into RNA genome replication
For positive-strand RNA [(+)RNA] viruses, the major target for antiviral therapies is genomic RNA replication, which occurs at poorly understood membrane-bound viral RNA replication complexes. Recent cryoelectron microscopy (cryo-EM) of nodavirus RNA replication complexes revealed that the viral double-stranded RNA replication template is coiled inside a 30- to 90-nm invagination of the outer mitochondrial membrane, whose necked aperture to the cytoplasm is gated by a 12-fold symmetric, 35-nm diameter “crown” complex that contains multifunctional viral RNA replication protein A. Here we report optimizing cryo-EM tomography and image processing to improve crown resolution from 33 to 8.5 Å. This resolves the crown into 12 distinct vertical segments, each with 3 major subdomains: A membrane-connected basal lobe and an apical lobe that together comprise the ~19-nm-diameter central turret, and a leg emerging from the basal lobe that connects to the membrane at ~35-nm diameter. Despite widely varying replication vesicle diameters, the resulting two rings of membrane interaction sites constrain the vesicle neck to a highly uniform shape. Labeling protein A with a His-tag that binds 5-nm Ni-nanogold allowed cryo-EM tomography mapping of the C terminus of protein A to the apical lobe, which correlates well with the predicted structure of the C-proximal polymerase domain of protein A. These and other results indicate that the crown contains 12 copies of protein A arranged basally to apically in an N-to-C orientation. Moreover, the apical polymerase localization has significant mechanistic implications for template RNA recruitment and (-) and (+)RNA synthesis.
Influence of Hyperfine Coupling Strain on Two-Dimensional ESEEM Spectra from I = 1/2 Nuclei
HYSCORE, a variant of two-dimensional ESEEM spectroscopy, is currently one of the most effective tools in high-resolution solid-state EPR spectroscopy. Our systematic studies of hydrogen bonds with semiquinones in 15 N-labeled proteins have previously provided us with an extensive collection of 15 N HYSCORE spectra produced by N–H…O nitrogen donors. Examination of these spectra has indicated in several cases lineshape distortions not described by available theoretical models. We were able to explain all observed artificial phenomena in the 15 N spectra of semiquinones considering a single mechanism—strain of the isotropic hyperfine interaction. In this review article, we introduce the HYSCORE experiment and the theoretical background for spectra of I = 1/2 nuclei in orientation-disordered samples. We then present a description of the observed lineshape distortions and an explanation of the hyperfine strain’s influence on the experimental spectra. In the final part of the article, we discuss (i) similar lineshape distortions found in previously published spectra from 15 N, 29 Si, 31 P, and 57 Fe nuclei that can be explained using the approach applied for the analysis of the 15 N spectra of semiquinones, and (ii) a recently published analysis of 31P spectra with a different type of lineshape distortion based on a more sophisticated method, where the strength of anisotropic coupling is correlated with the sign and value of the isotropic constant in different conformations of a Cu(II) complex.