Monitoring RNA dynamics in native transcriptional complexes.

Cotranscriptional RNA folding is crucial for the timely control of biological processes, but because of its transient nature, its study has remained challenging. While single-molecule Förster resonance energy transfer (smFRET) is unique to investigate transient RNA structures, its application to cotranscriptional studies has been limited to nonnative systems lacking RNA polymerase (RNAP)-dependent features, which are crucial for gene regulation. Here, we present an approach that enables site-specific labeling and smFRET studies of kilobase-length transcripts within native bacterial complexes. By monitoring Escherichia coli nascent riboswitches, we reveal an inverse relationship between elongation speed and metabolite-sensing efficiency and show that pause sites upstream of the translation start codon delimit a sequence hotspot for metabolite sensing during transcription. Furthermore, we demonstrate a crucial role of the bacterial RNAP actively delaying the formation, within the hotspot sequence, of competing structures precluding metabolite binding. Our approach allows the investigation of cotranscriptional regulatory mechanisms in bacterial and eukaryotic elongation complexes.

Fatty acids may influence insulin dynamics through modulation of albumin-Zn2+ interactions.

Insulin is stored within the pancreas in an inactive Zn2+ -bound hexameric form prior to release. Similarly, clinical insulins contain Zn2+ and form multimeric complexes. Upon release from the pancreas or upon injection, insulin only becomes active once Zn2+ disengages from the complex. In plasma and other extracellular fluids, the majority of Zn2+ is bound to human serum albumin (HSA), which plays a vital role in controlling insulin pharmacodynamics by enabling removal of Zn2+ . The Zn2+ -binding properties of HSA are attenuated by non-esterified fatty acids (NEFAs) also transported by HSA. Elevated NEFA concentrations are associated with obesity and type 2 diabetes. Here we present the hypothesis that higher NEFA levels in obese and/or diabetic individuals may contribute to insulin resistance and affect therapeutic insulin dose-response profiles, through modulation of HSA/Zn2+ dynamics. We envisage this novel concept to have important implications for personalized treatments and management of diabetes-related conditions in the future.

A structural intermediate pre-organizes the add adenine riboswitch for ligand recognition.

Riboswitches are RNA sequences that regulate gene expression by undergoing structural changes upon the specific binding of cellular metabolites. Crystal structures of purine-sensing riboswitches have revealed an intricate network of interactions surrounding the ligand in the bound complex. The mechanistic details about how the aptamer folding pathway is involved in the formation of the metabolite binding site have been previously shown to be highly important for the riboswitch regulatory activity. Here, a combination of single-molecule FRET and SHAPE assays have been used to characterize the folding pathway of the adenine riboswitch from Vibrio vulnificus. Experimental evidences suggest a folding process characterized by the presence of a structural intermediate involved in ligand recognition. This intermediate state acts as an open conformation to ensure ligand accessibility to the aptamer and folds into a structure nearly identical to the ligand-bound complex through a series of structural changes. This study demonstrates that the add riboswitch relies on the folding of a structural intermediate that pre-organizes the aptamer global structure and the ligand binding site to allow efficient metabolite sensing and riboswitch genetic regulation.

The influence of various regions of the FOXP2 sequence on its structure and DNA-binding function.

FOX proteins are a superfamily of transcription factors which share a DNA-binding domain referred to as the forkhead domain. Our focus is on the FOXP subfamily members, which are involved in language and cognition amongst other things. The FOXP proteins contain a conserved zinc finger and a leucine zipper motif in addition to the forkhead domain. The remainder of the sequence is predicted to be unstructured and includes an acidic C-terminal tail. In the present study, we aim to investigate how both the structured and unstructured regions of the sequence cooperate so as to enable FOXP proteins to perform their function. We do this by studying the effect of these regions on both oligomerisation and DNA binding. Structurally, the FOXP proteins appear to be comparatively globular with a high proportion of helical structure. The proteins multimerise via the leucine zipper, and the stability of the multimers is controlled by the unstructured interlinking sequence including the acid rich tail. FOXP2 is more compact than FOXP1, has a greater propensity to form higher order oligomers, and binds DNA with stronger affinity. We conclude that while the forkhead domain is necessary for DNA binding, the affinity of the binding event is attributable to the leucine zipper, and the unstructured regions play a significant role in the specificity of binding. The acid rich tail forms specific contacts with the forkhead domain which may influence oligomerisation and DNA binding, and therefore the acid rich tail may play an important regulatory role in FOXP transcription.

Asymmetric base-pair opening drives helicase unwinding dynamics.

The opening of a Watson-Crick double helix is required for crucial cellular processes, including replication, repair, and transcription. It has long been assumed that RNA or DNA base pairs are broken by the concerted symmetric movement of complementary nucleobases. By analyzing thousands of base-pair opening and closing events from molecular simulations, here, we uncover a systematic stepwise process driven by the asymmetric flipping-out probability of paired nucleobases. We demonstrate experimentally that such asymmetry strongly biases the unwinding efficiency of DNA helicases toward substrates that bear highly dynamic nucleobases, such as pyrimidines, on the displaced strand. Duplex substrates with identical thermodynamic stability are thus shown to be more easily unwound from one side than the other, in a quantifiable and predictable manner. Our results indicate a possible layer of gene regulation coded in the direction-dependent unwindability of the double helix.

Unveiling the multi-step solubilization mechanism of sub-micron size vesicles by detergents.

The solubilization of membranes by detergents is critical for many technological applications and has become widely used in biochemistry research to induce cell rupture, extract cell constituents, and to purify, reconstitute and crystallize membrane proteins. The thermodynamic details of solubilization have been extensively investigated, but the kinetic aspects remain poorly understood. Here we used a combination of single-vesicle Förster resonance energy transfer (svFRET), fluorescence correlation spectroscopy and quartz-crystal microbalance with dissipation monitoring to access the real-time kinetics and elementary solubilization steps of sub-micron sized vesicles, which are inaccessible by conventional diffraction-limited optical methods. Real-time injection of a non-ionic detergent, Triton X, induced biphasic solubilization kinetics of surface-immobilized vesicles labelled with the Dil/DiD FRET pair. The nanoscale sensitivity accessible by svFRET allowed us to unambiguously assign each kinetic step to distortions of the vesicle structure comprising an initial fast vesicle-swelling event followed by slow lipid loss and micellization. We expect the svFRET platform to be applicable beyond the sub-micron sizes studied here and become a unique tool to unravel the complex kinetics of detergent-lipid interactions.

Unprecedented tunability of riboswitch structure and regulatory function by sub-millimolar variations in physiological Mg2.

Riboswitches are cis-acting regulatory RNA biosensors that rival the efficiency of those found in proteins. At the heart of their regulatory function is the formation of a highly specific aptamer-ligand complex. Understanding how these RNAs recognize the ligand to regulate gene expression at physiological concentrations of Mg2+ ions and ligand is critical given their broad impact on bacterial gene expression and their potential as antibiotic targets. In this work, we used single-molecule FRET and biochemical techniques to demonstrate that Mg2+ ions act as fine-tuning elements of the amino acid-sensing lysC aptamer's ligand-free structure in the mesophile Bacillus subtilis. Mg2+ interactions with the aptamer produce encounter complexes with strikingly different sensitivities to the ligand in different, yet equally accessible, physiological ionic conditions. Our results demonstrate that the aptamer adapts its structure and folding landscape on a Mg2+-tunable scale to efficiently respond to changes in intracellular lysine of more than two orders of magnitude. The remarkable tunability of the lysC aptamer by sub-millimolar variations in the physiological concentration of Mg2+ ions suggests that some single-aptamer riboswitches have exploited the coupling of cellular levels of ligand and divalent metal ions to tightly control gene expression.

Real-time observation of conformational switching in single conjugated polymer chains.

Conjugated polymers (CPs) are an important class of organic semiconductors that combine novel optoelectronic properties with simple processing from organic solvents. It is important to study CP conformation in solution to understand the physics of these materials and because it affects the properties of solution-processed films. Single-molecule techniques are unique in their ability to extract information on a chain-to-chain basis; however, in the context of CPs, technical challenges have limited their general application to host matrices or semiliquid environments that constrain the conformational dynamics of the polymer. We introduce a conceptually different methodology that enables measurements in organic solvents using the single-end anchoring of polymer chains to avoid diffusion while preserving polymer flexibility. We explore the effect of organic solvents and show that, in addition to chain-to-chain conformational heterogeneity, collapsed and extended polymer segments can coexist within the same chain. The technique enables real-time solvent-exchange measurements, which show that anchored CP chains respond to sudden changes in solvent conditions on a subsecond time scale. Our results give an unprecedented glimpse into the mechanism of solvent-induced reorganization of CPs and can be expected to lead to a new range of techniques to investigate and conformationally manipulate CPs.

DNA binding and unwinding by Hel308 helicase requires dual functions of a winged helix domain.

Hel308 helicases promote genome stability linked to DNA replication in archaea, and have homologues in metazoans. In the crystal structure of archaeal Hel308 bound to a tailed DNA duplex, core helicase domains encircle single-stranded DNA (ssDNA) in a "ratchet" for directional translocation. A winged helix domain (WHD) is also present, but its function is mysterious. We investigated the WHD in full-length Hel308, identifying that mutations in a solvent exposed α-helix resulted in reduced DNA binding and unwinding activities. When isolated from the rest of Hel308, the WHD protein alone bound to duplex DNA but not ssDNA, and DNA binding by WHD protein was abolished by the same mutations as were analyzed in full-length Hel308. Isolated WHD from a human Hel308 homologue (HelQ) also bound to duplex DNA. By disrupting the interface between the Hel308 WHD and a RecA-like domain, a topology typical of Ski2 helicases, we show that this is crucial for ATPase and helicase activities. The data suggest a model in which the WHD promotes activity of Hel308 directly, through binding to duplex DNA that is distinct from ssDNA binding by core helicase, and indirectly through interaction with the RecA-like domain. We propose how the WHD may contribute to ssDNA translocation, resulting in DNA helicase activity or in removal of other DNA bound proteins by "reeling" ssDNA.

High-affinity RNA binding by a hyperthermophilic single-stranded DNA-binding protein.

Single-stranded DNA-binding proteins (SSBs), including replication protein A (RPA) in eukaryotes, play a central role in DNA replication, recombination, and repair. SSBs utilise an oligonucleotide/oligosaccharide-binding (OB) fold domain to bind DNA, and typically oligomerise in solution to bring multiple OB fold domains together in the functional SSB. SSBs from hyperthermophilic crenarchaea, such as Sulfolobus solfataricus, have an unusual structure with a single OB fold coupled to a flexible C-terminal tail. The OB fold resembles those in RPA, whilst the tail is reminiscent of bacterial SSBs and mediates interaction with other proteins. One paradigm in the field is that SSBs bind specifically to ssDNA and much less strongly to RNA, ensuring that their functions are restricted to DNA metabolism. Here, we use a combination of biochemical and biophysical approaches to demonstrate that the binding properties of S. solfataricus SSB are essentially identical for ssDNA and ssRNA. These features may represent an adaptation to a hyperthermophilic lifestyle, where DNA and RNA damage is a more frequent event.

Fluorescence-Based Strategies to Investigate the Structure and Dynamics of Aptamer-Ligand Complexes.

In addition to the helical nature of double-stranded DNA and RNA, single-stranded oligonucleotides can arrange themselves into tridimensional structures containing loops, bulges, internal hairpins and many other motifs. This ability has been used for more than two decades to generate oligonucleotide sequences, so-called aptamers, that can recognize certain metabolites with high affinity and specificity. More recently, this library of artificially-generated nucleic acid aptamers has been expanded by the discovery that naturally occurring RNA sequences control bacterial gene expression in response to cellular concentration of a given metabolite. The application of fluorescence methods has been pivotal to characterize in detail the structure and dynamics of these aptamer-ligand complexes in solution. This is mostly due to the intrinsic high sensitivity of fluorescence methods and also to significant improvements in solid-phase synthesis, post-synthetic labeling strategies and optical instrumentation that took place during the last decade. In this work, we provide an overview of the most widely employed fluorescence methods to investigate aptamer structure and function by describing the use of aptamers labeled with a single dye in fluorescence quenching and anisotropy assays. The use of 2-aminopurine as a fluorescent analog of adenine to monitor local changes in structure and fluorescence resonance energy transfer (FRET) to follow long-range conformational changes is also covered in detail. The last part of the review is dedicated to the application of fluorescence techniques based on single-molecule microscopy, a technique that has revolutionized our understanding of nucleic acid structure and dynamics. We finally describe the advantages of monitoring ligand-binding and conformational changes, one molecule at a time, to decipher the complexity of regulatory aptamers and summarize the emerging folding and ligand-binding models arising from the application of these single-molecule FRET microscopy techniques.

Morphology-Specific Inhibition of ß-Amyloid Aggregates by 17ß-Hydroxysteroid Dehydrogenase Type 10.

A major hallmark of Alzheimer's disease (AD) is the formation of toxic aggregates of the ß-amyloid peptide (Aß). Given that Aß peptides are known to localise within mitochondria and interact with 17ß-HSD10, a mitochondrial protein expressed at high levels in AD brains, we investigated the inhibitory potential of 17ß-HSD10 against Aß aggregation under a range of physiological conditions. Fluorescence self-quenching (FSQ) of Aß(1-42) labelled with HiLyte Fluor 555 was used to evaluate the inhibitory effect under conditions established to grow distinct Aß morphologies. 17ß-HSD10 preferentially inhibits the formation of globular and fibrillar-like structures but has no effect on the growth of amorphous plaque-like aggregates at endosomal pH 6. This work provides insights into the dependence of the Aß-17ß-HSD10 interaction with the morphology of Aß aggregates and how this impacts enzymatic function.

Mechanism of DNA loading by the DNA repair helicase XPD.

The xeroderma pigmentosum group D (XPD) helicase is a component of the transcription factor IIH complex in eukaryotes and plays an essential role in DNA repair in the nucleotide excision repair pathway. XPD is a 5' to 3' helicase with an essential iron-sulfur cluster. Structural and biochemical studies of the monomeric archaeal XPD homologues have aided a mechanistic understanding of this important class of helicase, but several important questions remain open. In particular, the mechanism for DNA loading, which is assumed to require large protein conformational change, is not fully understood. Here, DNA binding by the archaeal XPD helicase from Thermoplasma acidophilum has been investigated using a combination of crystallography, cross-linking, modified substrates and biochemical assays. The data are consistent with an initial tight binding of ssDNA to helicase domain 2, followed by transient opening of the interface between the Arch and 4FeS domains, allowing access to a second binding site on helicase domain 1 that directs DNA through the pore. A crystal structure of XPD from Sulfolobus acidocaldiarius that lacks helicase domain 2 has an otherwise unperturbed structure, emphasizing the stability of the interface between the Arch and 4FeS domains in XPD.

Binding dynamics of a monomeric SSB protein to DNA: a single-molecule multi-process approach.

Single-stranded DNA binding proteins (SSBs) are ubiquitous across all organisms and are characterized by the presence of an OB (oligonucleotide/oligosaccharide/oligopeptide) binding motif to recognize single-stranded DNA (ssDNA). Despite their critical role in genome maintenance, our knowledge about SSB function is limited to proteins containing multiple OB-domains and little is known about single OB-folds interacting with ssDNA. Sulfolobus solfataricus SSB (SsoSSB) contains a single OB-fold and being the simplest representative of the SSB-family may serve as a model to understand fundamental aspects of SSB:DNA interactions. Here, we introduce a novel approach based on the competition between Förster resonance energy transfer (FRET), protein-induced fluorescence enhancement (PIFE) and quenching to dissect SsoSSB binding dynamics at single-monomer resolution. We demonstrate that SsoSSB follows a monomer-by-monomer binding mechanism that involves a positive-cooperativity component between adjacent monomers. We found that SsoSSB dynamic behaviour is closer to that of Replication Protein A than to Escherichia coli SSB; a feature that might be inherited from the structural analogies of their DNA-binding domains. We hypothesize that SsoSSB has developed a balance between high-density binding and a highly dynamic interaction with ssDNA to ensure efficient protection of the genome but still allow access to ssDNA during vital cellular processes.

Single-Molecule Approaches for the Characterization of Riboswitch Folding Mechanisms.

Riboswitches are highly structured RNA molecules that control genetic expression by altering their structure as a function of metabolite binding. Accumulating evidence suggests that riboswitch structures are highly dynamic and perform conformational exchange between structural states that are important for the outcome of genetic regulation. To understand how ligand binding influences the folding of riboswitches, it is important to monitor in real time the riboswitch folding pathway as a function of experimental conditions. Single-molecule FRET (sm-FRET) is unique among biophysical techniques to study riboswitch conformational changes as it allows to both monitor steady-state populations of riboswitch conformers and associated interconversion dynamics. Since FRET fluorophores can be attached to virtually any nucleotide position, FRET assays can be adapted to monitor specific conformational changes, thus enabling to deduce complex riboswitch folding pathways. Herein, we show how to employ sm-FRET to study the folding pathway of the S-adenosylmethionine (SAM) and how this can be used to understand very specific conformational changes that are at the heart of riboswitch regulation mechanism.

Functional Studies of DNA-Protein Interactions Using FRET Techniques.

Protein-DNA interactions underpin life and play key roles in all cellular processes and functions including DNA transcription, packaging, replication, and repair. Identifying and examining the nature of these interactions is therefore a crucial prerequisite to understand the molecular basis of how these fundamental processes take place. The application of fluorescence techniques and in particular fluorescence resonance energy transfer (FRET) to provide structural and kinetic information has experienced a stunning growth during the past decade. This has been mostly promoted by new advances in the preparation of dye-labeled nucleic acids and proteins and in optical sensitivity, where its implementation at the level of individual molecules has opened a new biophysical frontier. Nowadays, the application of FRET-based techniques to the analysis of protein-DNA interactions spans from the classical steady-state and time-resolved methods averaging over large ensembles to the analysis of distances, conformational changes, and enzymatic reactions in individual protein-DNA complexes. This chapter introduces the practical aspects of applying these methods for the study of protein-DNA interactions.

Towards ratiometric sensing of amyloid fibrils in vitro.

The aggregation of amyloid-ß peptide and its accumulation in the human brain has an important role in the etiology of Alzheimer's disease. Thioflavin T has been widely used as a fluorescent marker for these amyloid aggregates. Nevertheless, its complex photophysical behavior, with strong wavelength dependencies of all its fluorescence properties, requires searching for new fluorescent probes. The use of 2-(2'-hydroxyphenyl)imidazo[4,5-b]pyridine (HPIP), which shows two emission bands and a rich excited-state behavior due to the existence of excited-state intramolecular processes of proton transfer and charge transfer, is proposed. These properties result in a high sensitivity of HPIP fluorescence to its microenvironment and cause a large differential fluorescence enhancement of the two bands upon binding to aggregates of the amyloid-ß peptide. Based on this behavior, a very sensitive ratiometric method is established for the detection and quantification of amyloid fibrils, which can be combined with the monitoring of fluorescence anisotropy. The binding selectivity of HPIP is discussed on the basis of the apparent binding equilibrium constants of this probe to amyloid-ß (1-42) fibrils and to the nonfibrillar protein bovine serum albumin. Finally, an exhaustive comparison between HPIP and thioflavin T is presented to discuss the sensitivity and specificity of these probes to amyloid aggregates and the significant advantages of the HPIP dye for quantitative determinations.

Using sm-FRET and denaturants to reveal folding landscapes.

RNA folding studies aim to clarify the relationship among sequence, tridimensional structure, and biological function. In the last decade, the application of single-molecule fluorescence resonance energy transfer (sm-FRET) techniques to investigate RNA structure and folding has revealed the details of conformational changes and timescale of the process leading to the formation of biologically active RNA structures with subnanometer resolution on millisecond timescales. In this review, we initially summarize the first wave of single-molecule FRET-based RNA techniques that focused on analyzing the influence of mono- and divalent metal ions on RNA function, and how these studies have provided very valuable information about folding pathways and the presence of intermediate and low-populated states. Next, we describe a second generation of single-molecule techniques that combine sm-FRET with the use of chemical denaturants as an emerging powerful approach to reveal information about the dynamics and energetics of RNA folding that remains hidden using conventional sm-FRET approaches. The main advantages of using the competing interplay between folding agents such as metal ions and denaturants to observe and manipulate the dynamics of RNA folding and RNA-ligand interactions is discussed in the context of the adenine riboswitch aptamer.

Single-molecule characterization of Fen1 and Fen1/PCNA complexes acting on flap substrates.

Flap endonuclease 1 (Fen1) is a highly conserved structure-specific nuclease that catalyses a specific incision to remove 5' flaps in double-stranded DNA substrates. Fen1 plays an essential role in key cellular processes, such as DNA replication and repair, and mutations that compromise Fen1 expression levels or activity have severe health implications in humans. The nuclease activity of Fen1 and other FEN family members can be stimulated by processivity clamps such as proliferating cell nuclear antigen (PCNA); however, the exact mechanism of PCNA activation is currently unknown. Here, we have used a combination of ensemble and single-molecule Förster resonance energy transfer together with protein-induced fluorescence enhancement to uncouple and investigate the substrate recognition and catalytic steps of Fen1 and Fen1/PCNA complexes. We propose a model in which upon Fen1 binding, a highly dynamic substrate is bent and locked into an open flap conformation where specific Fen1/DNA interactions can be established. PCNA enhances Fen1 recognition of the DNA substrate by further promoting the open flap conformation in a step that may involve facilitated threading of the 5' ssDNA flap. Merging our data with existing crystallographic and molecular dynamics simulations we provide a solution-based model for the Fen1/PCNA/DNA ternary complex.