Protein-nucleic Acid Interactions Research Articles

Probing Single Helicase Dynamics on Long Nucleic Acids Through Fluorescence-Force Measurement.

Helicases are nucleic acid-dependent ATPases which can bind and remodel nucleic acids, protein-nucleic acid complexes, or both. They are involved in almost every step in cells related to nucleic acid metabolisms, including DNA replication and repair, transcription, RNA maturation and splicing, and nuclear export processes. Using single-molecule fluorescence-force spectroscopy, we have previously directly observed helicase translocation on long single-stranded DNA and revealed that two monomers of UvrD helicase are required for the initiation of unwinding function. Here, we present the details of fluorescence-force spectroscopy instrumentation, calibration, and activity assays in detail for observing the biochemical activities of helicases in real time and revealing how mechanical forces are involved in protein-nucleic acid interaction. These single-molecule approaches are generally applicable to many other protein-nucleic acid systems.

Single-Molecule Characterization of DNA-Protein Interactions Using Nanopore Biosensors.

Detection and characterization of nucleic acid-protein interactions, particularly those involving DNA and proteins such as transcription factors, enzymes, and DNA packaging proteins, remain significant barriers to our understanding of genetic regulation. Nanopores are an extremely sensitive and versatile sensing platform for label-free detection of single biomolecules. Analyte molecules are drawn to and through a nanoscale aperture by an electrophoretic force, which acts upon their native charge while in the sensing region of the pore. When the nanopore's diameter is only slightly larger than the biopolymer's cross section (typically a few nm); the latter must translocate through the pore in a linear fashion due to the constricted geometry in this region. These features allow nanopores to interrogate protein-nucleic acids in multiple sensing modes: first, by scanning and mapping the locations of binding sites along an analyte molecule, and second, by probing the strength of the bond between a protein and nucleic acid, using the native charge of the nucleic acid to apply an electrophoretic force to the complex while the protein is geometrically prevented from passing through the nanopore. In this chapter, we describe progress toward nanopore sensing of protein-nucleic acid complexes in the context of both mapping binding sites and performing force spectroscopy to determine the strength of interactions. We conclude by reviewing the strengths and challenges of the nanopore technique in the context of studying DNA-protein interactions.

Direct Visualization of Helicase Dynamics Using Fluorescence Localization and Optical Trapping.

Helicases control the accessibility of single-stranded (ss) nucleic acid (NA) generated as a transient intermediate during almost every step in cells related to nucleic acid metabolisms. For subsequent processing, however, helicases need to adjust the pace of unwinding adequately to avoid ssNA exposure to nucleases. Therefore, understanding how the unwinding process of helicases is regulated is crucial to address genome integrity and repair mechanisms. Using single-molecule fluorescence-force spectroscopy with fluorescence localization, we recently observed the stoichiometry of UvrD helicase, which determines the functions of UvrD: translocation and unwinding. For the first time, we provide direct evidence that a UvrD dimer is required to initiate the unwinding pathway. Moreover, with subpixel precision of fluorescence localization, the dynamic parameters of helicases can be obtained directly. Here, we present detailed single-molecule assays for observing the biochemical activities of helicases in real time and revealing how mechanical forces are involved in protein-nucleic acid interactions. These single-molecule approaches are generally applicable to many other protein-nucleic acid systems.

Protein Dynamics and Contact Topology Reveal Protein-DNA Binding Orientation.

Structure-encoded conformational dynamics are crucial for biomolecular functions. However, there is insufficient evidence to support the notion that dynamics play a role in guiding protein-nucleic acid interactions. Here, we show that protein-DNA docking orientation is a function of protein intrinsic dynamics, but the binding site itself does not display unique patterns in the examined spectrum of motions. This revelation is made possible by a novel technique that locates "dynamics interfaces" in proteins across which protein parts are anticorrelated in their slowest dynamics. A striking statistic is that such interfaces intersect the DNA in 97% of the 104 examined cases. These findings were then used to screen decoys generated by rigid-body docking of DNA molecules onto DNA-binding proteins. Using our method, the chance to discern near-native poses from non-native decoys increased by 2.5- and 1.6-fold, as compared to a random guess and methods based on surface complementarity, respectively. Hence, dynamically allowed protein-DNA docking orientations can work as new filters to cull and rerank docking poses and therefore enhance the predictability of DNA-binding sites that themselves do not have distinct dynamics features. Computer software implementing the method can be accessed via http://dyn.life.nthu.edu.tw/IDD/DNA.htm .

Simulation of the M13 life cycle I: Assembly of a genetically-structured deterministic chemical kinetic simulation

To expand the quantitative, systems level understanding and foster the expansion of the biotechnological applications of the filamentous bacteriophage M13, we have unified the accumulated quantitative information on M13 biology into a genetically-structured, experimentally-based computational simulation of the entire phage life cycle. The deterministic chemical kinetic simulation explicitly includes the molecular details of DNA replication, mRNA transcription, protein translation and particle assembly, as well as the competing protein-protein and protein-nucleic acid interactions that control the timing and extent of phage production. The simulation reproduces the holistic behavior of M13, closely matching experimentally reported values of the intracellular levels of phage species and the timing of events in the M13 life cycle. The computational model provides a quantitative description of phage biology, highlights gaps in the present understanding of M13, and offers a framework for exploring alternative mechanisms of regulation in the context of the complete M13 life cycle.

Protein-Nukleinsäure-Interaktionen im Massenspektrometer

Protein-nucleic acid complexes play a significant role in all DNA- and RNA-related cellular processes. The identification of protein-nucleic acid interaction sites is important to understand the structure, function and regulation of these complexes. Here, new approaches in mass spectrometry using UV-induced cross-linking of nucleic acids with proteins have recently proven as an important technique to not only identify proteins but specific regions of proteins that interact with RNA or DNA.

Foamy Virus Protein-Nucleic Acid Interactions during Particle Morphogenesis.

Compared with orthoretroviruses, our understanding of the molecular and cellular replication mechanism of foamy viruses (FVs), a subfamily of retroviruses, is less advanced. The FV replication cycle differs in several key aspects from orthoretroviruses, which leaves established retroviral models debatable for FVs. Here, we review the general aspect of the FV protein-nucleic acid interactions during virus morphogenesis. We provide a summary of the current knowledge of the FV genome structure and essential sequence motifs required for RNA encapsidation as well as Gag and Pol binding in combination with details about the Gag and Pol biosynthesis. This leads us to address open questions in FV RNA engagement, binding and packaging. Based on recent findings, we propose to shift the point of view from individual glycine-arginine-rich motifs having functions in RNA interactions towards envisioning the FV Gag C-terminus as a general RNA binding protein module. We encourage further investigating a potential new retroviral RNA packaging mechanism, which seems more complex in terms of the components that need to be gathered to form an infectious particle. Additional molecular insights into retroviral protein-nucleic acid interactions help us to develop safer, more specific and more efficient vectors in an era of booming genome engineering and gene therapy approaches.

Development of a Quantitative BRET Affinity Assay for Nucleic Acid-Protein Interactions.

Protein-nucleic acid interactions play a crucial role in the regulation of diverse biological processes. Elucidating the roles that protein-nucleic acid complexes play in the regulation of transcription, translation, DNA replication, repair and recombination, and RNA processing continues to be a crucial aspect of understanding of cell biology and the mechanisms of disease. In addition, proteins have been demonstrated to interact with antisense oligonucleotide therapeutics in a sequence and chemistry dependent manner, influencing ASO potency and distribution in cells and in vivo. While many assays have been developed to measure protein-nucleic acid interactions, many suffer from lack of throughput and sensitivity, or challenges with protein purification and scalability. In this report we present a new BRET assay for the analysis of DNA-protein interactions which makes use of an extremely bright luciferase as a tag for the binding protein, along with a long-wavelength fluorophore conjugated to the nucleic acid. The resulting assay is high throughput, sensitive, does not require protein purification, and even allows for quantitative characterization of these interactions within the biologically relevant context of whole cells.

ChemInform Abstract: Multitarget Drugs: An Epigenetic Epiphany

AbstractReview: [71 refs.

High-resolution, hybrid optical trapping methods, and their application to nucleic acid processing proteins.

Optical tweezers have become a powerful tool to investigate nucleic-acid processing proteins at the single-molecule level. Recent advances in this technique have now enabled measurements resolving the smallest units of molecular motion, on the scale of a single base pair of DNA. In parallel, new instrumentation combining optical traps with other functionalities have been developed, incorporating mechanical manipulation along orthogonal directions or fluorescence imaging capabilities. Here, we review these technical advances, their capabilities, and limitations, focusing on benchmark studies of protein-nucleic acid interactions they have enabled. We highlight recent work that combines several of these advances together and its application to nucleic-acid processing enzymes. Finally, we discuss future prospects for these exciting developments. © 2016 Wiley Periodicals, Inc. Biopolymers 105: 704-714, 2016.

DBSI server: DNA binding site identifier.

: Protein-nucleic acid interactions are among the most important intermolecular interactions in the regulation of cellular events. Identifying residues involved in these interactions from protein structure alone is an important challenge. Here we introduce the webserver interface to DNA Binding Site Identifier (DBSI), a powerful structure-based SVM model for the prediction and visualization of DNA binding sites on protein structures. DBSI has been shown to be a top-performing model to predict DNA binding sites on the surface of a protein or peptide and shows promise in predicting RNA binding sites. Server is available at http://dbsi.mitchell-lab.org jcmitchell@wisc.edu Supplementary data are available at Bioinformatics online.

Binding by TRBP-dsRBD2 Does Not Induce Bending of Double-Stranded RNA

Protein-nucleic acid interactions are central to a variety of biological processes, many of which involve large-scale conformational changes that lead to bending of the nucleic acid helix. Here, we focus on the nonsequence-specific protein TRBP, whose double-stranded RNA-binding domains (dsRBDs) interact with the A-form geometry of double-stranded RNA (dsRNA). Crystal structures of dsRBD-dsRNA interactions suggest that the dsRNA helix must bend in such a way that its major groove expands to conform to the dsRBD’s binding surface. We show through isothermal titration calorimetry experiments that dsRBD2 of TRBP binds dsRNA with a temperature-independent observed binding affinity (KD ∼500 nM). Furthermore, a near-zero observed heat capacity change (ΔCp = 70 ± 40 cal·mol−1·K−1) suggests that large-scale conformational changes do not occur upon binding. This result is bolstered by molecular-dynamics simulations in which dsRBD-dsRNA interactions generate only modest bending of the RNA along its helical axis. Overall, these results suggest that this particular dsRBD-dsRNA interaction produces little to no change in the A-form geometry of dsRNA in solution. These results further support our previous hypothesis, based on extensive gel-shift assays, that TRBP preferentially binds to sites of nearly ideal A-form structure while being excluded from sites of local deformation in the RNA helical structure. The implications of this mechanism for efficient micro-RNA processing will be discussed.

Expected and unexpected features of protein-binding RNA aptamers.

RNA molecules with high affinity to specific proteins can be isolated from libraries of up to 1016 different RNA sequences by systematic evolution of ligands by exponential enrichment (SELEX). These so-called protein-binding RNA aptamers are often interesting, e.g., as modulators of protein function for therapeutic use, for probing the conformations of proteins, for studies of basic aspects of nucleic acid-protein interactions, etc. Studies on the interactions between RNA aptamers and proteins display a number of expected and unexpected features, including the chemical nature of the interacting RNA-protein surfaces, the conformation of protein-bound aptamer versus free aptamer, the conformation of aptamer-bound protein versus free protein, and the effects of aptamers on protein function. Here, we review current insights into the details of RNA aptamer-protein interactions. WIREs RNA 2016, 7:744-757. doi: 10.1002/wrna.1360 For further resources related to this article, please visit the WIREs website.

The DBHS proteins SFPQ, NONO and PSPC1: a multipurpose molecular scaffold.

Nuclear proteins are often given a concise title that captures their function, such as ‘transcription factor,’ ‘polymerase’ or ‘nuclear-receptor.’ However, for members of the Drosophila behavior/human splicing (DBHS) protein family, no such clean-cut title exists. DBHS proteins are frequently identified engaging in almost every step of gene regulation, including but not limited to, transcriptional regulation, RNA processing and transport, and DNA repair. Herein, we present a coherent picture of DBHS proteins, integrating recent structural insights on dimerization, nucleic acid binding modalities and oligomerization propensity with biological function. The emerging paradigm describes a family of dynamic proteins mediating a wide range of protein–protein and protein–nucleic acid interactions, on the whole acting as a multipurpose molecular scaffold. Overall, significant steps toward appreciating the role of DBHS proteins have been made, but we are only beginning to understand the complexity and broader importance of this family in cellular biology.

Single-molecule pull-down for investigating protein–nucleic acid interactions

The genome and transcriptome are constantly modified by proteins in the cell. Recent advances in single-molecule techniques allow for high spatial and temporal observations of these interactions between proteins and nucleic acids. However, due to the difficulty of obtaining functional protein complexes, it remains challenging to study the interactions between macromolecular protein complexes and nucleic acids. Here, we combined single-molecule fluorescence with various protein complex pull-down techniques to determine the function and stoichiometry of ribonucleoprotein complexes. Through the use of three examples of protein complexes from eukaryotic cells (Drosha, Dicer, and TUT4 protein complexes), we provide step-by-step guidance for using novel single-molecule techniques. Our single-molecule methods provide sub-second and nanometer resolution and can be applied to other nucleoprotein complexes that are essential for cellular processes.

Targeting Protein-Protein Interactions in the HIF System.

Animals respond to chronic hypoxia by increasing the levels of a transcription factor known as the hypoxia‐inducible factor (HIF). HIF upregulates multiple genes, the products of which work to ameliorate the effects of limited oxygen at cellular and systemic levels. Hypoxia sensing by the HIF system involves hydroxylase‐catalysed post‐translational modifications of the HIF α‐subunits, which 1) signal for degradation of HIF‐α and 2) limit binding of HIF to transcriptional coactivator proteins. Because the hypoxic response is relevant to multiple disease states, therapeutic manipulation of the HIF‐mediated response has considerable medicinal potential. In addition to modulation of catalysis by the HIF hydroxylases, the HIF system manifests other possibilities for therapeutic intervention involving protein–protein and protein–nucleic acid interactions. Recent advances in our understanding of the structural biology and biochemistry of the HIF system are facilitating medicinal chemistry efforts. Herein we give an overview of the HIF system, focusing on structural knowledge of protein–protein interactions and how this might be used to modulate the hypoxic response for therapeutic benefit.

Trifunctional cross-linker for mapping protein-protein interaction networks and comparing protein conformational states.

To improve chemical cross-linking of proteins coupled with mass spectrometry (CXMS), we developed a lysine-targeted enrichable cross-linker containing a biotin tag for affinity purification, a chemical cleavage site to separate cross-linked peptides away from biotin after enrichment, and a spacer arm that can be labeled with stable isotopes for quantitation. By locating the flexible proteins on the surface of 70S ribosome, we show that this trifunctional cross-linker is effective at attaining structural information not easily attainable by crystallography and electron microscopy. From a crude Rrp46 immunoprecipitate, it helped identify two direct binding partners of Rrp46 and 15 protein-protein interactions (PPIs) among the co-immunoprecipitated exosome subunits. Applying it to E. coli and C. elegans lysates, we identified 3130 and 893 inter-linked lysine pairs, representing 677 and 121 PPIs. Using a quantitative CXMS workflow we demonstrate that it can reveal changes in the reactivity of lysine residues due to protein-nucleic acid interaction.

Multitarget Drugs: an Epigenetic Epiphany.

Epigenetics refers to changes in a biological phenotype that are not due to an underlying change in genotype. In eukaryotes, epigenetics involves a set of chemical modifications of the DNA and the histone proteins in nucleosomes. These dynamic changes are carried out by enzymes and modulate protein-protein and protein-nucleic acid interactions to determine whether specific genes are expressed or silenced. Both the epigenetic enzymes and recognition domains are currently important drug discovery targets, particularly for the treatment of cancer. This review summarizes the progress of epigenetic targets that have reached a clinical stage: DNA methyltransferases, histone deacetylases, lysine methyltransferases, lysine demethylases, and bromodomains; this is followed by a comprehensive survey of multitarget drugs that have included an epigenetic target as one of their mechanisms of action.

Drugging Membrane Protein Interactions.

The majority of therapeutics target membrane proteins, accessible on the surface of cells, to alter cellular signaling. Cells use membrane proteins to transduce signals into cells, transport ions and molecules, bind cells to a surface or substrate, and catalyze reactions. Newly devised technologies allow us to drug conventionally "undruggable" regions of membrane proteins, enabling modulation of protein-protein, protein-lipid, and protein-nucleic acid interactions. In this review, we survey the state of the art of high-throughput screening and rational design in drug discovery, and we evaluate the advances in biological understanding and technological capacity that will drive pharmacotherapy forward against unorthodox membrane protein targets.

Binding of the Single-Stranded DNA Binding Protein (gp32) of T4 Bacteriophage Induces Position-Specific Local Conformational Changes in DNA Lattices that can be Monitored by Fluorescent Probes

The single-stranded (ss) DNA binding protein (gp32) of the bacteriophage T4 DNA replication system plays a major role in integrating the functions of the sub-assemblies of the replication complex by interacting with different replication proteins. Substitution of fluorescent base analogues at defined positions in nucleic acid constructs can be used to study local conformational changes of the nucleic acid bases at specific sites, and Cy3/Cy5 dye-pairs inserted into the sugar-phosphate backbones can track the backbone motions of the DNA lattice during gp32 binding. By monitoring the fluorescence, circular dichroism, absorbance and FRET signals of these base and backbone probes, equilibrium and dynamic measurements can provide information about the molecular details of replication mechanisms. In the present study we use 6-methyl isoxanthopterin (6-MI), a fluorescent base analogue of guanine, to monitor local conformational changes of DNA lattices on gp32 binding. We find that aromatic amino acid residues of gp32 stacks with the bases of ssDNA lattices, depending on the specific positions of the bases within the binding domain of the gp32 molecule, and that Molecular Dynamics simulations are consistent with the experimental results. These results may shed light on how the hydrophobic pockets found in many protein-nucleic acid interaction complexes may stabilize the intercalation or stacking of aromatic amino acid residues with nucleic acid bases. Using internally labeled cyanine dyes, we have also investigated the double-stranded DNA melting properties of gp32 protein at a replication fork junction, and our results suggest that gp32 can enhance breathing at a fork junction if the ssDNA arm at the fork is of sufficient length to permit formation of cooperatively-bound gp32 clusters at the junction.