Evaluation of FRET X for single-molecule protein fingerprinting.

Single-molecule protein identification is an unrealized concept with potentially ground-breaking applications in biological research. We propose a method called FRET X (Förster Resonance Energy Transfer via DNA eXchange) fingerprinting, in which the FRET efficiency is read out between exchangeable dyes on protein-bound DNA docking strands and accumulated FRET efficiencies constitute the fingerprint for a protein. To evaluate the feasibility of this approach, we simulated fingerprints for hundreds of proteins using a coarse-grained lattice model and experimentally demonstrated FRET X fingerprinting on model peptides. Measured fingerprints are in agreement with our simulations, corroborating the validity of our modeling approach. In a simulated complex mixture of >300 human proteins of which only cysteines, lysines, and arginines were labeled, a support vector machine was able to identify constituents with 95% accuracy. We anticipate that our FRET X fingerprinting approach will form the basis of an analysis tool for targeted proteomics.

Tetrameric architecture of an active phenol-bound form of the AAA+ transcriptional regulator DmpR.

The Pseudomonas putida phenol-responsive regulator DmpR is a bacterial enhancer binding protein (bEBP) from the AAA+ ATPase family. Even though it was discovered more than two decades ago and has been widely used for aromatic hydrocarbon sensing, the activation mechanism of DmpR has remained elusive. Here, we show that phenol-bound DmpR forms a tetramer composed of two head-to-head dimers in a head-to-tail arrangement. The DmpR-phenol complex exhibits altered conformations within the C-termini of the sensory domains and shows an asymmetric orientation and angle in its coiled-coil linkers. The structural changes within the phenol binding sites and the downstream ATPase domains suggest that the effector binding signal is propagated through the coiled-coil helixes. The tetrameric DmpR-phenol complex interacts with the σ54 subunit of RNA polymerase in presence of an ATP analogue, indicating that DmpR-like bEBPs tetramers utilize a mechanistic mode distinct from that of hexameric AAA+ ATPases to activate σ54-dependent transcription.

Resolving Chemical Modifications to a Single Amino Acid within a Peptide Using a Biological Nanopore.

While DNA sequencing is now amply available, fast, and inexpensive, protein sequencing remains a tremendous challenge. Nanopores may allow for developing a protein sequencer with single-molecule capabilities. As identification of 20 different amino acids currently presents an unsurmountable challenge, fingerprinting schemes are pursued, in which only a subset of amino acids is labeled and detected. This requires modification of amino acids with chemical structures that generate a distinct nanopore ionic current signal. Here, we use a model peptide and the fragaceatoxin C nanopore to characterize six potential tags for a fingerprinting approach using nanopores. We find that labeled and unlabeled proteins can be clearly distinguished and that sensitive detection is obtained for labels with a spectrum of different physicochemical properties such as mass (427-1275 Da), geometry, charge, and hydrophobicity. Additionally, information about the position of the label along the peptide chain can be obtained from individual current-blockade event features. The results represent an important advance toward the development of a single-molecule protein-fingerprinting device with nanopores.

Electro-Mechanical Conductance Modulation of a Nanopore Using a Removable Gate.

Ion channels form the basis of information processing in living cells by facilitating the exchange of electrical signals across and along cellular membranes. Applying the same principles to man-made systems requires the development of synthetic ion channels that can alter their conductance in response to a variety of external manipulations. By combining single-molecule electrical recordings with all-atom molecular dynamics simulations, we here demonstrate a hybrid nanopore system that allows for both a stepwise change of its conductance and a nonlinear current-voltage dependence. The conductance modulation is realized by using a short flexible peptide gate that carries opposite electric charge at its ends. We show that a constant transmembrane bias can position (and, in a later stage, remove) the peptide gate right at the most-sensitive sensing region of a biological nanopore FraC, thus partially blocking its channel and producing a stepwise change in the conductance. Increasing or decreasing the bias while having the peptide gate trapped in the pore stretches or compresses the peptide within the nanopore, thus modulating its conductance in a nonlinear but reproducible manner. We envision a range of applications of this removable-gate nanopore system, e.g. from an element of biological computing circuits to a test bed for probing the elasticity of intrinsically disordered proteins.

Viral suppressors of RNAi employ a rapid screening mode to discriminate viral RNA from cellular small RNA.

RNA interference (RNAi) is an indispensable mechanism for antiviral defense in insects, including mosquitoes that transmit human diseases. To escape this antiviral defense system, viruses encode suppressors of RNAi that prevent elimination of viral RNAs, and thus ensure efficient virus accumulation. Although the first animal Viral Suppressor of RNAi (VSR) was identified more than a decade ago, the molecular basis of RNAi suppression by these viral proteins remains unclear. Here, we developed a single-molecule fluorescence assay to investigate how VSRs inhibit the recognition of viral RNAs by Dcr-2, a key endoribonuclease enzyme in the RNAi pathway. Using VSRs from three insect RNA viruses (Culex Y virus, Drosophila X virus and Drosophila C virus), we reveal bimodal physical interactions between RNA molecules and VSRs. During initial interactions, these VSRs rapidly discriminate short RNA substrates from long dsRNA. VSRs engage nearly irreversible binding with long dsRNAs, thereby shielding it from recognition by Dcr-2. We propose that the length-dependent switch from rapid screening to irreversible binding reflects the main mechanism by which VSRs distinguish viral dsRNA from cellular RNA species such as microRNAs.

RecA filament maintains structural integrity using ATP-driven internal dynamics.

At the core of homologous DNA repair, RecA catalyzes the strand exchange reaction. This process is initiated by a RecA loading protein, which nucleates clusters of RecA proteins on single-stranded DNA. Each cluster grows to cover the single-stranded DNA but may leave 1- to 2-nucleotide (nt) gaps between the clusters due to three different structural phases of the nucleoprotein filaments. It remains to be revealed how RecA proteins eliminate the gaps to make a seamless kilobase-long filament. We develop a single-molecule fluorescence assay to observe the novel internal dynamics of the RecA filament. We directly observe the structural phases of individual RecA filaments and find that RecA proteins move their positions along the substrate DNA to change the phase of the filament. This reorganization process, which is a prerequisite step for interjoining of two adjacent clusters, requires adenosine triphosphate hydrolysis and is tightly regulated by the recombination hotspot, Chi. Furthermore, RecA proteins recognize and self-align to a 3-nt-period sequence pattern of TGG. This sequence-dependent phase bias may help the RecA filament to maintain structural integrity within the kilobase-long filament for accurate homology search and strand exchange reaction.

Water-mediated recognition of t1-adenosine anchors Argonaute2 to microRNA targets.

MicroRNAs (miRNAs) direct post-transcriptional regulation of human genes by guiding Argonaute proteins to complementary sites in messenger RNAs (mRNAs) targeted for repression. An enigmatic feature of many conserved mammalian miRNA target sites is that an adenosine (A) nucleotide opposite miRNA nucleotide-1 confers enhanced target repression independently of base pairing potential to the miRNA. In this study, we show that human Argonaute2 (Ago2) possesses a solvated surface pocket that specifically binds adenine nucleobases in the 1 position (t1) of target RNAs. t1A nucleotides are recognized indirectly through a hydrogen-bonding network of water molecules that preferentially interacts with the N6 amine on adenine. t1A nucleotides are not utilized during the initial binding of Ago2 to its target, but instead function by increasing the dwell time on target RNA. We also show that N6 adenosine methylation blocks t1A recognition, revealing a possible mechanism for modulation of miRNA target site potency.

TUT7 controls the fate of precursor microRNAs by using three different uridylation mechanisms.

Terminal uridylyl transferases (TUTs) function as integral regulators of microRNA (miRNA) biogenesis. Using biochemistry, single-molecule, and deep sequencing techniques, we here investigate the mechanism by which human TUT7 (also known as ZCCHC6) recognizes and uridylates precursor miRNAs (pre-miRNAs) in the absence of Lin28. We find that the overhang of a pre-miRNA is the key structural element that is recognized by TUT7 and its paralogues, TUT4 (ZCCHC11) and TUT2 (GLD2/PAPD4). For group II pre-miRNAs, which have a 1-nt 3' overhang, TUT7 restores the canonical end structure (2-nt 3' overhang) through mono-uridylation, thereby promoting miRNA biogenesis. For pre-miRNAs where the 3' end is further recessed into the stem (as in 3' trimmed pre-miRNAs), TUT7 generates an oligo-U tail that leads to degradation. In contrast to Lin28-stimulated oligo-uridylation, which is processive, a distributive mode is employed by TUT7 for both mono- and oligo-uridylation in the absence of Lin28. The overhang length dictates the frequency (but not duration) of the TUT7-RNA interaction, thus explaining how TUT7 differentiates pre-miRNA species with different overhangs. Our study reveals dual roles and mechanisms of uridylation in repair and removal of defective pre-miRNAs.

Distinct mechanisms regulating mechanical force-induced Ca²âº signals at the plasma membrane and the ER in human MSCs.

It is unclear that how subcellular organelles respond to external mechanical stimuli. Here, we investigated the molecular mechanisms by which mechanical force regulates Ca(2+) signaling at endoplasmic reticulum (ER) in human mesenchymal stem cells. Without extracellular Ca(2+), ER Ca(2+) release is the source of intracellular Ca(2+) oscillations induced by laser-tweezer-traction at the plasma membrane, providing a model to study how mechanical stimuli can be transmitted deep inside the cell body. This ER Ca(2+) release upon mechanical stimulation is mediated not only by the mechanical support of cytoskeleton and actomyosin contractility, but also by mechanosensitive Ca(2+) permeable channels on the plasma membrane, specifically TRPM7. However, Ca(2+) influx at the plasma membrane via mechanosensitive Ca(2+) permeable channels is only mediated by the passive cytoskeletal structure but not active actomyosin contractility. Thus, active actomyosin contractility is essential for the response of ER to the external mechanical stimuli, distinct from the mechanical regulation at the plasma membrane.

Cooperative conformational transitions keep RecA filament active during ATPase cycle.

The active, stretched conformation of the RecA filament bound to single-stranded DNA is required for homologous recombination. During this process, the RecA filament mediates the homology search and base pair exchange with a complementary sequence. Subsequently, the RecA filament dissociates from DNA upon reaction completion. ATP binding and hydrolysis is critical throughout these processes. Little is known about the timescale, order of conversion between different cofactor bound forms during ATP hydrolysis, and the associated changes in filament conformation. We used single-molecule fluorescence techniques to investigate how ATP hydrolysis is coupled with filament dynamics. For the first time, we observed real-time cooperative structural changes within the RecA filament. This cooperativity between neighboring monomers provides a time window for nucleotide cofactor exchange, which keeps the filament in the active conformation amidst continuous cycles of ATP hydrolysis.

Real-time observation of strand exchange reaction with high spatiotemporal resolution.

RecA binds to single-stranded (ss) DNA to form a helical filament that catalyzes strand exchange with a homologous double-stranded (ds) DNA. The study of strand exchange in ensemble assays is limited by the diffusion limited homology search process, which masks the subsequent strand exchange reaction. We developed a single-molecule fluorescence assay with a few base-pair and millisecond resolution that can separate initial docking from the subsequent propagation of joint molecule formation. Our data suggest that propagation occurs in 3 bp increments with destabilization of the incoming dsDNA and concomitant pairing with the reference ssDNA. Unexpectedly, we discovered the formation of a dynamic complex between RecA and the displaced DNA that remains bound transiently after joint molecule formation. This finding could have important implications for the irreversibility of strand exchange. Our model for strand exchange links structural models of RecA to its catalytic function.

TUT4 in concert with Lin28 suppresses microRNA biogenesis through pre-microRNA uridylation.

As key regulators in cellular functions, microRNAs (miRNAs) themselves need to be tightly controlled. Lin28, a pluripotency factor, was reported to downregulate let-7 miRNA by inducing uridylation of let-7 precursor (pre-let-7). But the enzyme responsible for the uridylation remained unknown. Here we identify a noncanonical poly (A) polymerase, TUTase4 (TUT4), as the uridylyl transferase for pre-let-7. Lin28 recruits TUT4 to pre-let-7 by recognizing a tetra-nucleotide sequence motif (GGAG) in the terminal loop. TUT4 in turn adds an oligouridine tail to the pre-let-7, which blocks Dicer processing. Other miRNAs with the same sequence motif (miR-107, -143, and -200c) are regulated through the same mechanism. Knockdown of TUT4 and Lin28 reduces the level of stem cell markers, suggesting that they are required for stem cell maintenance. This study uncovers the role of TUT4 and Lin28 as specific suppressors of miRNA biogenesis, which has implications for stem cell research and cancer biology.

Lin28 mediates the terminal uridylation of let-7 precursor MicroRNA.

The precise control of microRNA (miRNA) biogenesis is critical for embryonic development and normal cellular functions, and its dysregulation is often associated with human diseases. Though the birth and maturation pathway of miRNA has been established, the regulation and death pathway remains largely unknown. Here, we report the RNA-binding proteins, Lin28a and Lin28b, as posttranscriptional repressors of let-7 miRNA biogenesis. We observe that the Lin28 proteins act mainly in the cytoplasm by inducing uridylation of precursor let-7 (pre-let-7) at its 3' end. The uridylated pre-let-7 (up-let-7) fails Dicer processing and undergoes degradation. We provide a mechanism for the posttranscriptional regulation of miRNA biogenesis by Lin28 which is highly expressed in undifferentiated cells and certain cancer cells. The Lin28-mediated downregulation of let-7 may play a key role in development, stem cell programming, and tumorigenesis.

Fueling protein DNA interactions inside porous nanocontainers.

Vesicle encapsulation offers a biologically relevant environment for many soluble proteins and nucleic acids and an optimal immobilization medium for single-molecule fluorescence assays. Furthermore, the confinement of biomolecules within small volumes opens up new avenues to unique experimental configurations. Nevertheless, the vesicles' impermeability, even toward ions and other small molecules such as ATP, hinders more general applications. We therefore developed methods to induce pores into vesicles. Porous vesicles were then used to modulate the interaction between Escherichia coli RecA proteins and ssDNA by changing the extravesicular nucleotides. Repetitive binding and dissociation of the same RecA filament on the DNA was observed with a rebinding rate two orders of magnitude greater than in the absence of confinement, suggesting a previously unreported nucleation pathway for RecA filament. This method provides a biofriendly and simple alternative to surface tethering that is ideal for the study of transient and weakly interacting biological complexes.

Repetitive shuttling of a motor protein on DNA.

Many helicases modulate recombination, an essential process that needs to be tightly controlled. Mutations in some human disease helicases cause increased recombination, genome instability and cancer. To elucidate the potential mode of action of these enzymes, here we developed a single-molecule fluorescence assay that can visualize DNA binding and translocation of Escherichia coli Rep, a superfamily 1 DNA helicase homologous to Saccharomyces cerevisiae Srs2. Individual Rep monomers were observed to move on single-stranded (ss)DNA in the 3' to 5' direction using ATP hydrolysis. Strikingly, on hitting a blockade, such as duplex DNA or streptavidin, the protein abruptly snapped back close to its initial position, followed by further cycles of translocation and snapback. This repetitive shuttling is likely to be caused by a blockade-induced protein conformational change that enhances DNA affinity for the protein's secondary DNA binding site, thereby resulting in a transient DNA loop. Repetitive shuttling was also observed on ssDNA bounded by a stalled replication fork and an Okazaki fragment analogue, and the presence of Rep delayed formation of a filament of recombination protein RecA on ssDNA. Thus, the binding of a single Rep monomer to a stalled replication fork can lead to repetitive shuttling along the single-stranded region, possibly keeping the DNA clear of toxic recombination intermediates.