Short prokaryotic Argonaute systems trigger cell death upon detection of invading DNA.

Argonaute proteins use single-stranded RNA or DNA guides to target complementary nucleic acids. This allows eukaryotic Argonaute proteins to mediate RNA interference and long prokaryotic Argonaute proteins to interfere with invading nucleic acids. The function and mechanisms of the phylogenetically distinct short prokaryotic Argonaute proteins remain poorly understood. We demonstrate that short prokaryotic Argonaute and the associated TIR-APAZ (SPARTA) proteins form heterodimeric complexes. Upon guide RNA-mediated target DNA binding, four SPARTA heterodimers form oligomers in which TIR domain-mediated NAD(P)ase activity is unleashed. When expressed in Escherichia coli, SPARTA is activated in the presence of highly transcribed multicopy plasmid DNA, which causes cell death through NAD(P)+ depletion. This results in the removal of plasmid-invaded cells from bacterial cultures. Furthermore, we show that SPARTA can be repurposed for the programmable detection of DNA sequences. In conclusion, our work identifies SPARTA as a prokaryotic immune system that reduces cell viability upon RNA-guided detection of invading DNA.

Probing DNA - Transcription Factor Interactions Using Single-Molecule Fluorescence Detection in Nanofluidic Devices.

Single-molecule fluorescence detection offers powerful ways to study biomolecules and their complex interactions. Here, nanofluidic devices and camera-based, single-molecule Förster resonance energy transfer (smFRET) detection are combined to study the interactions between plant transcription factors of the auxin response factor (ARF) family and DNA oligonucleotides that contain target DNA response elements. In particular, it is shown that the binding of the unlabeled ARF DNA binding domain (ARF-DBD) to donor and acceptor labeled DNA oligonucleotides can be detected by changes in the FRET efficiency and changes in the diffusion coefficient of the DNA. In addition, this data on fluorescently labeled ARF-DBDs suggest that, at nanomolar concentrations, ARF-DBDs are exclusively present as monomers. In general, the fluidic framework of freely diffusing molecules minimizes potential surface-induced artifacts, enables high-throughput measurements, and proved to be instrumental in shedding more light on the interactions between ARF-DBDs monomers and between ARF-DBDs and their DNA response element.

Two-Component Nanoparticle Vaccine Displaying Glycosylated Spike S1 Domain Induces Neutralizing Antibody Response against SARS-CoV-2 Variants.

Vaccines pave the way out of the SARS-CoV-2 pandemic. Besides mRNA and adenoviral vector vaccines, effective protein-based vaccines are needed for immunization against current and emerging variants. We have developed a virus-like particle (VLP)-based vaccine using the baculovirus-insect cell expression system, a robust production platform known for its scalability, low cost, and safety. Baculoviruses were constructed encoding SARS-CoV-2 spike proteins: full-length S, stabilized secreted S, or the S1 domain. Since subunit S only partially protected mice from SARS-CoV-2 challenge, we produced S1 for conjugation to bacteriophage AP205 VLP nanoparticles using tag/catcher technology. The S1 yield in an insect-cell bioreactor was ∼11 mg/liter, and authentic protein folding, efficient glycosylation, partial trimerization, and ACE2 receptor binding was confirmed. Prime-boost immunization of mice with 0.5 µg S1-VLPs showed potent neutralizing antibody responses against Wuhan and UK/B.1.1.7 SARS-CoV-2 variants. This two-component nanoparticle vaccine can now be further developed to help alleviate the burden of COVID-19. IMPORTANCE Vaccination is essential to reduce disease severity and limit the transmission of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). Protein-based vaccines are useful to vaccinate the world population and to boost immunity against emerging variants. Their safety profiles, production costs, and vaccine storage temperatures are advantageous compared to mRNA and adenovirus vector vaccines. Here, we use the versatile and scalable baculovirus expression vector system to generate a two-component nanoparticle vaccine to induce potent neutralizing antibody responses against SARS-CoV-2 variants. These nanoparticle vaccines can be quickly adapted as boosters by simply updating the antigen component.

Characterization of a nitrite-reducing octaheme hydroxylamine oxidoreductase that lacks the tyrosine cross-link.

The hydroxylamine oxidoreductase (HAO) family consists of octaheme proteins that harbor seven bis-His ligated electron-transferring hemes and one 5-coordinate catalytic heme with His axial ligation. Oxidative HAOs have a homotrimeric configuration with the monomers covalently attached to each other via a unique double cross-link between a Tyr residue and the catalytic heme moiety of an adjacent subunit. This cross-linked active site heme, termed the P460 cofactor, has been hypothesized to modulate enzyme reactivity toward oxidative catalysis. Conversely, the absence of this cross-link is predicted to favor reductive catalysis. However, this prediction has not been directly tested. In this study, an HAO homolog that lacks the heme-Tyr cross-link (HAOr) was purified to homogeneity from the nitrite-dependent anaerobic ammonium-oxidizing (anammox) bacterium Kuenenia stuttgartiensis, and its catalytic and spectroscopic properties were assessed. We show that HAOr reduced nitrite to nitric oxide and also reduced nitric oxide and hydroxylamine as nonphysiological substrates. In contrast, HAOr was not able to oxidize hydroxylamine or hydrazine supporting the notion that cross-link-deficient HAO enzymes are reductases. Compared with oxidative HAOs, we found that HAOr harbors an active site heme with a higher (at least 80 mV) midpoint potential and a much lower degree of porphyrin ruffling. Based on the physiology of anammox bacteria and our results, we propose that HAOr reduces nitrite to nitric oxide in vivo, providing anammox bacteria with NO, which they use to activate ammonium in the absence of oxygen.

Architecture of DNA elements mediating ARF transcription factor binding and auxin-responsive gene expression in Arabidopsis.

The hormone auxin controls many aspects of the plant life cycle by regulating the expression of thousands of genes. The transcriptional output of the nuclear auxin signaling pathway is determined by the activity of AUXIN RESPONSE transcription FACTORs (ARFs), through their binding to cis-regulatory elements in auxin-responsive genes. Crystal structures, in vitro, and heterologous studies have fueled a model in which ARF dimers bind with high affinity to distinctly spaced repeats of canonical AuxRE motifs. However, the relevance of this "caliper" model, and the mechanisms underlying the binding affinities in vivo, have remained elusive. Here we biochemically and functionally interrogate modes of ARF-DNA interaction. We show that a single additional hydrogen bond in Arabidopsis ARF1 confers high-affinity binding to individual DNA sites. We demonstrate the importance of AuxRE cooperativity within repeats in the Arabidopsis TMO5 and IAA11 promoters in vivo. Meta-analysis of transcriptomes further reveals strong genome-wide association of auxin response with both inverted (IR) and direct (DR) AuxRE repeats, which we experimentally validated. The association of these elements with auxin-induced up-regulation (DR and IR) or down-regulation (IR) was correlated with differential binding affinities of A-class and B-class ARFs, respectively, suggesting a mechanistic basis for the distinct activity of these repeats. Our results support the relevance of high-affinity binding of ARF transcription factors to uniquely spaced DNA elements in vivo, and suggest that differential binding affinities of ARF subfamilies underlie diversity in cis-element function.

Design principles of a minimal auxin response system.

Auxin controls numerous growth processes in land plants through a gene expression system that modulates ARF transcription factor activity1-3. Gene duplications in families encoding auxin response components have generated tremendous complexity in most land plants, and neofunctionalization enabled various unique response outputs during development1,3,4. However, it is unclear what fundamental biochemical principles underlie this complex response system. By studying the minimal system in Marchantia polymorpha, we derive an intuitive and simple model where a single auxin-dependent A-ARF activates gene expression. It is antagonized by an auxin-independent B-ARF that represses common target genes. The expression patterns of both ARF proteins define developmental zones where auxin response is permitted, quantitatively tuned or prevented. This fundamental design probably represents the ancestral system and formed the basis for inflated, complex systems.

Discovery of a functional, contracted heme-binding motif within a multiheme cytochrome.

Anaerobic ammonium-oxidizing (anammox) bacteria convert nitrite and ammonium via nitric oxide (NO) and hydrazine into dinitrogen gas by using a diverse array of proteins, including numerous c-type cytochromes. Many new catalytic and spectroscopic properties of c-type cytochromes have been unraveled by studies on the biochemical pathways underlying the anammox process. The unique anammox intermediate hydrazine is produced by a multiheme cytochrome c protein, hydrazine synthase, through the comproportionation of ammonium and NO and the input of three electrons. It is unclear how these electrons are delivered to hydrazine synthase. Here, we report the discovery of a functional tetraheme c-type cytochrome from the anammox bacterium Kuenenia stuttgartiensis with a naturally-occurring contracted Cys-Lys-Cys-His (CKCH) heme-binding motif, which is encoded in the hydrazine synthase gene cluster. The purified tetraheme protein (named KsTH) exchanged electrons with hydrazine synthase. Complementary spectroscopic techniques revealed that this protein harbors four low-spin hexa-coordinated hemes with His/Lys (heme 1), His/Cys (heme 2), and two His/His ligations (hemes 3 and 4). A genomic database search revealed that c-type cytochromes with a contracted CXCH heme-binding motif are present throughout the bacterial and archaeal domains in the tree of life, suggesting that this heme recognition site may be employed by many different groups of microorganisms.

Iron assimilation and utilization in anaerobic ammonium oxidizing bacteria.

The most abundant transition metal in biological systems is iron. It is incorporated into protein cofactors and serves either catalytic, redox or regulatory purposes. Anaerobic ammonium oxidizing (anammox) bacteria rely heavily on iron-containing proteins - especially cytochromes - for their energy conservation, which occurs within a unique organelle, the anammoxosome. Both their anaerobic lifestyle and the presence of an additional cellular compartment challenge our understanding of iron processing. Here, we combine existing concepts of iron uptake, utilization and metabolism, and cellular fate with genomic and still limited biochemical and physiological data on anammox bacteria to propose pathways these bacteria may employ.

SMA-SH: Modified Styrene-Maleic Acid Copolymer for Functionalization of Lipid Nanodiscs.

Challenges in purification and subsequent functionalization of membrane proteins often complicate their biochemical and biophysical characterization. Purification of membrane proteins generally involves replacing the lipids surrounding the protein with detergent molecules, which can affect protein structure and function. Recently, it was shown that styrene-maleic acid copolymers (SMA) can dissolve integral membrane proteins from biological membranes into nanosized discs. Within these nanoparticles, proteins are embedded in a patch of their native lipid bilayer that is stabilized in solution by the amphipathic polymer that wraps the disc like a bracelet. This approach for detergent-free purification of membrane proteins has the potential to greatly simplify purification but does not facilitate conjugation of functional compounds to the membrane proteins. Often, such functionalization involves laborious preparation of protein variants and optimization of labeling procedures to ensure only minimal perturbation of the protein. Here, we present a strategy that circumvents several of these complications through modifying SMA by grafting the polymer with cysteamine. The reaction results in SMA that has solvent-exposed sulfhydrils (SMA-SH) and allows tuning of the coverage with SH groups. Size exclusion chromatography, dynamic light scattering, and transmission electron microscopy demonstrate that SMA-SH dissolves lipid bilayer membranes into lipid nanodiscs, just like SMA. In addition, we demonstrate that, just like SMA, SMA-SH solubilizes proteoliposomes into protein-loaded nanodiscs. We covalently modify SMA-SH-lipid nanodiscs using thiol-reactive derivatives of Alexa Fluor 488 and biotin. Thus, SMA-SH promises to simultaneously tackle challenges in purification and functionalization of membrane proteins.

Gradual Folding of an Off-Pathway Molten Globule Detected at the Single-Molecule Level.

Molten globules (MGs) are compact, partially folded intermediates that are transiently present during folding of many proteins. These intermediates reside on or off the folding pathway to native protein. Conformational evolution during folding of off-pathway MGs is largely unexplored. Here, we characterize the denaturant-dependent structure of apoflavodoxin's off-pathway MG. Using single-molecule fluorescence resonance energy transfer (smFRET), we follow conversion of unfolded species into MG down to denaturant concentrations that favor formation of native protein. Under strongly denaturing conditions, fluorescence resonance energy transfer histograms show a single peak, arising from unfolded protein. The smFRET efficiency distribution shifts to higher value upon decreasing denaturant concentration because the MG folds. Strikingly, upon approaching native conditions, the fluorescence resonance energy transfer efficiency of the MG rises above that of native protein. Thus, smFRET exposes the misfolded nature of apoflavodoxin's off-pathway MG. We show that conversion of unfolded into MG protein is a gradual, second-order-like process that simultaneously involves separate regions within the polypeptide.

Double Electron-Electron Spin Resonance Tracks Flavodoxin Folding.

Protein folding is one of the important challenges in biochemistry. Understanding the folding process requires mapping of protein structure as it folds. Here we test the potential of distance determination between paramagnetic spin-labels by a pulsed electron paramagnetic resonance method. We use double electron-electron spin resonance (DEER) to study the denaturant-dependent equilibrium folding of flavodoxin. This flavoprotein is spin-labeled with MTSL ((1-oxy-,2,2,5,5-tetramethyl-d-pyrroline-3-methyl)-methanethiosulfonate) at positions 69 and 131. We find that nativelike spin-label separation dominates the distance distributions up to 0.8 M guanidine hydrochloride. At 2.3 M denaturant, the distance distributions show an additional component, which we attribute to a folding intermediate. Upon further increase of denaturant concentration, the protein expands and evidence for a larger number of conformations than in the native state is found. We thus demonstrate that DEER is a versatile technique to expand the arsenal of methods for investigating how proteins fold.

Rise-time of FRET-acceptor fluorescence tracks protein folding.

Uniform labeling of proteins with fluorescent donor and acceptor dyes with an equimolar ratio is paramount for accurate determination of Förster resonance energy transfer (FRET) efficiencies. In practice, however, the labeled protein population contains donor-labeled molecules that have no corresponding acceptor. These FRET-inactive donors contaminate the donor fluorescence signal, which leads to underestimation of FRET efficiencies in conventional fluorescence intensity and lifetime-based FRET experiments. Such contamination is avoided if FRET efficiencies are extracted from the rise time of acceptor fluorescence upon donor excitation. The reciprocal value of the rise time of acceptor fluorescence is equal to the decay rate of the FRET-active donor fluorescence. Here, we have determined rise times of sensitized acceptor fluorescence to study the folding of double-labeled apoflavodoxin molecules and show that this approach tracks the characteristics of apoflavodoxin's complex folding pathway.

Illuminating the off-pathway nature of the molten globule folding intermediate of an α-ß parallel protein.

Partially folded protein species transiently form during folding of most proteins. Often, these species are molten globules, which may be on- or off-pathway to the native state. Molten globules are ensembles of interconverting protein conformers that have a substantial amount of secondary structure, but lack virtually all tertiary side-chain packing characteristics of natively folded proteins. Due to solvent-exposed hydrophobic groups, molten globules are prone to aggregation, which can have detrimental effects on organisms. The molten globule observed during folding of the 179-residue apoflavodoxin from Azotobacter vinelandii is off-pathway, as it has to unfold before native protein can form. Here, we study folding of apoflavodoxin and characterize its molten globule using fluorescence spectroscopy and Förster Resonance Energy Transfer (FRET). Apoflavodoxin is site-specifically labeled with fluorescent donor and acceptor dyes, utilizing dye-inaccessibility of Cys69 in cofactor-bound protein. Donor (i.e., Alexa Fluor 488) is covalently attached to Cys69 in all apoflavodoxin variants used. Acceptor (i.e., Alexa Fluor 568) is coupled to Cys1, Cys131 and Cys178, respectively. Our FRET data show that apoflavodoxin's molten globule forms in a non-cooperative manner and that its N-terminal 69 residues fold last. In addition, striking conformational differences between molten globule and native protein are revealed, because the inter-label distances sampled in the 111-residue C-terminal segment of the molten globule are shorter than observed for native apoflavodoxin. Thus, FRET sheds light on the off-pathway nature of the molten globule during folding of an α-ß parallel protein.

Fluorescence of Alexa fluor dye tracks protein folding.

Fluorescence spectroscopy is an important tool for the characterization of protein folding. Often, a protein is labeled with appropriate fluorescent donor and acceptor probes and folding-induced changes in Förster Resonance Energy Transfer (FRET) are monitored. However, conformational changes of the protein potentially affect fluorescence properties of both probes, thereby profoundly complicating interpretation of FRET data. In this study, we assess the effects protein folding has on fluorescence properties of Alexa Fluor 488 (A488), which is commonly used as FRET donor. Here, A488 is covalently attached to Cys69 of apoflavodoxin from Azotobacter vinelandii. Although coupling of A488 slightly destabilizes apoflavodoxin, the three-state folding of this protein, which involves a molten globule intermediate, is unaffected. Upon folding of apoflavodoxin, fluorescence emission intensity of A488 changes significantly. To illuminate the molecular sources of this alteration, we applied steady state and time-resolved fluorescence techniques. The results obtained show that tryptophans cause folding-induced changes in quenching of Alexa dye. Compared to unfolded protein, static quenching of A488 is increased in the molten globule. Upon populating the native state both static and dynamic quenching of A488 decrease considerably. We show that fluorescence quenching of Alexa Fluor dyes is a sensitive reporter of conformational changes during protein folding.

Distant residues mediate picomolar binding affinity of a protein cofactor.

Numerous proteins require cofactors to be active. Computer simulations suggest that cooperative interaction networks achieve optimal cofactor binding. There is a need for the experimental identification of the residues crucial for stabilizing these networks and thus for cofactor binding. Here we investigate the electron transporter flavodoxin, which contains flavin mononucleotide as non-covalently bound cofactor. We show that after binding flavin mononucleotide with nanomolar affinity, the protein relaxes extremely slowly (time constant ~5 days) to an energetically more favourable state with picomolar-binding affinity. Rare small-scale openings of this state are revealed through H/D exchange of N(3)H of flavin. We find that H/D exchange can pinpoint amino acids that cause tight cofactor binding. These hitherto unknown residues are dispersed throughout the structure, and many are located distantly from the flavin and seem irrelevant to flavodoxin's function. Quantification of the thermodynamics of ligand binding is important for understanding, engineering, designing and evolving ligand-binding proteins.

Cofactor binding protects flavodoxin against oxidative stress.

In organisms, various protective mechanisms against oxidative damaging of proteins exist. Here, we show that cofactor binding is among these mechanisms, because flavin mononucleotide (FMN) protects Azotobacter vinelandii flavodoxin against hydrogen peroxide-induced oxidation. We identify an oxidation sensitive cysteine residue in a functionally important loop close to the cofactor, i.e., Cys69. Oxidative stress causes dimerization of apoflavodoxin (i.e., flavodoxin without cofactor), and leads to consecutive formation of sulfinate and sulfonate states of Cys69. Use of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) reveals that Cys69 modification to a sulfenic acid is a transient intermediate during oxidation. Dithiothreitol converts sulfenic acid and disulfide into thiols, whereas the sulfinate and sulfonate forms of Cys69 are irreversible with respect to this reagent. A variable fraction of Cys69 in freshly isolated flavodoxin is in the sulfenic acid state, but neither oxidation to sulfinic and sulfonic acid nor formation of intermolecular disulfides is observed under oxidising conditions. Furthermore, flavodoxin does not react appreciably with NBD-Cl. Besides its primary role as redox-active moiety, binding of flavin leads to considerably improved stability against protein unfolding and to strong protection against irreversible oxidation and other covalent thiol modifications. Thus, cofactors can protect proteins against oxidation and modification.

Topological switching between an alpha-beta parallel protein and a remarkably helical molten globule.

Partially folded protein species transiently exist during folding of most proteins. Often these species are molten globules, which may be on- or off-pathway to native protein. Molten globules have a substantial amount of secondary structure but lack virtually all the tertiary side-chain packing characteristic of natively folded proteins. These ensembles of interconverting conformers are prone to aggregation and potentially play a role in numerous devastating pathologies, and thus attract considerable attention. The molten globule that is observed during folding of apoflavodoxin from Azotobacter vinelandii is off-pathway, as it has to unfold before native protein can be formed. Here we report that this species can be trapped under nativelike conditions by substituting amino acid residue F44 by Y44, allowing spectroscopic characterization of its conformation. Whereas native apoflavodoxin contains a parallel beta-sheet surrounded by alpha-helices (i.e., the flavodoxin-like or alpha-beta parallel topology), it is shown that the molten globule has a totally different topology: it is helical and contains no beta-sheet. The presence of this remarkably nonnative species shows that single polypeptide sequences can code for distinct folds that swap upon changing conditions. Topological switching between unrelated protein structures is likely a general phenomenon in the protein structure universe.

Macromolecular crowding compacts unfolded apoflavodoxin and causes severe aggregation of the off-pathway intermediate during apoflavodoxin folding.

To understand how proteins fold in vivo, it is important to investigate the effects of macromolecular crowding on protein folding. Here, the influence of crowding on in vitro apoflavodoxin folding, which involves a relatively stable off-pathway intermediate with molten globule characteristics, is reported. To mimic crowded conditions in cells, dextran 20 at 30% (w/v) is used, and its effects are measured by a diverse combination of optical spectroscopic techniques. Fluorescence correlation spectroscopy shows that unfolded apoflavodoxin has a hydrodynamic radius of 37+/-3 A at 3 M guanidine hydrochloride. Förster resonance energy transfer measurements reveal that subsequent addition of dextran 20 leads to a decrease in protein volume of about 29%, which corresponds to an increase in protein stability of maximally 1.1 kcal mol(-1). The compaction observed is accompanied by increased secondary structure, as far-UV CD spectroscopy shows. Due to the addition of crowding agent, the midpoint of thermal unfolding of native apoflavodoxin rises by 2.9 degrees C. Although the stabilization observed is rather limited, concomitant compaction of unfolded apoflavodoxin restricts the conformational space sampled by the unfolded state, and this could affect kinetic folding of apoflavodoxin. Most importantly, crowding causes severe aggregation of the off-pathway folding intermediate during apoflavodoxin folding in vitro. However, apoflavodoxin can be over expressed in the cytoplasm of Escherichia coli, where it efficiently folds to its functional native form at high yield without noticeable problems. Apparently, in the cell, apoflavodoxin requires the help of chaperones like Trigger Factor and the DnaK system for efficient folding.