Dual film-like organelles enable spatial separation of orthogonal eukaryotic translation.

Engineering new functionality into living eukaryotic systems by enzyme evolution or de novo protein design is a formidable challenge. Cells do not rely exclusively on DNA-based evolution to generate new functionality but often utilize membrane encapsulation or formation of membraneless organelles to separate distinct molecular processes that execute complex operations. Applying this principle and the concept of two-dimensional phase separation, we develop film-like synthetic organelles that support protein translation on the surfaces of various cellular membranes. These sub-resolution synthetic films provide a path to make functionally distinct enzymes within the same cell. We use these film-like organelles to equip eukaryotic cells with dual orthogonal expanded genetic codes that enable the specific reprogramming of distinct translational machineries with single-residue precision. The ability to spatially tune the output of translation within tens of nanometers is not only important for synthetic biology but has implications for understanding the function of membrane-associated protein condensation in cells.

Beyond the Transport Function of Import Receptors: What's All the FUS about?

Nuclear import receptors are central players in transporting protein cargoes into the nucleus. Moving beyond this role, four newly published articles describe a function in regulating supramolecular assemblies by fine-tuning the phase separating properties of RNA-binding proteins, which has implications for a variety of devastating neurodegenerative disorders.

Direct Visualization of the Conformational Dynamics of Single Influenza Hemagglutinin Trimers.

Influenza hemagglutinin (HA) is the canonical type I viral envelope glycoprotein and provides a template for the membrane-fusion mechanisms of numerous viruses. The current model of HA-mediated membrane fusion describes a static "spring-loaded" fusion domain (HA2) at neutral pH. Acidic pH triggers a singular irreversible conformational rearrangement in HA2 that fuses viral and cellular membranes. Here, using single-molecule Förster resonance energy transfer (smFRET)-imaging, we directly visualized pH-triggered conformational changes of HA trimers on the viral surface. Our analyses reveal reversible exchange between the pre-fusion and two intermediate conformations of HA2. Acidification of pH and receptor binding shifts the dynamic equilibrium of HA2 in favor of forward progression along the membrane-fusion reaction coordinate. Interaction with the target membrane promotes irreversible transition of HA2 to the post-fusion state. The reversibility of HA2 conformation may protect against transition to the post-fusion state prior to arrival at the target membrane.

Shining a Light on Phase Separation in the Cell.

Phase separation of proteins is recognized as an important aspect of cellular organization and disease mechanisms. Shin et al. introduce a novel optogenetic tool, which enables different phase-space regimes inside living cells to be assessed and the transition paths between them to be studied with unprecedented spatiotemporal control.

Intrinsic disorder: A term to define the specific physicochemical characteristic of protein conformational heterogeneity.

In his commentary in this issue of Molecular Cell,1 Struhl reasons that the term "intrinsically disordered regions" represents a vague and confusing concept for protein function. However, the term "intrinsically disordered" highlights the important physicochemical characteristic of conformational heterogeneity. Thus, "intrinsically disordered" is the counterpart to the term "folded, " with neither term having specific functional implications.

Stressing the role of a short linear motif in ataxin-2 condensation.

Ataxin-2, an RNA-binding protein that is conserved across eukaryotes, is involved in stress granule assembly and age-associated neurodegenerative diseases. In this issue of Molecular Cell, Boeynaems et al.1 identify a short linear motif in ataxin-2 as a condensation switch, providing molecular insights into its essential role in cellular stress response.

Plasticity of an ultrafast interaction between nucleoporins and nuclear transport receptors.

The mechanisms by which intrinsically disordered proteins engage in rapid and highly selective binding is a subject of considerable interest and represents a central paradigm to nuclear pore complex (NPC) function, where nuclear transport receptors (NTRs) move through the NPC by binding disordered phenylalanine-glycine-rich nucleoporins (FG-Nups). Combining single-molecule fluorescence, molecular simulations, and nuclear magnetic resonance, we show that a rapidly fluctuating FG-Nup populates an ensemble of conformations that are prone to bind NTRs with near diffusion-limited on rates, as shown by stopped-flow kinetic measurements. This is achieved using multiple, minimalistic, low-affinity binding motifs that are in rapid exchange when engaging with the NTR, allowing the FG-Nup to maintain an unexpectedly high plasticity in its bound state. We propose that these exceptional physical characteristics enable a rapid and specific transport mechanism in the physiological context, a notion supported by single molecule in-cell assays on intact NPCs.

Visualizing the disordered nuclear transport machinery in situ.

The approximately 120 MDa mammalian nuclear pore complex (NPC) acts as a gatekeeper for the transport between the nucleus and cytosol1. The central channel of the NPC is filled with hundreds of intrinsically disordered proteins (IDPs) called FG-nucleoporins (FG-NUPs)2,3. Although the structure of the NPC scaffold has been resolved in remarkable detail, the actual transport machinery built up by FG-NUPs-about 50 MDa-is depicted as an approximately 60-nm hole in even highly resolved tomograms and/or structures computed with artificial intelligence4-11. Here we directly probed conformations of the vital FG-NUP98 inside NPCs in live cells and in permeabilized cells with an intact transport machinery by using a synthetic biology-enabled site-specific small-molecule labelling approach paired with highly time-resolved fluorescence microscopy. Single permeabilized cell measurements of the distance distribution of FG-NUP98 segments combined with coarse-grained molecular simulations of the NPC allowed us to map the uncharted molecular environment inside the nanosized transport channel. We determined that the channel provides-in the terminology of the Flory polymer theory12-a 'good solvent' environment. This enables the FG domain to adopt expanded conformations and thus control transport between the nucleus and cytoplasm. With more than 30% of the proteome being formed from IDPs, our study opens a window into resolving disorder-function relationships of IDPs in situ, which are important in various processes, such as cellular signalling, phase separation, ageing and viral entry.

Phase Separation Comes of Age: From Phenomenology to Single Molecules.

In this issue of Molecular Cell, Pitchiaya et al. (2019) use high-resolution single-molecule microscopy to dissect the localization of different types of RNAs with processing bodies (PBs) in cells, revealing novel insights about their dynamic recruitment to PBs.

Reliability and accuracy of single-molecule FRET studies for characterization of structural dynamics and distances in proteins.

Single-molecule Förster-resonance energy transfer (smFRET) experiments allow the study of biomolecular structure and dynamics in vitro and in vivo. We performed an international blind study involving 19 laboratories to assess the uncertainty of FRET experiments for proteins with respect to the measured FRET efficiency histograms, determination of distances, and the detection and quantification of structural dynamics. Using two protein systems with distinct conformational changes and dynamics, we obtained an uncertainty of the FRET efficiency ≤0.06, corresponding to an interdye distance precision of ≤2 Å and accuracy of ≤5 Å. We further discuss the limits for detecting fluctuations in this distance range and how to identify dye perturbations. Our work demonstrates the ability of smFRET experiments to simultaneously measure distances and avoid the averaging of conformational dynamics for realistic protein systems, highlighting its importance in the expanding toolbox of integrative structural biology.

Associating HIV-1 envelope glycoprotein structures with states on the virus observed by smFRET.

The HIV-1 envelope glycoprotein (Env) trimer mediates cell entry and is conformationally dynamic1-8. Imaging by single-molecule fluorescence resonance energy transfer (smFRET) has revealed that, on the surface of intact virions, mature pre-fusion Env transitions from a pre-triggered conformation (state 1) through a default intermediate conformation (state 2) to a conformation in which it is bound to three CD4 receptor molecules (state 3)8-10. It is currently unclear how these states relate to known structures. Breakthroughs in the structural characterization of the HIV-1 Env trimer have previously been achieved by generating soluble and proteolytically cleaved trimers of gp140 Env that are stabilized by a disulfide bond, an isoleucine-to-proline substitution at residue 559 and a truncation at residue 664 (SOSIP.664 trimers)5,11-18. Cryo-electron microscopy studies have been performed with C-terminally truncated Env of the HIV-1JR-FL strain in complex with the antibody PGT15119. Both approaches have revealed similar structures for Env. Although these structures have been presumed to represent the pre-triggered state 1 of HIV-1 Env, this hypothesis has never directly been tested. Here we use smFRET to compare the conformational states of Env trimers used for structural studies with native Env on intact virus. We find that the constructs upon which extant high-resolution structures are based predominantly occupy downstream conformations that represent states 2 and 3. Therefore, the structure of the pre-triggered state-1 conformation of viral Env that has been identified by smFRET and that is preferentially stabilized by many broadly neutralizing antibodies-and thus of interest for the design of immunogens-remains unknown.

Comparative analysis of the coordinated motion of Hsp70s from different organelles observed by single-molecule three-color FRET.

Cellular function depends on the correct folding of proteins inside the cell. Heat-shock proteins 70 (Hsp70s), being among the first molecular chaperones binding to nascently translated proteins, aid in protein folding and transport. They undergo large, coordinated intra- and interdomain structural rearrangements mediated by allosteric interactions. Here, we applied a three-color single-molecule Förster resonance energy transfer (FRET) combined with three-color photon distribution analysis to compare the conformational cycle of the Hsp70 chaperones DnaK, Ssc1, and BiP. By capturing three distances simultaneously, we can identify coordinated structural changes during the functional cycle. Besides the known conformations of the Hsp70s with docked domains and open lid and undocked domains with closed lid, we observed additional intermediate conformations and distance broadening, suggesting flexibility of the Hsp70s in adopting the states in a coordinated fashion. Interestingly, the difference of this distance broadening varied between DnaK, Ssc1, and BiP. Study of their conformational cycle in the presence of substrate peptide and nucleotide exchange factors strengthened the observation of additional conformational intermediates, with BiP showing coordinated changes more clearly compared to DnaK and Ssc1. Additionally, DnaK and BiP were found to differ in their selectivity for nucleotide analogs, suggesting variability in the recognition mechanism of their nucleotide-binding domains for the different nucleotides. By using three-color FRET, we overcome the limitations of the usual single-distance approach in single-molecule FRET, allowing us to characterize the conformational space of proteins in higher detail.

Cargo transport through the nuclear pore complex at a glance.

Bidirectional transport of macromolecules across the nuclear envelope is a hallmark of eukaryotic cells, in which the genetic material is compartmentalized inside the nucleus. The nuclear pore complex (NPC) is the major gateway to the nucleus and it regulates nucleocytoplasmic transport, which is key to processes including transcriptional regulation and cell cycle control. Accordingly, components of the nuclear transport machinery are often found to be dysregulated or hijacked in diseases. In this Cell Science at a Glance article and accompanying poster, we provide an overview of our current understanding of cargo transport through the NPC, from the basic transport signals and machinery to more emerging aspects, all from a 'cargo perspective'. Among these, we discuss the transport of large cargoes (>15 nm), as well as the roles of different cargo properties to nuclear transport, from size and number of bound nuclear transport receptors (NTRs), to surface and mechanical properties.

PED in 2021: a major update of the protein ensemble database for intrinsically disordered proteins.

The Protein Ensemble Database (PED) (https://proteinensemble.org), which holds structural ensembles of intrinsically disordered proteins (IDPs), has been significantly updated and upgraded since its last release in 2016. The new version, PED 4.0, has been completely redesigned and reimplemented with cutting-edge technology and now holds about six times more data (162 versus 24 entries and 242 versus 60 structural ensembles) and a broader representation of state of the art ensemble generation methods than the previous version. The database has a completely renewed graphical interface with an interactive feature viewer for region-based annotations, and provides a series of descriptors of the qualitative and quantitative properties of the ensembles. High quality of the data is guaranteed by a new submission process, which combines both automatic and manual evaluation steps. A team of biocurators integrate structured metadata describing the ensemble generation methodology, experimental constraints and conditions. A new search engine allows the user to build advanced queries and search all entry fields including cross-references to IDP-related resources such as DisProt, MobiDB, BMRB and SASBDB. We expect that the renewed PED will be useful for researchers interested in the atomic-level understanding of IDP function, and promote the rational, structure-based design of IDP-targeting drugs.

Synthesis, Biochemical Characterization, and Genetic Encoding of a 1,2,4-Triazole Amino Acid as an Acetyllysine Mimic for Bromodomains of the BET Family.

Lysine acetylation is a charge-neutralizing post-translational modification of proteins bound by bromodomains (Brds). A 1,2,4-triazole amino acid (ApmTri) was established as acetyllysine (Kac) mimic recruiting Brds of the BET family in contrast to glutamine commonly used for simulating this modification. Optimization of triazole substituents and side chain spacing allowed BET Brd recruitment to ApmTri-containing peptides with affinities similar to native substrates. Crystal structures of ApmTri-containing peptides in complex with two BET Brds revealed the binding mode which mirrored that of Kac ligands. ApmTri was genetically encoded and recombinant ApmTri-containing proteins co-enriched BRD3(2) from cellular lysates. This interaction was blocked by BET inhibitor JQ1. With genetically encoded ApmTri, biochemistry is now provided with a stable Kac mimic reflecting charge neutralization and Brd recruitment, allowing new investigations into BET proteins in vitro and in vivo.

Role of Solvent Compatibility in the Phase Behavior of Binary Solutions of Weakly Associating Multivalent Polymers.

Condensate formation of biopolymer solutions, prominently those of various intrinsically disordered proteins (IDPs), is often driven by "sticky" interactions between associating residues, multivalently present along the polymer backbone. Using a ternary mean-field "stickers-and-spacers" model, we demonstrate that if sticker association is of the order of a few times the thermal energy, a delicate balance between specific binding and nonspecific polymer-solvent interactions gives rise to a particularly rich ternary phase behavior under physiological circumstances. For a generic system represented by a solution comprising multiassociative scaffold and client polymers, the difference in solvent compatibility between the polymers modulates the nature of isothermal liquid-liquid phase separation (LLPS) between associative and segregative. The calculations reveal regimes of dualistic phase behavior, where both types of LLPS occur within the same phase diagram, either associated with the presence of multiple miscibility gaps or a flip in the slope of the tie-lines belonging to a single coexistence region.

Physics of the Nuclear Pore Complex: Theory, Modeling and Experiment.

The hallmark of eukaryotic cells is the nucleus that contains the genome, enclosed by a physical barrier known as the nuclear envelope (NE). On the one hand, this compartmentalization endows the eukaryotic cells with high regulatory complexity and flexibility. On the other hand, it poses a tremendous logistic and energetic problem of transporting millions of molecules per second across the nuclear envelope, to facilitate their biological function in all compartments of the cell. Therefore, eukaryotes have evolved a molecular "nanomachine" known as the Nuclear Pore Complex (NPC). Embedded in the nuclear envelope, NPCs control and regulate all the bi-directional transport between the cell nucleus and the cytoplasm. NPCs combine high molecular specificity of transport with high throughput and speed, and are highly robust with respect to molecular noise and structural perturbations. Remarkably, the functional mechanisms of NPC transport are highly conserved among eukaryotes, from yeast to humans, despite significant differences in the molecular components among various species. The NPC is the largest macromolecular complex in the cell. Yet, despite its significant complexity, it has become clear that its principles of operation can be largely understood based on fundamental physical concepts, as have emerged from a combination of experimental methods of molecular cell biology, biophysics, nanoscience and theoretical and computational modeling. Indeed, many aspects of NPC function can be recapitulated in artificial mimics with a drastically reduced complexity compared to biological pores. We review the current physical understanding of the NPC architecture and function, with the focus on the critical analysis of experimental studies in cells and artificial NPC mimics through the lens of theoretical and computational models. We also discuss the connections between the emerging concepts of NPC operation and other areas of biophysics and bionanotechnology.

Publisher Correction: Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

This paper was originally published under standard Springer Nature copyright. As of the date of this correction, the Analysis is available online as an open-access paper with a CC-BY license. No other part of the paper has been changed.

Precision and accuracy of single-molecule FRET measurements-a multi-laboratory benchmark study.

Single-molecule Förster resonance energy transfer (smFRET) is increasingly being used to determine distances, structures, and dynamics of biomolecules in vitro and in vivo. However, generalized protocols and FRET standards to ensure the reproducibility and accuracy of measurements of FRET efficiencies are currently lacking. Here we report the results of a comparative blind study in which 20 labs determined the FRET efficiencies (E) of several dye-labeled DNA duplexes. Using a unified, straightforward method, we obtained FRET efficiencies with s.d. between ±0.02 and ±0.05. We suggest experimental and computational procedures for converting FRET efficiencies into accurate distances, and discuss potential uncertainties in the experiment and the modeling. Our quantitative assessment of the reproducibility of intensity-based smFRET measurements and a unified correction procedure represents an important step toward the validation of distance networks, with the ultimate aim of achieving reliable structural models of biomolecular systems by smFRET-based hybrid methods.

Synthesis and Evaluation of Novel Ring-Strained Noncanonical Amino Acids for Residue-Specific Bioorthogonal Reactions in Living Cells.

Bioorthogonal reactions are ideally suited to selectively modify proteins in complex environments, even in vivo. Kinetics and product stability of these reactions are crucial parameters to evaluate their usefulness for specific applications. Strain promoted inverse electron demand Diels-Alder cycloadditions (SPIEDAC) between tetrazines and strained alkenes or alkynes are particularly popular, as they allow ultrafast labeling inside cells. In combination with genetic code expansion (GCE)-a method that allows to incorporate noncanonical amino acids (ncAAs) site-specifically into proteins in vivo. These reactions enable residue-specific fluorophore attachment to proteins in living mammalian cells. Several SPIEDAC capable ncAAs have been presented and studied under diverse conditions, revealing different instabilities ranging from educt decomposition to product loss due to ß-elimination. To identify which compounds yield the best labeling inside living mammalian cells has frequently been difficult. In this study we present a) the synthesis of four new SPIEDAC reactive ncAAs that cannot undergo ß-elimination and b) a fluorescence flow cytometry based FRET-assay to measure reaction kinetics inside living cells. Our results, which at first sight can be seen conflicting with some other studies, capture GCE-specific experimental conditions, such as long-term exposure of the ring-strained ncAA to living cells, that are not taken into account in other assays.