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Cyclic-phospholipids-based vesicles can play a role in facilitating the chemical evolution of protocells from the structurally simple to the functionally more complex form. Here, we present a protocol for preparing decanoic acid-derived cyclic phospholipid and glyceryl-diester phosphate-containing vesicles. We describe steps for sample preparation, equilibration, and image acquisition using confocal microscopy. This protocol has the potential for preparing a wide variety of these phospholipid-based artificial cell constructs. For complete details on the use and execution of this protocol, please refer to Pulletikurti et al. 1 ., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

The cyclic AMP response element (CRE) binding protein (CREB) is a transcription factor that contains a 280-residue N-terminal transactivation domain and a basic leucine zipper that mediates interaction with DNA. The transactivation domain comprises three subdomains, the glutamine-rich domains Q1 and Q2 and the kinase inducible activation domain (KID). NMR chemical shifts show that the isolated subdomains are intrinsically disordered but have a propensity to populate local elements of secondary structure. The Q1 and Q2 domains exhibit a propensity for formation of short β-hairpin motifs that function as binding sites for glutamine-rich sequences. These motifs mediate intramolecular interactions between the CREB Q1 and Q2 domains as well as intermolecular interactions with the glutamine-rich Q1 domain of the TATA-box binding protein associated factor 4 (TAF4) subunit of transcription factor IID (TFIID). Using small-angle X-ray scattering, NMR, and single-molecule Förster resonance energy transfer, we show that the Q1, Q2, and KID regions remain dynamically disordered in a full-length CREB transactivation domain (CREB TAD ) construct. The CREB TAD polypeptide chain is largely extended although some compaction is evident in the KID and Q2 domains. Paramagnetic relaxation enhancement reveals transient long-range contacts both within and between the Q1 and Q2 domains while the intervening KID domain is largely devoid of intramolecular interactions. Phosphorylation results in expansion of the KID domain, presumably making it more accessible for binding the CBP/p300 transcriptional coactivators. Our study reveals the complex nature of the interactions within the intrinsically disordered transactivation domain of CREB and provides molecular-level insights into dynamic and transient interactions mediated by the glutamine-rich domains.

Ebola virus (EBOV) infection induces the formation of membrane-less, cytoplasmic compartments termed viral factories, in which multiple viral proteins gather and coordinate viral transcription, replication, and assembly. Key to viral factory function is the recruitment of EBOV polymerase, a multifunctional machine that mediates transcription and replication of the viral RNA genome. We show that intracellularly reconstituted EBOV viral factories are biomolecular condensates, with composition-dependent internal exchange dynamics that likely facilitates viral replication. Within the viral factory, we found the EBOV polymerase clusters into foci. The distance between these foci increases when viral replication is enabled. In addition to the typical droplet-like viral factories, we report the formation of network-like viral factories during EBOV infection. Unlike droplet-like viral factories, network-like factories are inactive for EBOV nucleocapsid assembly. This unique view of EBOV propagation suggests a form-to-function relationship that describes how physical properties and internal structures of biomolecular condensates influence viral biogenesis., (© 2023. The Author(s).)

Currently, use of cannabinoids is limited to improve adverse effects of chemotherapy and their palliative administration during treatment is curiously concomitant with improved prognosis and regressed progression in patients with different tumor types. Although, non-psychoactive cannabidiol (CBD) and cannabigerol (CBG) display antineoplastic effects by repressing tumor growth and angiogenesis both in cell line and animal models, their use as chemotherapeutic agents is awaiting further investigation. Both clinical and epidemiological evidence supported by experimental findings suggest that micronutrients such as curcumin and piperine may present a safer strategy in preventing tumorigenesis and its recurrence. Recent studies demonstrated that piperine potentiates curcumin's inhibitory effect on tumor progression via enhancing its delivery and therapeutic activity. In this study, we investigated a plausible therapeutic synergism of a triple combination of CBD/CBG, curcumin, and piperine in the colon adenocarcinoma using HCT116 and HT29 cell lines. Potential synergistic effects of various combinations including these compounds were tested by measuring cancer cell proliferation and apoptosis. Our findings revealed that different genetic backgrounds of HCT116 and HT29 cell lines resulted in divergent responses to the combination treatments. Triple treatment showed synergism in terms of exhibiting anti-tumorigenic effects by activating the Hippo YAP signaling pathway in the HCT116 cell line., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Yüksel, Hızlı Deniz, Şahin, Sahin and Türkel.)

Background: Eukaryotic elongation factor 2 kinase (eukaryotic elongation factor 2 kinase, eEF2K) is a calcium calmodulin dependent protein kinase that keeps the highest energy consuming cellular process of protein synthesis under check through negative regulation. eEF2K pauses global protein synthesis rates at the translational elongation step by phosphorylating its only kown substrate elongation factor 2 (eEF2), a unique translocase activity in ekaryotic cells enabling the polypeptide chain elongation. Therefore, eEF2K is thought to preserve cellular energy pools particularly upon acute development of cellular stress conditions such as nutrient deprivation, hypoxia, or infections. Recently, high expression of this enzyme has been associated with poor prognosis in an array of solid tumor types. Therefore, in a growing number of studies tremendous effort is being directed to the development of treatment methods aiming to suppress eEF2K as a novel therapeutic approach in the fight against cancer., Methods: In our study, we aimed to investigate the changes in the tumorigenicity of chordoma cells in presence of gene silencing for eEF2K. Taking a transient gene silencing approach using siRNA particles, eEF2K gene expression was suppressed in chordoma cells., Results: Silencing eEF2K expression was associated with a slight increase in cellular proliferation and a decrease in death rates. Furthermore, no alteration in the sensitivity of chordoma cells to chemotherapy was detected in response to the decrease in eEF2K expression which intriguingly promoted suppression of cell migratory and invasion related properties., Conclusion: Our findings indicate that the loss of eEF2K expression in chordoma cell lines results in the reduction of metastatic capacity., (© 2023. The Author(s), under exclusive licence to Springer Nature B.V.)

The cyclic AMP response element binding protein (CREB) contains a basic leucine zipper motif (bZIP) that forms a coiled coil structure upon dimerization and specific DNA binding. Although this state is well characterized, key features of CREB bZIP binding and folding are not well understood. We used single-molecule Förster resonance energy transfer (smFRET) to probe conformations of CREB bZIP subdomains. We found differential folding of the basic region and leucine zipper in response to different binding partners; a strong and previously unreported DNA-independent dimerization affinity; folding upon binding to nonspecific DNA; and evidence of long-range interdomain interactions in full-length CREB that modulate DNA binding. These studies provide new insights into DNA binding and dimerization and have implications for CREB function., (© 2022 The Authors. FEBS Letters published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Biomolecular condensation and phase separation are increasingly understood to play crucial roles in cellular compartmentalization and spatiotemporal regulation of cell machinery implicated in function and pathology. A key aspect of current research is to gain insight into the underlying physical mechanisms of these processes. Accordingly, concepts of soft matter and polymer physics, the thermodynamics of mixing, and material science have been utilized for understanding condensation mechanisms of multivalent macromolecules resulting in viscoelastic mesoscopic supramolecular assemblies. Here, we focus on two topological concepts that have recently been providing key mechanistic understanding in the field. First, we will discuss how percolation provides a network-topology-related framework that offers an interesting paradigm to understand the complex networking of dense 'connected' condensate structures and, therefore, their phase behavior. Second, we will discuss the idea of entanglement as another topological concept that has deep roots in polymer physics and important implications for biomolecular condensates. We will first review some historical developments and fundamentals of these concepts, then we will discuss current advancements and recent examples. Our discussion ends with a few open questions and the challenges to address them, hinting at unveiling fresh possibilities for the modification of existing knowledge as well as the development of new concepts relevant to condensate science.

RNA-RNA interactions have increasingly been recognized for their potential to shape the mesoscale properties of biomolecular condensates, influencing morphology, organization, and material state through networking interactions. While most studies have focused on networking via Watson-Crick base pairing interactions, previous work has suggested a potential for noncanonical RNA-RNA interactions to also give rise to condensation and alter overall material state. Here, we test the phase separation of short polyA RNA (polyrA) homopolymers. We discover and characterize the potential for short polyrA sequences to form RNA condensates at lower Mg 2+ concentrations than previously observed, which appear as internally arrested droplets with slow polyrA diffusion despite continued fusion. Our work also reveals a negative cooperativity effect between the effects of Mg 2+ and Na + on polyrA condensation. Finally, we observe that polyrA sequences can act as promoters of phase separation in mixed sequences. These results demonstrate the potential for noncanonical interactions to act as networking stickers, leading to specific condensation properties inherent to polyrA composition and structure, with implications for the fundamental physical chemistry of the system and function of polyA RNA in biology.

The emergence of biomolecular condensation and liquid-liquid phase separation (LLPS) introduces a new layer of complexity into our understanding of cell and molecular biology. Evidence steadily grows indicating that condensates are not only implicated in physiology but also human disease. Macro- and mesoscale characterization of condensates as a whole have been instrumental in understanding their biological functions and dysfunctions. By contrast, the molecular level characterization of condensates and how condensates modify the properties of the molecules that constitute them thus far remain comparably scarce. In this minireview we summarize and discuss the findings of several recent studies that have focused on structure, dynamics, and interactions of proteins undergoing condensation. The mechanistic insights they provide help us identify the relevant properties nature and scientists can leverage to modulate the behavior of condensate systems. We also discuss the unique environment of the droplet surface and speculate on effects of topological constraints and physical exclusion on condensate properties., Competing Interests: Declaration of interests The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2021 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Single-molecule FRET (smFRET) has become a mainstream technique for studying biomolecular structural dynamics. The rapid and wide adoption of smFRET experiments by an ever-increasing number of groups has generated significant progress in sample preparation, measurement procedures, data analysis, algorithms and documentation. Several labs that employ smFRET approaches have joined forces to inform the smFRET community about streamlining how to perform experiments and analyze results for obtaining quantitative information on biomolecular structure and dynamics. The recent efforts include blind tests to assess the accuracy and the precision of smFRET experiments among different labs using various procedures. These multi-lab studies have led to the development of smFRET procedures and documentation, which are important when submitting entries into the archiving system for integrative structure models, PDB-Dev. This position paper describes the current 'state of the art' from different perspectives, points to unresolved methodological issues for quantitative structural studies, provides a set of 'soft recommendations' about which an emerging consensus exists, and lists openly available resources for newcomers and seasoned practitioners. To make further progress, we strongly encourage 'open science' practices., Competing Interests: EL, AB, JH, BA, VB, SB, RB, HS, TC, TC, AD, JD, JF, RG, IG, TH, CH, GH, NH, SH, SH, TH, AI, CJ, AK, HK, TL, NL, TL, EL, EM, JM, XM, SM, DN, TP, EP, YR, NR, BS, HS, CT, RV, DL, CS, SW No competing interests declared

Cellular liquid-liquid phase separation (LLPS) plays a key role in the dynamics and function of RNA-protein condensates like stress granules. In this issue of Cell, Yang et al., Guillén-Boixet et al., and Sanders et al. use a combination of experiment and modeling to provide an exciting mechanistic insight into the relationship between stress granules and LLPS, for example, in the context of protein disorder, switchable interactions, graph theory, and multiple interacting dense phases., (Copyright © 2020 Elsevier Inc. All rights reserved.)

Over the past few decades, numerous examples have demonstrated that intrinsic disorder in proteins lies at the heart of many vital processes, including transcriptional regulation, stress response, cellular signaling, and most recently protein liquid-liquid phase separation. The so-called intrinsically disordered proteins (IDPs) involved in these processes have presented a challenge to the classic protein "structure-function paradigm," as their functions do not necessarily involve well-defined structures. Understanding the mechanisms of IDP function is likewise challenging because traditional structure determination methods often fail with such proteins or provide little information about the diverse array of structures that can be related to different functions of a single IDP. Single-molecule fluorescence methods can overcome this ensemble-average masking, allowing the resolution of subpopulations and dynamics and thus providing invaluable insights into IDPs and their function. In this protocol, we describe a ratiometric single-molecule Förster resonance energy transfer (smFRET) routine that permits the investigation of IDP conformational subpopulations and dynamics. We note that this is a basic protocol, and we provide brief information and references for more complex analysis schemes available for in-depth characterization. This protocol covers optical setup preparation and protein handling and provides insights into experimental design and outcomes, together with background information about theory and a brief discussion of troubleshooting. © 2020 by John Wiley & Sons, Inc. Basic Protocol: Ratiometric smFRET detection and analysis of IDPs Support Protocol 1: Fluorophore labeling of a protein through maleimide chemistry Support Protocol 2: Sample chamber preparation Support Protocol 3: Determination of direct excitation of acceptor by donor excitation and leakage of donor emission to acceptor emission channel., (© 2020 John Wiley & Sons, Inc.)

Intrinsically disordered proteins (IDPs) are ubiquitous in proteomes and serve in a range of cellular functions including signaling, regulation, transport and enzyme function. IDP misfunction and aggregation are also associated with several diseases including neurodegenerative diseases and cancer. During the past decade, single-molecule methods have become popular for detailed biophysical and structural studies of these complex proteins. This work has included recent applications to cellular liquid-liquid phase separation (LLPS), relevant for functional dynamics of membraneless organelles such as the nucleolus and stress granules. In this concise review, we cover the conceptual motivations for development and application of single-molecule fluorescence methods for such IDP studies. We follow with a few key examples of systems and biophysical problems that have been addressed, and conclude with thoughts for emerging and future directions., (Copyright © 2019 The Authors. Published by Elsevier B.V. All rights reserved.)

Liquid-liquid phase separation (LLPS) of RNA-protein complexes plays a major role in the cellular function of membraneless organelles (MLOs). MLOs are sensitive to changes in cellular conditions, such as fluctuations in cytoplasmic ion concentrations. To investigate the effect of these changes on MLOs, we studied the influence of divalent cations on the physical and chemical properties of RNA coacervates. Using a model system comprised of an arginine-rich peptide and RNA, we predicted and observed that variations in signaling cations exert interaction-dependent effects on RNA LLPS. Changing the ionic environment has opposing effects on the propensity for heterotypic peptide-RNA and homotypic RNA LLPS, which results in a switch between coacervate types. Furthermore, divalent ion variations continuously tune the microenvironments and fluid properties of heterotypic and homotypic droplets. Our results may provide a general mechanism for modulating the biochemical environment of RNA coacervates in a cellular context.

Compartmentalization of biochemical processes is essential for cell function. Although membrane-bound organelles are well studied in this context, recent work has shown that phase separation is a key contributor to cellular compartmentalization through the formation of liquid-like membraneless organelles (MLOs). In this Minireview, the key mechanistic concepts that underlie MLO dynamics and function are first briefly discussed, including the relevant noncovalent interaction chemistry and polymer physical chemistry. Next, a few examples of MLOs and relevant proteins are given, along with their functions, which highlight the relevance of the above concepts. The developing area of active matter and non-equilibrium systems, which can give rise to unexpected effects in fluctuating cellular conditions, are also discussed. Finally, our thoughts for emerging and future directions in the field are discussed, including in vitro and in vivo studies of MLO physical chemistry and function., (© 2019 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

In the past decade a number of different stem cell types have entered the clinical applications increasingly as a therapeutic option, due to their tissue maintenance capacity at the site where they localize. Although it was initially thought that conferral of resilience to damaged tissue largely depends on the stem cells themselves through orchestration of signaling among the local epithelial and immune systems at the injury site, recent findings point out that the remarkable regenerative capacity of stem cells is rather due to their nanovesicular products that emerge as the new active players of tissue repair processes. Among these extracellular vesicles exosomes generated particularly by stem cells have been receiving a substantial interest both in the fields of stem cell biology and extracellular vesicles. In this chapter fundamental facts about stem cell biology, biogenesis of extracellular vesicles and exosomes, their structure, and function are summarized. Moreover, properties of both tumor-derived exosomes as well as those derived from stem cells are discussed relatively in-depth in terms of their influence on proximal and distal tissue physiology. Last but not the least, among countless studies in an exploding field, we summarize those that attempt to unravel the complex signaling networks through which stem cell-derived exosomes alter the fate of differentiating stem cells as well as the molecular make-up of exosomes released from differentiating stem cells by conducting thorough proteomic and genomic analyses with the ultimate goal of identifying effector gene products mediating exosomal cues in stem cell biology.

Single-molecule fluorescence is widely used to study conformational complexity in proteins, and has proven especially valuable with intrinsically disordered proteins (IDPs). Protein studies using dual-color single-molecule Förster resonance energy transfer (smFRET) are now quite common, but many could benefit from simultaneous measurement of multiple distances through multi-color labeling. Such studies, however, have suffered from limitations in site-specific incorporation of more than two dyes per polypeptide. Here we present a fully site-specific three-color labeling scheme for α-synuclein, an IDP with important putative functions and links to Parkinson disease. The convergent synthesis combines native chemical ligation with regiospecific cysteine protection of expressed protein fragments to permit highly controlled labeling via standard cysteine-maleimide chemistry, enabling more global smFRET studies. Furthermore, this modular approach is generally compatible with recombinant proteins and expandable to accommodate even more complex experiments, such as by labeling with additional colors., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Compartmentalization of biochemical components, interactions, and reactions is critical for the function of cells. While intracellular partitioning of molecules via membranes has been extensively studied, there has been an expanding focus in recent years on the critical cellular roles and biophysical mechanisms of action of membraneless organelles (MLOs) such as the nucleolus. In this context, a substantial body of recent work has demonstrated that liquid-liquid phase separation plays a key role in MLO formation. However, less is known about MLO dissociation, with phosphorylation being the primary mechanism demonstrated thus far. In this Perspective, we focus on another mechanism for MLO dissociation that has been described in recent work, namely a reentrant phase transition (RPT). This concept, which emerges from the polymer physics field, provides a mechanistic basis for both formation and dissolution of MLOs by monotonic tuning of RNA concentration, which is an outcome of cellular processes such as transcription. Furthermore, the RPT model also predicts the formation of dynamic substructures (vacuoles) of the kind that have been observed in cellular MLOs. We end with a discussion of future directions in terms of open questions and methods that can be used to answer them, including further exploration of RPTs in vitro, in cells, and in vivo using ensemble and single-molecule methods as well as theory and computation. We anticipate that continued studies will further illuminate the important roles of reentrant phase transitions and associated non-equilibrium dynamics in the spatial patterning of the biochemistry and biology of the cell.

Nucleophosmin (NPM1) is an abundant, oligomeric protein in the granular component of the nucleolus with roles in ribosome biogenesis. Pentameric NPM1 undergoes liquid-liquid phase separation (LLPS) via heterotypic interactions with nucleolar components, including ribosomal RNA (rRNA) and proteins which display multivalent arginine-rich linear motifs (R-motifs), and is integral to the liquid-like nucleolar matrix. Here we show that NPM1 can also undergo LLPS via homotypic interactions between its polyampholytic intrinsically disordered regions, a mechanism that opposes LLPS via heterotypic interactions. Using a combination of biophysical techniques, including confocal microscopy, SAXS, analytical ultracentrifugation, and single-molecule fluorescence, we describe how conformational changes within NPM1 control valency and switching between the different LLPS mechanisms. We propose that this newly discovered interplay between multiple LLPS mechanisms may influence the direction of vectorial pre-ribosomal particle assembly within, and exit from the nucleolus as part of the ribosome biogenesis process.

Protein thermodynamic stability is intricately linked to cellular function, and altered stability can lead to dysfunction and disease. The linear extrapolation model (LEM) is commonly used to obtain protein unfolding free energies ([Formula: see text]) by extrapolation of solvent denaturation data to zero denaturant concentration. However, for some proteins, different denaturants result in non-coincident LEM-derived [Formula: see text] values, raising questions about the inherent assumption that the obtained [Formula: see text] values are intrinsic to the protein. Here, we used single-molecule FRET measurements to better understand such discrepancies by directly probing changes in the dimensions of the protein G B1 domain (GB1), a well-studied protein folding model, upon urea and guanidine hydrochloride denaturation. A comparison of the results for the two denaturants suggests denaturant-specific structural energetics in the GB1 denatured ensemble, revealing a role of the denatured state in the variable thermodynamic behavior of proteins.

Intracellular ribonucleoprotein (RNP) granules are membrane-less droplet organelles that are thought to regulate posttranscriptional gene expression. While liquid-liquid phase separation may drive RNP granule assembly, the mechanisms underlying their supramolecular dynamics and internal organization remain poorly understood. Herein, we demonstrate that RNA, a primary component of RNP granules, can modulate the phase behavior of RNPs by controlling both droplet assembly and dissolution in vitro. Monotonically increasing the RNA concentration initially leads to droplet assembly by complex coacervation and subsequently triggers an RNP charge inversion, which promotes disassembly. This RNA-mediated reentrant phase transition can drive the formation of dynamic droplet substructures (vacuoles) with tunable lifetimes. We propose that active cellular processes that can create an influx of RNA into RNP granules, such as transcription, can spatiotemporally control the organization and dynamics of such liquid-like organelles., (© 2017 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The intrinsically disordered protein (IDP), α-synuclein (αS), is well-known for phospholipid membrane binding-coupled folding into tunable helical conformers. Here, using single-molecule experiments in conjunction with ensemble assays and a theoretical model, we present a unique case demonstrating that the interaction-folding landscape of αS can be tuned by two-dimensional (2D) crowding through simultaneous binding of a second protein on the bilayer surface. Unexpectedly, the experimental data show a clear deviation from a simple competitive inhibition model, but are consistent with a bimodal inhibition mechanism wherein membrane binding of a second protein (a membrane interacting chaperone, Hsp27, in this case) differentially inhibits two distinct modules of αS-membrane interaction. As a consequence, αS molecules are forced to access a hidden conformational state on the phospholipid bilayer in which only the higher-affinity module remains membrane-bound. Our results demonstrate that macromolecular crowding in two dimensions can play a significant role in shaping the conformational landscape of membrane-binding IDPs with multiple binding modes., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Many cellular functions are critically dependent on the folding of complex multimeric proteins, such as p97, a hexameric multidomain AAA+ chaperone. Given the complex architecture of p97, single-molecule (sm) FRET would be a powerful tool for studying folding while avoiding ensemble averaging. However, dual site-specific labeling of such a large protein for smFRET is a significant challenge. Here, we address this issue by using bioorthogonal azide-alkyne chemistry to attach an smFRET dye pair to site-specifically incorporated unnatural amino acids, allowing us to generate p97 variants reporting on inter- or intradomain structural features. An initial proof-of-principle set of smFRET results demonstrated the strengths of this labeling method. Our results highlight this as a powerful tool for structural studies of p97 and other large protein machines., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The nucleolus is a membrane-less organelle formed through liquid-liquid phase separation of its components from the surrounding nucleoplasm. Here, we show that nucleophosmin (NPM1) integrates within the nucleolus via a multi-modal mechanism involving multivalent interactions with proteins containing arginine-rich linear motifs (R-motifs) and ribosomal RNA (rRNA). Importantly, these R-motifs are found in canonical nucleolar localization signals. Based on a novel combination of biophysical approaches, we propose a model for the molecular organization within liquid-like droplets formed by the N-terminal domain of NPM1 and R-motif peptides, thus providing insights into the structural organization of the nucleolus. We identify multivalency of acidic tracts and folded nucleic acid binding domains, mediated by N-terminal domain oligomerization, as structural features required for phase separation of NPM1 with other nucleolar components in vitro and for localization within mammalian nucleoli. We propose that one mechanism of nucleolar localization involves phase separation of proteins within the nucleolus.

The structural features and dynamics of biological macromolecules underlie the molecular biology and correct functioning of cells. However, heterogeneity and other complexity of these molecules and their interactions often lead to loss of important information in traditional biophysical experiments. Single-molecule methods have dramatically altered the conceptual thinking and experimental tests available for such studies, leveraging their ability to avoid ensemble averaging. Here, I discuss briefly the rise of fluorescence single-molecule methods over the past two decades, a few key applications, and end with a view to challenges and future prospects., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

As for many intrinsically disordered proteins, order-disorder transitions in the N-terminal oligomerization domain of the multifunctional nucleolar protein nucleophosmin (Npm-N) are central to its function, with phosphorylation and partner binding acting as regulatory switches. However, the mechanism of this transition and its regulation remain poorly understood. In this study, single-molecule and ensemble experiments revealed pathways with alternative sequences of folding and assembly steps for Npm-N. Pathways could be switched by altering the ionic strength. Phosphorylation resulted in pathway-specific effects, and decoupled folding and assembly steps to facilitate disorder. Conversely, binding to a physiological partner locked Npm-N in ordered pentamers and counteracted the effects of phosphorylation. The mechanistic plasticity found in the Npm-N order-disorder transition enabled a complex interplay of phosphorylation and partner-binding steps to modulate its folding landscape., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Intrinsically disordered proteins (IDPs) are involved in diverse cellular functions. Many IDPs can interact with multiple binding partners, resulting in their folding into alternative ligand-specific functional structures. For such multi-structural IDPs, a key question is whether these multiple structures are fully encoded in the protein sequence, as is the case in many globular proteins. To answer this question, here we employed a combination of single-molecule and ensemble techniques to compare ligand-induced and osmolyte-forced folding of α-synuclein. Our results reveal context-dependent modulation of the protein's folding landscape, suggesting that the codes for the protein's native folds are partially encoded in its primary sequence, and are completed only upon interaction with binding partners. Our findings suggest a critical role for cellular interactions in expanding the repertoire of folds and functions available to disordered proteins., (© 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A substantial fraction of the human proteome encodes disordered proteins. Protein disorder is associated with a variety of cellular functions and misfunction, and is therefore of clear import to biological systems. However, disorder lends itself to conformational flexibility and heterogeneity, rendering proteins which feature prominent disorder difficult to study using conventional structural biology methods. Here we discuss a few examples of how single-molecule methods are providing new insight into the biophysics and complexity of these proteins by avoiding ensemble averaging, thereby providing direct information about the complex distributions and dynamics of this important class of proteins. Examples of note include characterization of isolated IDPs in solution as collapsed and dynamic species, detailed insight into complex IDP folding landscapes, and new information about how tunable regulation of structure-mediated binding cooperativity and consequent function can be achieved through protein disorder. With these exciting advances in view, we conclude with a discussion of a few complementary and emerging single-molecule efforts of particular promise, including complementary and enhanced methodologies for studying disorder in proteins, and experiments to investigate the potential role for IDP-induced phase separation as a critical functional element in biological systems., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

The temperature-jump technique, in which the sample is rapidly heated by a powerful laser pulse, has been widely used to probe the fast dynamics of folding of proteins and nucleic acids. However, the existing temperature-jump setups tend to involve sophisticated and expensive instrumentation, while providing only modest temperature changes of ~10-15 °C, and the temperature changes are only rapid for heating, but not cooling. Here we present a setup comprising a thermally conductive sapphire substrate with light-absorptive nano-coating, a microfluidic device and a rapidly switched moderate-power infrared laser with the laser beam focused on the nano-coating, enabling heating and cooling of aqueous solutions by ~50 °C on a 1-μs time scale. The setup is used to probe folding and unfolding dynamics of DNA hairpins after direct and inverse temperature jumps, revealing low-pass filter behaviour during periodic temperature variations.

Single-molecule (SM) fluorescence methods have been increasingly instrumental in our current understanding of a number of key aspects of protein folding and aggregation landscapes over the past decade. With the advantage of a model free approach and the power of probing multiple subpopulations and stochastic dynamics directly in a heterogeneous structural ensemble, SM methods have emerged as a principle technique for studying complex systems such as intrinsically disordered proteins (IDPs), globular proteins in the unfolded basin and during folding, and early steps of protein aggregation in amyloidogenesis. This review highlights the application of these methods in investigating the free energy landscapes, folding properties and dynamics of individual protein molecules and their complexes, with an emphasis on inherently flexible systems such as IDPs.

Allostery is an intrinsic property of many globular proteins and enzymes that is indispensable for cellular regulatory and feedback mechanisms. Recent theoretical and empirical observations indicate that allostery is also manifest in intrinsically disordered proteins, which account for a substantial proportion of the proteome. Many intrinsically disordered proteins are promiscuous binders that interact with multiple partners and frequently function as molecular hubs in protein interaction networks. The adenovirus early region 1A (E1A) oncoprotein is a prime example of a molecular hub intrinsically disordered protein. E1A can induce marked epigenetic reprogramming of the cell within hours after infection, through interactions with a diverse set of partners that include key host regulators such as the general transcriptional coactivator CREB binding protein (CBP), its paralogue p300, and the retinoblastoma protein (pRb; also called RB1). Little is known about the allosteric effects at play in E1A-CBP-pRb interactions, or more generally in hub intrinsically disordered protein interaction networks. Here we used single-molecule fluorescence resonance energy transfer (smFRET) to study coupled binding and folding processes in the ternary E1A system. The low concentrations used in these high-sensitivity experiments proved to be essential for these studies, which are challenging owing to a combination of E1A aggregation propensity and high-affinity binding interactions. Our data revealed that E1A-CBP-pRb interactions have either positive or negative cooperativity, depending on the available E1A interaction sites. This striking cooperativity switch enables fine-tuning of the thermodynamic accessibility of the ternary versus binary E1A complexes, and may permit a context-specific tuning of associated downstream signalling outputs. Such a modulation of allosteric interactions is probably a common mechanism in molecular hub intrinsically disordered protein function.

Protein structure and function depend on a close interplay between intrinsic folding energy landscapes and the chemistry of the protein environment. Osmolytes are small-molecule compounds that can act as chemical chaperones by altering the environment in a cellular context. Despite their importance, detailed studies on the role of these chemical chaperones in modulating structure and dimensions of intrinsically disordered proteins have been limited. Here, we used single-molecule Förster resonance energy transfer to test the counteraction hypothesis of counterbalancing effects between the protecting osmolyte trimethylamine-N-oxide (TMAO) and denaturing osmolyte urea for the case of α-synuclein, a Parkinson's disease-linked protein whose monomer exhibits significant disorder. The single-molecule experiments, which avoid complications from protein aggregation, do not exhibit clear solvent-induced cooperative protein transitions for these osmolytes, unlike results from previous studies on globular proteins. Our data demonstrate the ability of TMAO and urea to shift α-synuclein structures towards either more compact or expanded average dimensions. Strikingly, the experiments directly reveal that a 21 [urea][TMAO] ratio has a net neutral effect on the protein's dimensions, a result that holds regardless of the absolute osmolyte concentrations. Our findings shed light on a surprisingly simple aspect of the interplay between urea and TMAO on α-synuclein in the context of intrinsically disordered proteins, with potential implications for the biological roles of such chemical chaperones. The results also highlight the strengths of single-molecule experiments in directly probing the chemical physics of protein structure and disorder in more chemically complex environments.

Single molecule observation of fluorescence resonance energy transfer can be used to provide insight into the structure and dynamics of proteins. Using a straightforward triple-colour labelling strategy, we present a measurement and analysis scheme that can simultaneously study multiple regions within single intrinsically disordered proteins.

Some amyloid-forming polypeptides are associated with devastating human diseases and others provide important biological functions. For both, oligomeric intermediates appear during amyloid assembly. Currently we have few tools for characterizing these conformationally labile intermediates and discerning what governs their benign versus toxic states. Here, we examine intermediates in the assembly of a normal, functional amyloid, the prion-determining region of yeast Sup35 (NM). During assembly, NM formed a variety of oligomers with different sizes and conformation-specific antibody reactivities. Earlier oligomers were less compact and reacted with the conformational antibody A11. More mature oligomers were more compact and reacted with conformational antibody OC. We found we could arrest NM in either of these two distinct oligomeric states with small molecules or crosslinking. The A11-reactive oligomers were more hydrophobic (as measured by Nile Red binding) and were highly toxic to neuronal cells, while OC-reactive oligomers were less hydrophobic and were not toxic. The A11 and OC antibodies were originally raised against oligomers of Aβ, an amyloidogenic peptide implicated in Alzheimer's disease (AD) that is completely unrelated to NM in sequence. Thus, this natural yeast prion samples two conformational states similar to those sampled by Aβ, and when assembly stalls at one of these two states, but not the other, it becomes extremely toxic. Our results have implications for selective pressures operating on the evolution of amyloid folds across a billion years of evolution. Understanding the features that govern such conformational transitions will shed light on human disease and evolution alike.

We use a form of "freeze-trap, kinetic crystallography" to explore the migration of Xe atoms away from the dinuclear heme a(3)/Cu(B) center in Thermus thermophilus cytochrome ba(3) oxidase. This enzyme is a member of the heme-copper oxidase superfamily and is thus crucial for dioxygen-dependent life. The mechanisms involved in the migration of oxygen, water, electrons, and protons into and/or out of the specialized channels of the heme-copper oxidases are generally not well understood. Pressurization of crystals with Xe gas previously revealed a O(2) diffusion channel in cytochrome ba(3) oxidase that is continuous, Y-shaped, 18-20 Å in length and comprised of hydrophobic residues, connecting the protein surface within the bilayer to the a(3)-Cu(B) center in the active site. To understand movement of gas molecules within the O(2) channel, we performed crystallographic analysis of 19 Xe laden crystals freeze-trapped in liquid nitrogen at selected times between 0 and 480 s while undergoing outgassing at room temperature. Variation in Xe crystallographic occupancy at five discrete sites as a function of time leads to a kinetic model revealing relative degrees of mobility of Xe atoms within the channel. Xe egress occurs primarily through the channel formed by the Xe1 → Xe5 → Xe3 → Xe4 sites, suggesting that ingress of O(2) is likely to occur by the reverse of this process. The channel itself appears not to undergo significant structural changes during Xe migration, thereby indicating a passive role in this important physiological function.

Structural studies of intrinsically disordered proteins (IDPs) entail unique experimental challenges due in part to the lack of well-defined three-dimensional structures exhibited by this class of proteins. Although IDPs can be studied in their native disordered conformations using a variety of ensemble and single-molecule biophysical techniques, one particularly informative experimental strategy is to probe protein disordered states as part of folding-unfolding transitions. In this chapter, we describe solution methods for probing conformational properties of IDPs (and unfolded proteins, in general), including the use of naturally occurring osmolytes to force protein folding, the quantification of coupled folding and ligand binding of IDPs, and the structural interrogation of solvent- and/or binding-induced folded conformations by thermal perturbations.

The antiviral role of RNA interference (RNAi) in humans remains to be better understood. In RNAi, Ago2 proteins and microRNAs (miRNAs) or small interfering RNAs (siRNAs) form endonucleolytically active complexes which down-regulate expression of target mRNAs. P-bodies, cytoplasmic centers of mRNA decay, are involved in these pathways. Evidence exists that hepatitis C virus (HCV) utilizes host cellular RNAi machinery, including miRNA-122, Ago1-4, and Dicer proteins for replication and viral genome translation in Huh7 cells by, so far, nebulous mechanisms. Conversely, synthetic siRNAs have been used to suppress HCV replication. Here, using a combination of biochemical, transfection, confocal imaging, and digital image analysis approaches, we reveal that replication of HCV RNA depends on recruitment of Ago2 and miRNA-122 to lipid droplets, while suppression of HCV RNA by siRNA and Ago2 involves interaction with P-bodies. Such partitioning of Ago2 proteins into different complexes and separate subcellular domains likely results in modulation of their activity by different reaction partners. We propose a model in which partitioning of host RNAi and viral factors into physically and functionally distinct subcellular compartments emerges as a mechanism regulating the dual interaction of cellular RNAi with HCV RNA.

The protein folding reaction carries great significance for cellular function and hence continues to be the research focus of a large interdisciplinary protein science community. Single-molecule methods are providing new and powerful tools for dissecting the mechanisms of this complex process by virtue of their ability to provide views of protein structure and dynamics without associated ensemble averaging. This review briefly introduces common FRET and force methods, and then explores several areas of protein folding where single-molecule experiments have yielded insights. These include exciting new information about folding landscapes, dynamics, intermediates, unfolded ensembles, intrinsically disordered proteins, assisted folding and biomechanical unfolding. Emerging and future work is expected to include advances in single-molecule techniques aimed at such investigations, and increasing work on more complex systems from both the physics and biology standpoints, including folding and dynamics of systems of interacting proteins and of proteins in cells and organisms. This article is part of a Special Issue entitled: Protein Dynamics: Experimental and Computational Approaches., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Under "permissive" conditions at 25°C, the chaperonin substrate protein DM-MBP refolds 5-10 times more rapidly in the GroEL/GroES folding chamber than in free solution. This has been suggested to indicate that the chaperonin accelerates polypeptide folding by entropic effects of close confinement. Here, using native-purified DM-MBP, we show that the different rates of refolding are due to reversible aggregation of DM-MBP while folding free in solution, slowing its kinetics of renaturation: the protein exhibited concentration-dependent refolding in solution, with aggregation directly observed by dynamic light scattering. When refolded in chloride-free buffer, however, dynamic light scattering was eliminated, refolding became concentration-independent, and the rate of refolding became the same as that in GroEL/GroES. The GroEL/GroES chamber thus appears to function passively toward DM-MBP., (Copyright © 2011 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.)

We combined rapid microfluidic mixing with single-molecule fluorescence resonance energy transfer to study the folding kinetics of the intrinsically disordered human protein α-synuclein. The time-resolution of 0.2 ms revealed initial collapse of the unfolded protein induced by binding with lipid mimics and subsequent rapid formation of transient structures in the encounter complex. The method also enabled analysis of rapid dissociation and unfolding of weakly bound complexes triggered by massive dilution.

Proper protein function in cells, tissues and organisms depends critically on correct protein folding or interaction with partners. Over the last decade, single-molecule FRET (smFRET) has emerged as a powerful tool to probe complex distributions, dynamics, pathways and landscapes in protein folding and binding reactions, leveraging its ability to avoid averaging over an ensemble of molecules. While smFRET was practiced in a two-color form until recently, the last few years have seen the development of enhanced multicolor smFRET methods that provide additional structural information permitting us to probe more complex mechanisms. In this review, we provide a brief introduction to the smFRET technique, then follow with advanced multicolor measurements and end with ongoing methodology developments in microfluidics and protein labeling that are beginning to make these techniques more broadly applicable to answering a number of key questions about folding and binding.

Intrinsically disordered proteins (IDPs) (also referred to as natively unfolded proteins) play critical roles in a variety of cellular processes such as transcription and translation and also are linked to several human diseases. Biophysical studies of IDPs present unusual experimental challenges due in part to their broad conformational heterogeneity and potentially complex binding-induced folding behavior. By minimizing the averaging over an ensemble (which is typical of most conventional experiments), single-molecule fluorescence (SMF) techniques have recently begun to add advanced capabilities for structural studies to the experimental arsenal of IDP investigators. Here, we briefly discuss a few common SMF methods that are particularly useful for IDP studies, including SMF resonance energy transfer and fluorescence correlation spectroscopy, along with site-specific protein-labeling methods that are essential for application of these methods to IDPs. We then present an overview of a few studies in this area, highlighting how SMF methods are being used to gain valuable information about two amyloidogenic IDPs, the Parkinson's disease-linked alpha-synuclein and the NM domain of the yeast prion protein Sup 35. SMF experiments provided new information about the proteins' rapidly fluctuating IDP forms, and the complex alpha-synuclein folding behavior upon its binding to lipid and membrane mimics. We anticipate that SMF and single-molecule methods, in general, will find broad application for structural and mechanistic studies of a wide variety of IDPs, both of their disordered conformations, and their ordered ensembles relevant for function and disease., (Copyright 2010 Elsevier Inc. All rights reserved.)

A microfluidic device made of polydimethylsiloxane (PDMS) addresses key limitations in single-molecule fluorescence experiments by providing high dye photostability and low sample sticking. Photobleaching is dramatically reduced by deoxygenation via gas diffusion through porous channel walls. Rapid buffer exchange in a laminar sheath flow followed by optical interrogation minimizes surface-sample contacts and allows the in situ addition and combination of other reagents.

We present a microfluidic device for rapid and efficient determination of protein conformations in a range of medium conditions and temperatures. The device generates orthogonal gradients of concentration and temperature in an interrogation area that fits into the field of view of an objective lens with a numerical aperture of 0.45. A single Förster resonance energy transfer (FRET) image of the interrogation area containing a dual-labeled protein provides a 100 x 100 point map of the FRET efficiency that corresponds to a diagram of protein conformations in the coordinates of temperature and medium conditions. The device is used to explore the conformations of alpha-synuclein, an intrinsically disordered protein linked to Parkinson's and Alzheimer's diseases, in the presence of a binding partner, the lipid-mimetic sodium dodecyl sulfate (SDS). The experiment provides a diagram of conformations of alpha-synuclein with 10,000 individual data points in a range of 21-47 degrees C and 0-2.5 mM SDS. The diagram is consistent with previous reports but also reveals new conformational transitions that would be difficult to detect with conventional techniques. The microfluidic device can potentially be used to study other biomolecular and soft-matter systems.

Biological activity in proteins requires them to share the energy landscape for folding and global conformational motions, 2 key determinants of function. Although most structural studies to date have focused on fluctuations around a single structural basin, we directly observe the coexistence of 2 symmetrically opposed conformations for a mutant of the Rop-homodimer (Repressor of Primer) in single-molecule fluorescence resonance energy transfer (smFRET) measurements. We find that mild denaturing conditions can affect the sensitive balance between the conformations, generating an equilibrium ensemble consisting of 2 equally occupied structural basins. Despite the need for large-scale conformational rearrangement, both native structures are dynamically and reversibly adopted for the same paired molecules without separation of the constituent monomers. Such an ability of some proteins or protein complexes to switch between conformations by thermal fluctuations and/or minor environmental changes could be central to their ability to control biological function.

We studied the coupled binding and folding of alpha-synuclein, an intrinsically disordered protein linked with Parkinson's disease. Using single-molecule fluorescence resonance energy transfer and correlation methods, we directly probed protein membrane association, structural distributions, and dynamics. Results revealed an intricate energy landscape on which binding of alpha-synuclein to amphiphilic small molecules or membrane-like partners modulates conformational transitions between a natively unfolded state and multiple alpha-helical structures. Alpha-synuclein conformation is not continuously tunable, but instead partitions into 2 main classes of folding landscape structural minima. The switch between a broken and an extended helical structure can be triggered by changing the concentration of binding partners or by varying the curvature of the binding surfaces presented by micelles or bilayers composed of the lipid-mimetic SDS. Single-molecule experiments with lipid vesicles of various composition showed that a low fraction of negatively charged lipids, similar to that found in biological membranes, was sufficient to drive alpha-synuclein binding and folding, resulting here in the induction of an extended helical structure. Overall, our results imply that the 2 folded structures are preencoded by the alpha-synuclein amino acid sequence, and are tunable by small-molecule supramolecular states and differing membrane properties, suggesting novel control elements for biological and amyloid regulation of alpha-synuclein.