Folding Kinetics Research Articles

Toward protein fingerprinting with a solid-state nanopore.

Solid-state nanopores have shown great potential for single-molecule protein identification in recent years. As a first step toward single-molecule protein fingerprinting using a solid-state nanopore device, thorough characterization of the passage of proteins under the different conformational states is required. For this purpose, we use green fluorescent protein (GFP), antibiotin immunoglobulin G (IgG), and cytochrome c (cyt c) as model proteins, and show results from field-driven translocations through nanopores with diameters ranging from 2 to 10 nm in SiN membranes of thicknesses from 10 to 20 nm. As expected, the distinguishable current blockades observed during individual translocations allow the identification of different protein conformations adopted throughout its passage, ranging from partially unfolded to completely unfolded conformations. Here we show how different temperatures and voltages facilitate protein unfolding and alter their folding kinetics. This is achieved by varying voltages from 0.1 V to 1 V and by varying the temperature from room temperature to 60 oC while maintaining low-noise ionic current recording at 1 MHz bandwidth using a custom instrument with thermal control offering ±0.2 oC precision and ±1 oC repeatability. These results will serve to better understand the accessible states in the protein energy landscape and to benchmark future studies furthering protein fingerprinting.

Investigating flexible and semi-flexible polymers using solid-state nanopores.

Following the rise of nanopore DNA sequencing, nanopore sensors are showing great potential for characterizing and ultimately decoding information from other types of biotic and abiotic polymers. In particular, nanostructured DNA molecules are being proposed to store digital information for archival applications or as barcodes for multiplexed biomarker detection and solid-state nanopores are being used to decode them. However, progress in this area requires better understanding of the molecular mechanisms for translocation both at the theoretical and experimental level to optimize these applications, even with recent progress. Here we investigate the influence of polymer flexibility on translocation times and folding dynamics during polymer translocation through solid-state nanopores ranging in size from 5 to 15 nm on 10–15-nm-thick SiN membranes. We design DNA with modulated flexibilities by leaving gaps in one of the strands making up the DNA double helix. In doing so we create block-polymers consisting of rigid double stranded pieces held together by flexible single stranded parts of DNA. As expected, we observe that folding happens preferentially on these ssDNA sections in the structured polymer and propose this as a mechanism to control the folding of longer polymers by restricting folding to the ends of the molecule, leaving large internal blocks of the polymer unfolded. This strategy to limit polymer folding inside a pore can be used to tackle one of the major challenges of data storage and barcode readout using solid-state nanopores.

The PCDDB project: Tools and resources for CD analyses, archiving, display, and sharing.

The Protein Circular Dichroism Data Bank Project was initiated more than 20 years ago as a means of creating an integrated set of software and tools to aid in analyses, interpretations, visual displays, and data sharing of circular dichroism (CD) and synchrotron radiation circular dichroism (SRCD) spectra and metadata. It has been a basis for the creation of a wide range of novel biophysical and computational methods, and has resulted from the coordinated effort of our two labs and many collaborators. At its heart has been the DichroWeb analysis server, which has been used for ∼2 M secondary structure analyses by >9000 users, and which has included the creation of new computational methods and associated reference databases for examining soluble, membrane, and intrinsically-disordered proteins. The PCDDB itself was created as a repository for CD data and metadata with links to a wide range of bioinformatics, computational, and structural databases and tools, encouraging interoperability of CD and other biophysical methods. It includes links to primary sequence (UniProt) and structural (PDB) databases, as well as to secondary databases such as the Enzyme Commission functional database and the CATH fold database. In addition, tools were created for data processing, and display of CD spectra, for validation of spectral quality (ValiDichro), for integration with other methods such as PDB2CD, PDBMD2CD, 2StructCompare. and DichroCompare, plus novel methods of visualisation, including kinetics of protein folding. Software created is available online via GitHub. In addition to providing widely-used research tools, this project has also been the core teaching tool for CD summer schools and courses worldwide, and has benefitted from the contributions of many biophysicis groups worldwide. [Supported by grants from the UK Biotechnology and Biological Resources programme of the BBSRC).

Unraveling coupled folding and binding dynamics of ribonuclease S with transient infrared spectroscopy

Molecular interactions of proteins consisting of coupled disorder-to-order transitions and spontaneous binding have a crucial role in understanding their biological function and working mechanism. Here we investigate a well-known globular binding system, ribonuclease S (RNase S), to study the dynamics of protein-peptide interaction. Various biophysical approaches have investigated the binding mechanism of two proteolytic fragments, S-peptide and S-protein, which non-covalently bind to form an active enzyme RNase S. Due to challenges in dissecting the coupled nature of protein binding and folding, molecular level details remain elusive. Here, we use amide-I vibrations as a label-free probe of protein secondary structure and temperature as a control variable. By temperature-jump two-dimensional infrared spectroscopy (t-2DIR), we track the thermally-induced conformational changes associated with relaxation time that span from nanoseconds to many minute timescales. Additionally, we compare the results of an unlabeled complex with one containing a 13C=18O isotope-label of a residue buried within the helical segment of S-peptide. t‑2DIR measurements of labeled- and unlabeled- RNase S and isolated S-protein reveal two kinetic components on millisecond (fast) and second (slow) time scales. Both slow and fast phases are dominated by β-sheet disordering signatures that indicate S-protein unfolding. The isotope-label exhibits significant amplitude in the slow phase, suggesting this time window corresponds to both unbinding and unfolding of the S-peptide. Together, these results indicate that thermally-induced partial unfolding of the S-protein destabilizes the hydrophobic binding pocket containing S-peptide that, in turn, promotes unbinding and unfolding of the S-peptide. Our direct observations from transient spectroscopic data demonstrate the role of a conformational transition state during assembly of the RNase S complex.

Development of an aptamer-based screening for identification and characterizationof novel RNA therapeutics using switchSENSE® technology.

Approximately 85 % of the genes are transcribed into RNA but only 3 % are translated into proteins. This is not reflected in the amount of available drugs, as most of them target proteins which relate to 0.05 % of nucleic acids. Thus, an effort must be made to fill this gap and focus on the transcriptome level of diseases. Non-coding RNAs are especially interesting as they account for the major share of the transcriptome and fulfill important regulatory functions as small interfering (siRNA), transfer-RNA (tRNA) and other unknown ones. Due to their importance, they could be explored as novel drug targets, e.g. to develop alternative antibiotics. A successful example in procaryotes is targeting the synthesis of the highly conserved and essential survival factor flavin mononucleotide (FMN) by its riboswitch which can modulate the expression over structural re-arrangement. Identifying small molecules binding to the aptamer region of these riboswitches to disrupt its synthesis is an innovative approach for developing alternative antibiotics. Different methods are used to identify small molecules, like structural approaches by NMR, thermodynamic changes with ITC or radius of gyration by SAXS. Herein, we developed biochips to overcome pricy sample preparation and consumption, with which we measure binding kinetics of molecules targeting aptamers using switchSENSE® biosensing technology. switchSENSE® is based on electro-switchable DNA-layers immobilized on a gold surface. The 2-color optical detection system allows on-chip FRET experiments to monitor not only binding, but also structural re-arrangements. As proof of concept we studied the binding-induced folding of the cocaine-binding aptamer with different small molecules. This tool delivers fast results on small molecule screenings for detailed binding kinetics and binding-induced folding kinetics which can improve the search of novel drugs at the transcriptome level.

Dynamics of the bypass polymerase, dinB homolog.

The dinB homolog (Dbh) polymerase is a member of the Y-family of translesion DNA polymerases, able to synthesize DNA across damaged DNA template. Since Dbh is derived from the thermophilic archaeon Sulfolobus acidocaldarius, it is capable of functioning at high temperatures where mesophilic proteins would be denatured. Here we use NMR and Circular Dichroism (CD) to compare protein stability at a wide range of temperatures. 1H-15N HSQC spectra show a change of tertiary structures starting at 25 °C and progression at colder temperatures (15, 5 °C). CD show retentions of secondary structure with a slight decrease percent helicity and an increase in percent beta strand as temperature decrease. We have analyzed hydrogen exchange measurement at 35°C and 50°C to investigate the folding dynamics of Dbh. We observe the moderate modulation of dynamics upon heating. Core residues in the palm and little finger domain, especially those in the interior of the β-sheets and interior face of α-helices, are key in maintaining the stability of Dbh; with the palm providing a stable platform for the active site residues. We report on regions of the protein that become more dynamic as the temperature is increased toward the functional temperature.

BICePs repx: Force field validation by a Bayesian/maximum entropy approach.

Bayesian Inference of Conformational Populations (BICePs) is a reweighting algorithm that reconciles simulated ensembles with sparse and/or noisy observables. Typically, force fields are ranked using a chi-squared metric by comparing simulated observables with experimental measurements, using some estimate of the experimental uncertainty, while neglecting systematic error. In contrast to this approach, BICePs has the significant advantage of performing objective model selection by sampling the full posterior distribution of conformational populations as well as uncertainties due to random and systematic error. To demonstrate this, we used BICePs to perform Bayesian model selection for nine different force fields used to simulate the folding dynamics of the mini-protein chignolin in TIP3P solvent. The simulated ensembles were compared against 158 experimental measurements (139 NOE distances, 13 chemical shifts, and 6 vicinal J-coupling constants for HN and Hα). A significant advantage of our approach is the BICePs score, which is a metric that quantifies the agreement between simulation and experiment by computing a free energy-like quantity that reflects the amount of adjustment/reweighting necessary for the populations to match the experimental restraints. For the nine force fields tested (ff14SB, ff99SB-ildn, ff99, ff99SBnmr1-ildn, ff99SB, CHARMM22star, CHARMM27, CHARMM36, OPLS-aa), our results are consistent with previous work that used a conventional chi-squared metric for model selection for small polypeptides and ubiquitin, with ff99SBnmr1-ildn remaining as the best-scoring model.

The Role of Transient Intermediate Structures in the Unfolding of the Trp-Cage Fast-Folding Protein: Generating Ensembles from Time-Resolved X-ray Solution Scattering with Genetic Algorithms.

The Trp-cage miniprotein is one of the smallest systems to exhibit a stable secondary structure and fast-folding dynamics, serving as an apt model system to study transient intermediates with both experimental and computational analyses. Previous spectroscopic characterizations that have been done on Trp-cage have inferred a single stable intermediate on a pathway from folded to unfolded basins. We aim to bridge the understanding of Trp-cage structural folding dynamics on microsecond-time scales, by utilizing time-resolved X-ray solution scattering to probe the temperature-induced unfolding pathway. Our results indicate the formation of a conformationally extended intermediate on the time scale of 1 μs, which undergoes complete unfolding within 5 μs. We further investigated the atomistic structural details of the unfolding pathway using a genetic algorithm to generate ensemble model fits to the scattering profiles. This analysis paves the way for direct benchmarking of theoretical models of protein folding ensembles produced with molecular dynamics simulations.

The Deuterium Calculator: An Open-Source Tool for Hydrogen-Deuterium Exchange Mass Spectrometry Analysis.

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful protein footprinting technique to study protein dynamics and binding; however, HDX-MS data analysis is often challenging and time-consuming. Moreover, the HDX community is expanding to investigate multiprotein and highly complex protein systems which further complicates data analysis. Thus, a simple, open-source software package designed to analyze large and highly complex protein systems is needed. In this vein, we have developed "The Deuterium Calculator", a Python-based software package for HDX-MS data analysis. The Deuterium Calculator is capable of differential and nondifferential HDX-MS analysis, produces standardized data files according to recommendations from the International Conference on Hydrogen-Exchange Mass Spectrometry (IC-HDX) to increase transparency in data analysis, and generates Woods' plots for statistical analysis and data visualization. This standard output can be used to perform time dependent deuteration studies and for the study of protein folding kinetics or differential uptake. Moreover, The Deuterium Calculator is capable of performing these analyses on large HDX-MS data sets (e.g., LC-HDX-MS from cell lysate digest). The Deuterium Calculator is freely available for download at https://github.com/OUWuLab/TheDeuteriumCalculator.git. Data are available via ProteomeXchange with identifier PXD036813.

Compliant mechanical response of the ultrafast folding protein EnHD under force

Ultrafast folding proteins have become an important paradigm in the study of protein folding dynamics. Due to their low energetic barriers and fast kinetics, they are amenable for study by both experiment and simulation. However, single molecule force spectroscopy experiments on these systems are challenging as these proteins do not provide the mechanical fingerprints characteristic of more mechanically stable proteins, which makes it difficult to extract information about the folding dynamics of the molecule. Here, we investigate the unfolding of the ultrafast protein Engrailed Homeodomain (EnHD) by single-molecule atomic force microscopy experiments. Constant speed experiments on EnHD result in featureless transitions typical of compliant proteins. However, in the force-ramp mode we recover sigmoidal curves that we interpret as a very compliant protein that folds and unfolds many times over a marginal barrier. This is supported by a simple theoretical model and coarse-grained molecular simulations. Our results show the ability of force to modulate the unfolding dynamics of ultrafast folding proteins.

Structure-activity correlations for peptaibols obtained from clade Longibrachiatum of Trichoderma: A combined experimental and computational approach

Integrated disease management and plant protection have been discussed with much fervor in the past decade due to the rising environmental concerns of using industrially produced pesticides. Members of the genus Trichoderma are a subject of considerable research today due to their several properties as biocontrol agents. In our study, the peptaibol production of Trichoderma longibrachiatum SZMC 1775, T. longibrachiatum f. bissettii SZMC 12546, T. reesei SZMC 22616, T. reesei SZMC 22614, T. saturnisporum SZMC 22606 and T. effusum SZMC 22611 were investigated to elucidate structure-activity relationships (SARs) between the properties of peptaibols and their 3D structures. The effects of peptaibol mixtures obtained from every Trichoderma strain were examined against nine commonly known bacteria. The lowest minimum inhibitory concentrations (MIC, mg ml−1) were exerted by T. longibrachiatum f. bissettii SZMC 12546 against Gram-positive bacteria, which was also able to inhibit the plant pathogenic Gram-negative Rhizobium radiobacter. Accelerated molecular dynamics (aMD) simulations were performed in aqueous solvent to explore the folding dynamics of 12 selected peptaibol sequences. The most characteristic difference between the peptaibols from group A and B relies in the ‘Gly-Leu-Aib-Pro’ and ‘Gly-Aib-Aib-Pro’ motifs (‘Aib’ stands for α-aminoisobutyric acid), which imparted a significant effect on the folding dynamics in water and might be correlated with their expressed bioactivity. In our aMD simulation experiments, Group A peptaibols showed more restricted folding dynamics with well-folded helical conformations as the most stable representative structures. This structural stability and dynamics may contribute to their bioactivity against the selected bacterial species.

Qualitative Bioinformatics: Towards a Public Understanding of Neurodegenerative Disease Research through BioArt, Data-Art, Hands-on BioMedia Workshops, Immersive Environments, and Artists in Labs.

Neurodegenerative disease (ND) research is producing new pharmaceutical compounds for chronic diseases utilizing bioinformatics. Algorithmic data mining of high-throughput omics systems is conjoined with the study of the in vitro dynamics of protein folding through visualization techniques, i.e., Bio-AFM. Novel models of disease pathway diagrams are being produced and the processes are in place to enhance the validation of these models. From bench work to BLAST to bedside, globally standardized research is led by collaborative clusters, working on cloud-based platforms, with crowd sourced human cohort collections, pairing pure, curiosity-based neurological research with deep learning data analysis techniques. ND research is leading the way towards clinical applications for preventing, curing, or ameliorating major diseases of neurological dysfunction, such as Alzheimer's disease (AD) and Parkinson's disease (PD).Most of this is too complex for the average citizen to comprehend. Can bioart and data-art contribute to the public understanding of genome sequencing and bioinformatics databases in the context of ND research? Informal studies towards demystifying bioinformatics through creative practices have been tested, revealing significant qualitative benefits for public well-being while introducing bioinformatics databases, for instance, previous projects: the VASTAL Bioinformatics and Literary Studies: (De)Mystified Genetic Code Lab, held at the Waag in Amsterdam, NL, 2009 and more recently the Creative Germline Constructs Bank (CGCB) of the transgenic human Genome Alternatives Project (thGAP), held at Hackteria ZET, Zurich, Switzerland, 2021.For the Hub of Art Laboratories (HAL), of the Department of Audio & Visual Arts (AVArts), Ionian University in Corfu, Greece, the animation node is challenged to create audiovisual, immersive, and interactive environments that highlight natural processes and phenomena of the microcosm and macrocosm through nature/data interface experimentation. It is the intention of the node's researchers to expand these notions to be inclusive of the processes of the biotechnological and bioinformatics interrogation of human disease as a part of local clades and global nature. In our productions, we would like to emphasize: cryogenic storage, genome analysis, bioinformatic database management, biomarkers for polygenic abnormalities, genotyping/phenotyping, gene expression patterns over time, AI reading of cloud-based research, experiencial DNA/RNA synthesis, the development of novel DNA, RNA and protein-based therapeutic agents for biomedical applications and CRISPR construct design for model organisms. It is the HAL animation node's goal to design and explore bioart-based, hands-on public workshops that mix bioinformatics and data-art with in-depth knowledge of the scientific community around ND research bodies.To develop these labs, dedicated artistic research is required to be undertaken through laboratory immersion, interactions with scientists, and hands-on experience in the lab. Artists must experience scientific processes through residencies in labs to learn both how to convey the techniques and the social implications of novel methodologies. Artists developing bioart workshops that can introduce non-specialists to a dizzying array of research methodologies need to know what they are talking about. It takes experience and dedication to convey simplified versions of real-time biosensor data, automated histology, clinical databases of medical case studies (anonymized clients), real-time PCR, novel diagnostic biomarker development, etc. As qualitative researchers, artists in labs ensure creative yet informed art and science (SciArt) outcomes able to stimulate public debate through unorthodox education and accurate playfulness. Obviously, the immersive experience of lab research provides an artist's projects with both nuance and knowledge. How can artists in labs also add to the scientific potentials of a laboratory's research goals?

Towards de novo design of transmembrane α-helical assemblies using structural modelling and molecular dynamics simulation.

Computational de novo protein design involves iterative processes consisting of amino acid sequence design, structural modelling and scoring, and design validation by synthesis and experimental characterisation. Recent advances in protein structure prediction and modelling methods have enabled the highly efficient and accurate design of water-soluble proteins. However, the design of membrane proteins remains a major challenge. To advance membrane protein design, considering the higher complexity of membrane protein folding, stability, and dynamic interactions between water, ions, lipids, and proteins is an important task. For introducing explicit solvents and membranes to these design methods, all-atom molecular dynamics (MD) simulations of designed proteins provide useful information that cannot be obtained experimentally. In this review, we first describe two major approaches to designing transmembrane α-helical assemblies, consensus and de novo design. We further illustrate recent MD studies of membrane protein folding related to protein design, as well as advanced treatments in molecular models and conformational sampling techniques in the simulations. Finally, we discuss the possibility to introduce MD simulations after the existing static modelling and screening of design decoys as an additional step for refinement of the design, which considers membrane protein folding dynamics and interactions with explicit membranes.

Monomeric and dimeric states of human ZO1-PDZ2 are functional partners of the SARS-CoV-2 E protein

The Envelope (E) protein of SARS-CoV-2 plays a key role in virus maturation, assembly, and virulence mechanisms. The E protein is characterized by the presence of a PDZ-binding motif (PBM) at its C-terminus that allows it to interact with several PDZ-containing proteins in the intracellular environment. One of the main binding partners of the SARS-CoV-2 E protein is the PDZ2 domain of ZO1, a protein with a crucial role in the formation of epithelial and endothelial tight junctions (TJs). In this work, through a combination of analytical ultracentrifugation analysis and equilibrium and kinetic folding experiments, we show that ZO1-PDZ2 domain is able to fold in a monomeric state, an alternative form to the dimeric conformation that is reported to be functional in the cell for TJs assembly. Importantly, surface plasmon resonance (SPR) data indicate that the PDZ2 monomer is fully functional and capable of binding the C-terminal portion of the E protein of SARS-CoV-2, with a measured affinity in the micromolar range. Moreover, we present a detailed computational analysis of the complex between the C-terminal portion of E protein with ZO1-PDZ2, both in its monomeric conformation (computed as a high confidence AlphaFold2 model) and dimeric conformation (obtained from the Protein Data Bank), by using both polarizable and nonpolarizable simulations. Together, our results indicate both the monomeric and dimeric states of PDZ2 to be functional partners of the E protein, with similar binding mechanisms, and provide mechanistic and structural information about a fundamental interaction required for the replication of SARS-CoV-2.

Mechanism of β-hairpin formation in AzoChignolin and Chignolin.

AzoChignolin is a photoswitchable variant of the mini-protein Chignolin with an azobenzene (AMPP) replacing the central loop. AzoChignolin is unfolded with AMPP in the trans-isomer. Transition to the cis-isomer causes β-hairpin folding similar to Chignolin. The AzoChignolin system is excellently suited for comprehensive analysis of folding nucleation kinetics. Utilizing multiple long-time MD simulations of AzoChignolin and Chignolin in MeOH and water, we estimated Markov models to examine folding kinetics of both peptides. We show that while AzoChignolin mimics Chignolin's structure well, the folding kinetics are quite different. Not only folding times but also intermediate states differ, particularly Chignolin is able to fold in MeOH into an α-helical intermediate which is impossible to form in AzoChignolin. The Markov models demonstrate that AzoChignolin's kinetics are generally faster, specifically when comparing the two main microfolding processes of hydrophobic collapse and turn formation. Photoswitchable loops are used frequently to understand the kinetics of elementary protein folding nucleation. However, our results indicate that intermediates and folding kinetics may differ between natural loops and photoswitchable variants.

Metal-binding and circular permutation-dependent thermodynamic and kinetic stability of azurin.

Native topology is known to determine the folding kinetics and the energy landscape of proteins. Furthermore, the circular permutation (CP) of proteins alters the order of the secondary structure connectivity while retaining the three-dimensional structure, making it an elegant and powerful approach to altering native topology. Previous studies elucidated the influence of CP in proteins with different folds such as Greek key β-barrel, β-sandwich, β-α-β, and all α-Greek key. CP mainly affects the protein stability and unfolding kinetics, while folding kinetics remains mostly unaltered. However, the effect of CP on metalloproteins is yet to be elaborately studied. The active site of metalloproteins poses an additional complexity in studying protein folding. Here, we investigate a CP variant (cpN42) of azurin-in both metal-free and metal-bound (holo) forms. As observed earlier in other proteins, apo-forms of wild-type (WT) and cpN42 fold with similar rates. In contrast, zinc-binding accelerates the folding of WT but decelerates the folding of cpN42. On zinc-binding, the spontaneous folding rate of WT increases by >250 times that of cpN42, which is unprecedented and the highest for any CP to date. On the other hand, zinc-binding reduces the spontaneous unfolding rate of cpN42 by ~100 times, making the WT and CP azurins unfold at similar rates. Our study demonstrates metal binding as a novel way to modulate the unfolding and folding rates of CPs compared to their WT counterparts. We hope our study increases the understanding of the effect of CP on the folding mechanism and energy landscape of metalloproteins.

Evolutionary conservation of amino acids contributing to the protein folding transition state.

The question of whether amino acids critical to protein folding kinetics are evolutionarily conserved has been investigated intensively in the past, but no consensus has yet been reached. Recently, we have demonstrated that the transition state, dictating folding kinetics, is characterized as the state of maximum dynamic cooperativity, i.e., the state of maximum correlations between amino acid contact formations. Here, we investigate the evolutionary conservation of those amino acids contributing significantly to the dynamic cooperativity. We find a strong indication of a new kind of relationship-necessary but not sufficient causality-between the evolutionary conservation and the dynamic cooperativity: larger contributions to the dynamic cooperativity arise from more conserved residues, but not vice versa. This holds for all the protein systems for which long folding simulation trajectories are available. To our knowledge, this is the first systematic demonstration of any kind of evolutionary conservation of amino acids relevant to folding kinetics.

Single Molecule FRET Analysis of CRISPR Cas9 Single Guide RNA Folding Dynamics.

CRISPR Cas9 is an RNA guided endonuclease that is part of a bacterial adaptive immune system. Single guide RNA (sgRNA) can be designed to target genomic DNA, making Cas9 a programmable DNA binding/cutting enzyme and allowing applications such as epigenome editing, controlling transcription, and targeted DNA insertion. Some of the main hurdles against an even wider adoption are off-target effects and variability in Cas9 editing outcomes. Most studies that aim to understand the mechanisms that underlie these two areas have focused on Cas9 DNA binding, DNA unwinding, and target cleavage. The assembly of Cas9 RNA ribonucleoprotein complex (RNP) precedes all these steps and includes sgRNA folding and Cas9 binding to sgRNA. We know from the crystal structure of the Cas9 RNP what the final sgRNA conformation is. However, the assembly dynamics has not been studied in detail and a better understanding of RNP assembly could lead to better-designed sgRNAs and better editing outcomes. To study this process, we developed a single molecule FRET assay to monitor the conformation of the sgRNA and the binding of Cas9 to sgRNA. We labeled the sgRNA with a donor fluorophore and an acceptor fluorophore such that when the sgRNA folds, there are changes in FRET efficiency. We measured sgRNA folding dynamics under different ion conditions, under various methods of folding (refolding vs vectorial), and with or without Cas9. sgRNA that closely mimics the sgRNA construct used for high resolution structural analysis of the Cas9-gRNA complex showed two main FRET states without Cas9, and Cas9 addition shifted the distribution toward the higher FRET state attributed to the properly assembled complex. Even in the absence of Cas9, folding the sgRNA vectorially using a superhelicase-dependent release of the sgRNA in the direction of transcription resulted in almost exclusively high FRET state. An addition of Cas9 during vectorial folding greatly reduced a slow-folding fraction. Our studies shed light on the heterogeneous folding dynamics of sgRNA and the impact of co-transcriptional folding and Cas9 binding in sgRNA folding. Further studies of sequence dependence may inform rational design of sgRNAs for optimal function.

Cohesin and CTCF control the dynamics of chromosome folding

In mammals, interactions between sequences within topologically associating domains enable control of gene expression across large genomic distances. Yet it is unknown how frequently such contacts occur, how long they last and how they depend on the dynamics of chromosome folding and loop extrusion activity of cohesin. By imaging chromosomal locations at high spatial and temporal resolution in living cells, we show that interactions within topologically associating domains are transient and occur frequently during the course of a cell cycle. Interactions become more frequent and longer in the presence of convergent CTCF sites, resulting in suppression of variability in chromosome folding across time. Supported by physical models of chromosome dynamics, our data suggest that CTCF-anchored loops last around 10 min. Our results show that long-range transcriptional regulation might rely on transient physical proximity, and that cohesin and CTCF stabilize highly dynamic chromosome structures, facilitating selected subsets of chromosomal interactions.

Folding Dynamics of 3,4,3-LI(1,2-HOPO) in Its Free and Bound State with U4+ Implicated by MD Simulations

The octadentate hydroxypyridonate ligand 3,4,3-LI(1,2-HOPO) (t-HOPO) shows strong binding affinity with actinide cations and is considered as a promising decorporation agent used to eliminate in vivo actinides, while its dynamics in its unbound and bound states in the condensed phase remain unclear. In this work, by means of MD simulations, the folding dynamics of intact t-HOPO in its neutral (t-HOPO0) and in its deprotonated state (t-HOPO4-) were studied. The results indicated that the deprotonation of t-HOPO in the aqueous phase significantly narrowed the accessible conformational space under the simulated conditions, and it was prepared in a conformation that could conveniently clamp the cations. The simulation of UIV-t-HOPO showed that the tetravalent uranium ion was deca-coordinated with eight ligating O atoms from the t-HOPO4- ligand, and two from aqua ligands. The strong electrostatic interaction between the U4+ ion and t-HOPO4- further diminished the flexibility of t-HOPO4- and confined it in a limited conformational space. The strong interaction between the U4+ ion and t-HOPO4- was also implicated in the shortened residence time of water molecules.