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SummaryUnderstanding how the nuclear pore complex (NPC) assembles is of fundamental importance to grasp the mechanisms behind its essential function and understand its role during evolution of eukaryotes1–4. While at least two NPC assembly pathways exist, one during exit from mitosis and one during nuclear growth in interphase, we currently lack a quantitative map of the molecular assembly events. Here, we use fluorescence correlation spectroscopy (FCS) calibrated live imaging of endogenously fluorescently-tagged nucleoporins to map the changes in composition and stoichiometry of seven major modules of the human NPC during its assembly in single dividing cells. This systematic quantitative map reveals that the two assembly pathways employ strikingly different molecular mechanisms, inverting the order of addition of two large structural components, the central ring complex and nuclear filaments. The dynamic stoichiometry data underpinned integrative spatiotemporal modeling of the NPC assembly pathway, predicting the structures of postmitotic NPC assembly intermediates.

Inside prokaryotic cells, passive translational diffusion is fundamental to most cellular processes because it allows the sorting of macromolecules, proteins in particular, and it sets the upper limit for biochemical reactions to happen. In the highly crowded, confined, and non-homogeneous environment of a bacterial cell, protein diffusion is typically hindered. Drawing general conclusions about the size dependence of protein diffusion from previous studies was hampered by the limited number of protein probes investigated and by the differences in the strain, the growth conditions, or the measurement technique. Furthermore, while the impact of several physiological perturbations on protein diffusion has been established, most of the previous studies used either large particles or free GFP as probes. How these perturbations affect the diffusional properties of the cytoplasm over the entire physiological range of protein sizes remains unknown. Here, we address these limitations by systematically analyzing the mobility of a large number of differently-sized cytoplasmic fluorescent protein constructs in the cytoplasm of Escherichia coli by fluorescence correlation spectroscopy (FCS) under standardized conditions. By combining experimental evidences with simulations and theoretical modeling, we concluded that protein mobility in the cytoplasm could be well described by Brownian diffusion in the confined geometry of the bacterial cell and at the high viscosity imposed by macromolecular crowding. Pronounced sub-diffusion and hindered mobility were only observed for proteins with extensive interactions within the cytoplasm. We observed that the size dependence of protein diffusion for the majority of tested proteins, whether native or foreign to E. coli, it was well consistent with the Stokes-Einstein relation once the specific dumbbell shape of protein fusions is taken into account. Furthermore, such size dependence was conserved over the different spatiotemporal scales explored by FCS and fluorescence recovery after photobleaching (FRAP). A subset of constructs, spanning over a wide range of physiologically relevant protein masses, was then used to probe the effect on diffusion of diverse, biologically meaningful, physicochemical perturbations and of cell growth. In particular, protein diffusion became markedly faster in actively growing cells, at high temperature, or upon treatment with rifampicin, and slower at high osmolarity. Importantly, all of these perturbations affected proteins of different sizes in the same proportions, which could thus be described as changes of a well-defined cytoplasmic viscosity. We then observed that the reactivation of ATP-dependent enzymes in diffusionally-impaired cells is sufficient to promote cytoplasmic stirring and diffusion of inert sfGFP. Lastly, we demonstrated that catalytically-induced enhanced diffusion, previously observed only in vitro, can be detected for some ATP-dependent enzymes also in living systems.

In this thesis I describe the results of Pulsed Interleaved Excitation and Fluorescence (Cross) Correlation Spectroscopy (PIE-F(C)CS) combined with single-pair Förster Resonance Energy Transfer (spFRET) used to study dynamics in single nucleosomes, which depends on subtle differences in the length of DNA ends, DNA sequence, histone variants and specific and non-specific protein interactions. This technique, which can resolve distances between two fluorophores of only a few nanometers, is an excellent technique to monitor changes in nucleosomal compaction, as the nucleosome is only ten nanometers in diameter. In combination with F(C)CS and PIE, spFRET makes it possible to monitor conformational dynamics on a timescale of micro- to milliseconds.

Background The cytoskeleton is a key component of the system responsible for transmitting mechanical cues from the cellular environment to the nucleus, where they trigger downstream responses. This communication is particularly relevant in embryonic stem (ES) cells since forces can regulate cell fate and guide developmental processes. However, little is known regarding cytoskeleton organization in ES cells, and thus, relevant aspects of nuclear-cytoskeletal interactions remain elusive. Results We explored the three-dimensional distribution of the cytoskeleton in live ES cells and show that these filaments affect the shape of the nucleus. Next, we evaluated if cytoskeletal components indirectly modulate the binding of the pluripotency transcription factor OCT4 to chromatin targets. We show that actin depolymerization triggers OCT4 binding to chromatin sites whereas vimentin disruption produces the opposite effect. In contrast to actin, vimentin contributes to the preservation of OCT4-chromatin interactions and, consequently, may have a pro-stemness role. Conclusions Our results suggest roles of components of the cytoskeleton in shaping the nucleus of ES cells, influencing the interactions of the transcription factor OCT4 with the chromatin and potentially affecting pluripotency and cell fate.

International audience; The identification and characterization of bona fide abiotic stress signaling proteins can occur at different levels of the complete in vivo signaling cascade or network. Knowledge of a particular abiotic stress signaling protein could theoretically lead to the characterization of complete networks through the analysis of unknown proteins that interact with the previously known protein. Such signaling proteins of interest can indeed be experimentally used as bait proteins to catch interacting prey proteins, provided that the association of bait proteins and prey proteins should yield a biochemical or biophysical signal that can be detected. To this end, several biochemical and biophysical techniques are available to provide experimental evidence for specific protein-protein interactions, such as co-immunoprecipitation, bimolecular fluorescence complementation, tandem affinity purification coupled to mass spectrometry, yeast two hybrid, protein microarrays, Förster resonance energy transfer, or fluorescence correlation spectroscopy. This array of methods can be implemented to establish the biochemical reality of putative protein-protein interactions between two proteins of interest or to identify previously unknown partners related to an initially known protein of interest. The ultimate validity of these methods however depends on the in vitro/in vivo nature of the approach and on the heterologous/homologous context of the analysis. This chapter will review the application and success of some classical methods of protein-protein interaction analysis in the field of plant abiotic stress signaling.

The high volatility, water‐immiscibility, and light/oxygen‐sensitivity of most aroma compounds represent a challenge to their incorporation in liquid consumer products. Current encapsulation methods entail the use of petroleum‐based materials, initiators, and crosslinkers as well as mixing, heating, and purification steps. Hence, more efficient and eco‐friendly approaches to encapsulation must be sought. Herein, we propose a simple method by making use of a pre‐formed amphiphilic polymer and employing the Hansen Solubility Parameters approach to determine which fragrances could be encapsulated by spontaneous coacervation in water. The coacervates do not precipitate as solids but they remain suspended as colloidally stable liquid microcapsules, as demonstrated by fluorescence correlation spectroscopy. The effective encapsulation of fragrance is proven through confocal Raman spectroscopy, while the structure of the capsules is investigated by means of cryo FIB/SEM, confocal laser scanning microscopy, and small‐angle X‐ray scattering., The use of a PEG‐g‐PVAc amphiphilic graft copolymer together with hydrophobic fragrances in aqueous medium leads to the spontaneous formation of colloidally stable suspensions of liquid coacervate droplets. Arrows indicate the local turbulences sustaining the spontaneous mixing of the components. The bottom panel shows the confocal Raman microspectroscopy mapping of a multicompartment coacervate droplet with carvone as fragrance.

Nanotechnologies hold great promise for various applications. To predict and guarantee the safety of novel nanomaterials, it is essential to understand their mechanism of action in an organism, causally connecting adverse outcomes with early molecular events. This is best investigated using noninvasive advanced optical methods, such as high-resolution live-cell fluorescence microscopy, which require stable labeling of nanoparticles with fluorescent dyes. However, as shown here, when the labeling is performed inadequately, unbound fluorescent dyes and inadvertently altered chemical and physical properties of the nanoparticles can result in experimental artefacts and erroneous conclusions. To prevent such unintentional errors, we introduce a tested minimal combination of experimental methods to enable artefact-free fluorescent labeling of metal-oxide nanoparticles-the largest subpopulation of nanoparticles by industrial production and applications-and demonstrate its application in the case of TiO2 nanotubes. We (1) characterize potential changes of the nanoparticles' surface charge and morphology that might occur during labeling by using zeta potential measurements and transmission electron microscopy, respectively, and (2) assess stable binding of the fluorescent dye to the nanoparticles with either fluorescence intensity measurements or fluorescence correlation spectroscopy, which ensures correct nanoparticle localization. Together, these steps warrant the reliability and reproducibility of advanced optical tracking, which is necessary to explore nanomaterials' mechanism of action and will foster widespread and safe use of new nanomaterials.

Dynamic compartmentalization is a prevailing principle regulating the spatiotemporal organization of the living cell membrane from the nano- up to the mesoscale. This non-arbitrary organization is intricately linked to cell function. On living cell membranes, dynamic domains or ‘membrane rafts' enriched with cholesterol, sphingolipids and other certain proteins exist at the nanoscale serving as signaling and sorting platforms. Moreover, it has been postulated that other local organizers of the cell membrane such as intrinsic protein interactions, the extracellular matrix and/or the actin cytoskeleton synergize with rafts to provide spatiotemporal hierarchy to the membrane. Elucidating the intricate coupling of multiple spatial and temporal scales requires the application of correlative techniques, with a particular need for simultaneous nanometer spatial precision and microsecond temporal resolution. Here, we review novel fluorescence-based techniques that readily allow to decode nanoscale membrane dynamics with unprecedented spatiotemporal resolution and single-molecule sensitivity. We particularly focus on correlative approaches from the field of nanophotonics. Notably, we introduce a versatile planar nanoantenna platform combined with fluorescence correlation spectroscopy to study spatiotemporal heterogeneities on living cell membranes at the nano- up to the mesoscale. Finally, we outline remaining future technological challenges and comment on potential directions to advance our understanding of cell membrane dynamics under the influence of the actin cytoskeleton and extracellular matrix in uttermost detail.

Journal of fluorescence (2021). doi:10.1007/s10895-021-02803-3, Published by Springer Science + Business Media B.V., New York, NY

Ribonucleic acid (RNA) is exceedingly sensitive to degradation compared to DNA. The current protocol for storage of purified RNA requires freezing conditions below −20 °C. Recent advancements in biological chemistry have identified amino acid-based ionic liquids as suitable preservation media for RNA, even in the presence of degrading enzymes. However, the mechanistic insight into the interaction between ILs and RNA is unclear. To the best of our knowledge, no attempts are made so far to provide a molecular view. This work aims to establish a detailed understanding of how ILs enable structural stability to RNA sourced from Torula yeast. Herein, we manifest the hypothesis of multimodal binding of IL and its minimal perturbation to the macromolecular structure, with several spectroscopic techniques such as time-resolved fluorescence and fluorescence correlation spectroscopy (FCS) aided with molecular dynamics at microsecond time scales. Relevant structural and thermodynamic details from biophysical experiments confirm that even long-term RNA preservation with ILs is a possible alternative devoid of any structural deformation. These results establish a unifying mechanism of how ILs are maintaining conformational integrity and thermal stability. The atomistic insights are transferable for their potential applications in drug delivery and biomaterials by considering the advantages of having maximum structural retention and minimum toxicity., Electrostatic interactions drive the multimodal binding between ChAAILs and RNA. Biocompatible ChAAILs can be used as a green preservative medium for long-term storage of RNA at ambient conditions.

Antibody-based therapeutics are the fastest growing drug class on the market, used to treat aggressive forms of cancer, chronic autoimmune conditions, and numerous other disease states. While the specificity, affinity, and versatility of therapeutic antibodies can provide an advantage over traditional small molecule drugs, their development and optimization can be much more challenging and time-consuming. This is, in part, because the ideal formulation buffer systems used for in vitro characterization inadequately reflect the crowded biological environments (serum, endosomal lumen, etc.) that these drugs experience once administered to a patient. Such environments can perturb the binding of antibodies to their antigens and receptors, as well as homo- and hetero-aggregation, in ways that are incompletely understood, thereby altering therapeutic effect and disposition. While excluded volume effects are classically thought to favor binding, weak interactions with co-solutes in crowded conditions can inhibit binding. The second virial coefficient (B2) parameter quantifies such weak interactions and can be determined by a variety of techniques in dilute solution, but analogous methods in complex biological fluids are not well established. Here, we demonstrate that fluorescence correlation spectroscopy (FCS) is able to measure diffusive B2 values directly in undiluted serum. Apparent second virial coefficient (B2,app) measurements of antibodies in serum reveal that changes in the balance between attractive and repulsive interactions can dramatically impact global nonideality. Furthermore, our findings suggest that the common approach of isolating specific components and completing independent cross-term virial coefficient measurements is an incomplete representation of nonideality in serum. The approach presented here could enrich our understanding of the effects of biological environments on proteins in general, and advance the development of therapeutic antibodies and other protein-based therapeutics.STATEMENT OF SIGNIFICANCEWe present FCS as an orthogonal method to traditional methods for characterizing weak, nonspecific homo- and hetero-interactions through determination of self- and cross-term second virial coefficients, respectively. We also characterize weak interactions between therapeutic antibodies and serum components through determination of an apparent second virial coefficient (B2,app) directly in undiluted serum. Our results suggest that global nonideality effects are antibody-dependent, and that attractive and repulsive interactions with co-solutes are occurring simultaneously. This approach could advance our understanding of the impact of nonideality to the biophysical and pharmacological properties of therapeutic antibodies and other engineered proteins in relevant biological environments, and could accelerate the development and optimization of protein-based therapeutics.

Numerous studies have been devoted to understand the reaction kinetics in micelles, where the accessible kinetic time window is often limited by the dynamic range of the employed spectroscopic technique. This is usually accompanied by a selection of probes that comfortably explore time scales where slow solute exchange kinetics is negligible, as compared to the fast excited state reactions. This has led to an undervaluation of the role played by dynamic partitioning of hydrophilic solutes in microheterogeneous media. Here, we employ fluorescence correlation spectroscopy (FCS) and the zwitterionic dye Rhodamine 110 to quantitatively explore the impact of solute exchange on the photoinduced electron transfer between this dye and N,N-dimethylaniline in micellar media. Our study elucidates the coupling and interplay between the kinetics of photophysics, quenching, and solute exchange through a quantitative unified molecular-state quenching-kinetic model that describes the steady-state ensemble and FCS data from subnanosecond photon antibunching to millisecond diffusions.

Compartmentalization and integration of molecular processes through diffusion are basic mechanisms through which cells perform biological functions. To characterize these mechanisms in live cells, quantitative and ultrasensitive analytical methods with high spatial and temporal resolution are needed. Here, we present quantitative scanning-free confocal microscopy with single-molecule sensitivity, high temporal resolution (∼10 μs/frame), and fluorescence lifetime imaging capacity, developed by integrating massively parallel fluorescence correlation spectroscopy with fluorescence lifetime imaging microscopy (mpFCS/FLIM); we validate the method, use it to map in live cell location-specific variations in the concentration, diffusion, homodimerization, DNA binding, and local environment of the oligodendrocyte transcription factor 2 fused with the enhanced Green Fluorescent Protein (OLIG2-eGFP), and characterize the effects of an allosteric inhibitor of OLIG2 dimerization on these determinants of OLIG2 function. In particular, we show that cytoplasmic OLIG2-eGFP is largely monomeric and freely diffusing, with the fraction of freely diffusing OLIG2-eGFP molecules being fD,freecyt = (0.75 ± 0.10) and the diffusion time τD,freecyt = (0.5 ± 0.3) ms. In contrast, OLIG2-eGFP homodimers are abundant in the cell nucleus, constituting ∼25% of the nuclear pool, some fD,boundnuc = (0.65 ± 0.10) of nuclear OLIG2-eGFP is bound to chromatin DNA, whereas freely moving OLIG2-eGFP molecules diffuse at the same rate as those in the cytoplasm, as evident from the lateral diffusion times τD,freenuc = τD,freecyt = (0.5 ± 0.3) ms. OLIG2-eGFP interactions with chromatin DNA, revealed through their influence on the apparent diffusion behavior of OLIG2-eGFP, τD,boundnuc (850 ± 500) ms, are characterized by an apparent dissociation constant Kd,appOLIG2-DNA = (45 ± 30) nM. The apparent dissociation constant of OLIG2-eGFP homodimers was estimated to be Kd,app(OLIG2-eGFP)2 ≈ 560 nM. The allosteric inhibitor of OLIG2 dimerization, compound NSC 50467, neither affects OLIG2-eGFP properties in the cytoplasm nor does it alter the overall cytoplasmic environment. In contrast, it significantly impedes OLIG2-eGFP homodimerization in the cell nucleus, increasing five-fold the apparent dissociation constant, Kd,app,NSC50467(OLIG2-eGFP)2 ≈ 3 μM, thus reducing homodimer levels to below 7% and effectively abolishing OLIG2-eGFP specific binding to chromatin DNA. The mpFCS/FLIM methodology has a myriad of applications in biomedical research and pharmaceutical industry. For example, it is indispensable for understanding how biological functions emerge through the dynamic integration of location-specific molecular processes and invaluable for drug development, as it allows us to quantitatively characterize the interactions of drugs with drug targets in live cells.

Glycans of cellular glycoconjugates serve as biochemical signals for a multitude of (patho)physiological processes via binding to their receptors (e.g. lectins). In the case of human adhesion/growth-regulatory galectin-1 (Gal-1), small angle neutron scattering and fluorescence correlation spectroscopy have revealed a significant decrease of its gyration radius and increase of its diffusion coefficient upon binding lactose, posing the pertinent question on the nature and region(s) involved in the underlying structural alterations. Requiring neither a neutron source nor labeling, diffusion measurements by 1H NMR spectroscopy are shown here to be sufficiently sensitive to detect this ligand-induced change. In order to figure out which region(s) of Gal-1 is (are) affected at the level of peptides, we first explored the use of H/D exchange mass spectrometry (HDX MS). Hereby, we found a reduction in proton exchange kinetics beyond the lactose-binding site. The measurement of fast HN/H2O exchange by phase-modulated NMR clean chemical exchange (CLEANEX) NMR on 15N-labeled Gal-1 then increased the spatial resolution to the level of individual amino acids. The mapped regions with increased protection from HN/H2O (D2O) exchange that include the reduction of solvent exposure around the interface can underlie the protein's compaction. These structural changes have potential to modulate this galectin's role in lattice formation on the cell surface and its interaction(s) with protein(s) at the F-face.

Nanocomposite hydrogels containing two-dimensional nanosilicates (NS) have emerged as a new technology for the prolonged delivery of biopharmaceuticals. However, little is known about the physical–chemical properties governing the interaction between NS and proteins and the release profiles of NS–protein complexes in comparison to traditional poly(ethylene glycol) (PEG) hydrogel technologies. To fill this gap in knowledge, we fabricated a nanocomposite hydrogel composed of PEG and laponite and identified simple but effective experimental conditions to obtain sustained protein release, up to 23 times slower as compared to traditional PEG hydrogels, as determined by bulk release experiments and fluorescence correlation spectroscopy. Slowed protein release was attributed to the formation of NS–protein complexes, as NS–protein complex size was inversely correlated with protein diffusivity and release rates. While protein electrostatics, protein concentration, and incubation time were important variables to control protein–NS complex formation, we found that one of the most significant and less appreciated variable to obtain a sustained release of bioactive proteins was the buffer chosen for preparing the initial suspension of NS particles. The buffer was found to control the size of nanoparticles, the absorption potential, morphology, and stiffness of hydrogels. From these studies, we conclude that the PEG–laponite composite fabricated is a promising new platform for sustained delivery of positively charged protein therapeutics.

Many deep eutectic solvents (DESs) are currently being explored as environment-friendly media for biorelated applications. As an understanding of the effect of these solvents on the structure of biomolecules is crucial for these applications, we study how two DESs comprising trimethylglycine (TMG) and ethylene glycol (EG) or glycerol (GL) influence the structural stability and conformational dynamics of cytochrome c (Cytc) using single-molecule-based fluorescence correlation spectroscopy (FCS) technique and several other ensemble-based biophysical methods. The FCS studies on A488-labeled Cytc enable an estimation of the size (20.5 ± 1.5 Å) of the protein and capture its conformational dynamics (54 ± 2 μs) in aqueous buffered solution. It is observed that both size and conformational dynamics of the protein are influenced in the presence of the DESs, but this effect is more pronounced in the case of TMG-EG. The ensemble measurements on both labeled and wild-type Cytc reveal that the protein structure is unfolded completely by TMG-EG, whereas the structure is slightly altered by TMG-GL. The results suggest that the behavior of Cytc in hydrated DESs is determined by the strength of interactions between the DES constituents as well as that between the constituents and the water molecules present in the system.

Macromolecular crowding (MMC) in cells is a hot topic in biology; therefore, well-characterized measurement standards for the evaluation of the nano-environment in MMC solutions are necessary. We propose to use polarization-dependent fluorescence correlation spectroscopy (Pol-FCS) for evaluation of macromolecular crowding in solutions. Pol-FCS can simultaneously measure the relaxation times of rotational and translational diffusion of fluorescent molecules at the same position, even in living cells with low damage. In this report, the differences in the nano-environment among solutions of small molecules, gels, and MMC solutions were evaluated by comparing their rotational and translational diffusion using Pol-FCS. Moreover, this method could distinguish the phase shift in the polyethylene glycol solution. Finally, we separately evaluated the nano-environment in the cytosol and nucleus of living cells in different cell lines and cell cycles. We expect this evaluation method to be useful in characterizing the nano-environment in MMC studies. In addition, the proposed method may be useful for other nano-environments such as liquid–liquid phase separation.

Polymersomes are vesicular structures self-assembled from amphiphilic block copolymers and are considered an alternative to liposomes for applications in drug delivery, immunotherapy, biosensing, and as nanoreactors and artificial organelles. However, the limited availability of systematic stability, protein fouling (protein corona formation), and blood circulation studies hampers their clinical translation. Poly(2-oxazoline)s (POx) are valuable antifouling hydrophilic polymers that can replace the current gold-standard, poly(ethylene glycol) (PEG), yet investigations of POx functionality on nanoparticles are relatively sparse. Herein, a systematic study is reported of the structural, dynamic and antifouling properties of polymersomes made of poly(2-methyl-2-oxazoline)-block-poly(dimethylsiloxane)-block-poly(2-methyl-2-oxazoline) (PMOXA-b-PDMS-b-PMOXA). The study relates in vitro antifouling performance of the polymersomes to atomistic molecular dynamics simulations of polymersome membrane hydration behavior. These observations support the experimentally demonstrated benefit of maximizing the length of PMOXA (degree of polymerization (DP) > 6) while keeping PDMS at a minimal length that still provides sufficient membrane stability (DP > 19). In vitro macrophage association and in vivo blood circulation evaluation of polymersomes in zebrafish embryos corroborate these findings. They further suggest that single copolymer presentation on polymersomes is outperformed by blends of varied copolymer lengths. This study helps to rationalize design rules for stable and low-fouling polymersomes for future medical applications.

Fluorescence-based single-molecule techniques, mainly including fluorescence correlation spectroscopy (FCS) and single-molecule fluorescence resonance energy transfer (smFRET), are able to analyze the conformational dynamics and diversity of biological macromolecules. They have been applied to analysis of the dynamics of membrane proteins, such as membrane receptors and membrane transport proteins, due to their superior ability in resolving spatio-temporal heterogeneity and the demand of trace amounts of analytes. In this review, we first introduced the basic principle involved in FCS and smFRET. Then we summarized the labeling and immobilization strategies of membrane protein molecules, the confocal-based and TIRF-based instrumental configuration, and the data processing methods. The applications to membrane protein dynamics analysis are described in detail with the focus on how to select suitable fluorophores, labeling sites, experimental setup, and analysis methods. In the last part, the remaining challenges to be addressed and further development in this field are also briefly discussed.

Protein oligomerization plays a very important role in many physiological processes. p53 acts as a key tumor suppressor by regulating cell cycle arrest, DNA repair, and apoptosis, and its antitumor activity is regulated by the hetero- and homo-oligomerization of MDMX and MDM2 proteins. So far, some traditional methods have been utilized to study the oligomerization of MDMX and MDM2 in vitro, but they have not clarified some controversial issues or whether the extracellular results can represent the intracellular results. Here, we put forward an in situ method for studying protein homo- and hetero-oligomerization in single living cells by using fluorescence correlation spectroscopy. In this study, MDMX and MDM2 were labeled with fluorescent proteins using lentiviral transfection. Autocorrelation spectroscopy and cross-correlation spectroscopy methods were used to study the oligomerization of MDMX and MDM2 in situ and the effect of regulation of MDMX oligomerization on p53-MDMX interactions in single living cells. We observed the homo- and hetero-oligomerization of MDMX and MDM2 in living cells. Meanwhile, the levels of the homo-oligomers of MDMX and MDM2 were increased due to the lack of hetero-oligomerization. Finally, the binding affinity of MDMX for p53 was improved with an increase in the level of MDMX hetero-oligomerization.

The insertion of protein domains into membranes is an important step in many membrane remodeling processes, for example in vesicular transport. The membrane area taken up by the protein insertion influences the protein binding affinity as well as the mechanical stress induced in the membrane and thereby its curvature. Total area changes in lipid monolayers can be measured on a Langmuir film balance. Finding the area per inserted protein however proves challenging for two reasons: The number of inserted proteins must be determined without disturbing the binding equilibrium and the change in the film area can be very small. Here we address both issues using Fluorescence Correlation Spectroscopy (FCS): Firstly, by labeling a fraction of the protein molecules fluorescently and performing FCS experiments directly on the monolayer, the number of inserted proteins is determinedin situwithout having to rely on invasive techniques, such as collecting the monolayer by aspiration. Secondly, by using another FCS color channel and adding a small fraction of fluorescent lipids, the reduction in fluorescent lipid density accompanying protein insertion can be monitored to determine the total area increase. Here, we use this method to determine the insertion area per molecule of Sar1, a protein of the COPII complex, which is involved in transport vesicle formation, in a lipid monolayer. Sar1 has an N-terminal amphipathic helix, which is responsible for membrane binding and curvature generation. An insertion area of (3.4 ± 0.8) nm2was obtained for Sar1 in monolayers from a lipid mixture typically used in reconstitution, in good agreement with the expected insertion area of the Sar1 amphipathic helix. By using the two-color approach, determining insertion areas relies only on local fluorescence measurements. No macroscopic area measurements are needed, giving the method the potential to be applied also to laterally heterogeneous monolayers and bilayers.Statement of SignificanceWe show that two color Fluorescence Correlation Spectroscopy (FCS) measurements can be applied to the binding of a protein to a lipid monolayer on a Langmuir film balance in order to determine the protein insertion area. One labelling color was used to determine the number of bound proteins and the other one to monitor the area expansion of the lipid monolayer upon protein binding. A strategy for the FCS data analysis is provided, which includes focal area calibration by raster image correlation spectroscopy and a framework for applying z-scan FCS and including free protein in the aqueous subphase. This approach allows determining an area occupied by a protein in a quasi-planar model membrane from a local, non-invasive, optical measurement.

The transient interactions between cellular components, particularly on membrane surfaces, are critical in the proper function of many biochemical reactions. For example, many signaling pathways involve dimerization, oligomerization, or other types of clustering of signaling proteins as a key step in the signaling cascade. However, it is often experimentally challenging to directly observe and characterize the molecular mechanisms such interactions-the greatest difficulty lies in the fact that living cells have an unknown number of background processes that may or may not participate in the molecular process of interest, and as a consequence, it is usually impossible to definitively correlate an observation to a well-defined cellular mechanism. One of the experimental methods that can quantitatively capture these interactions is through membrane reconstitution, whereby a lipid bilayer is fabricated to mimic the membrane environment, and the biological components of interest are systematically introduced, without unknown background processes. This configuration allows the extensive use of fluorescence techniques, particularly fluorescence fluctuation spectroscopy and single-molecule fluorescence microscopy. In this review, we describe how the equilibrium diffusion of two proteins, K-Ras4B and the PH domain of Bruton's tyrosine kinase (Btk), on fluid lipid membranes can be used to determine the kinetics of homodimerization reactions. [BMB Reports 2021; 54(3): 157-163].

Supramolecular self-assembly of small organic molecules has emerged as a powerful tool to construct well-defined micro- and nanoarchitecture through fine-tuning a range of intermolecular interactions. The size, shape, and optical properties of these nanostructures largely depend on the specific assembly of the molecular building units, temperature and polarity of the medium, and external stimuli. The engineering of supramolecular self-assembled nanostructures with morphology-dependent tunable emission is in high demand due to the promising scope in nanodevices and molecular machines. However, probing the evolution of molecular aggregates from the solution and directing the self-assembly process in a pre-defined fashion are challenging. In the present study, we have deciphered the sequential evolution of supramolecular nanofibers from solution to spherical and oblong-shaped nanoparticles through the variation of solvent polarity, tuning the hydrophobic–hydrophilic interactions. An intriguing case of molecular self-assembly has been elucidated employing a newly designed π-conjugated thiophene derivative (TPAn) through a combination of steady-state absorption, emission measurements, fluorescence correlation spectroscopy (FCS), and electron microscopy. The FCS analysis and microscopy results revealed that the small-sized nanofibers in the dispersion further agglomerated upon solvent evaporation, resulting in a network of nanofibers. Stimuli-responsive reversible interconversion between a network of nanofibers and spherical nanoaggregates was probed both in dispersion and solvent-evaporated state. The evolution of organic nanofibers and a subtle control over the self-assembly process demonstrated in the current investigation provide a general paradigm to correlate the size, shape, and emission properties of fluorescent molecular aggregates in complex heterogeneous media, including a human cell., Supramolecular nanofiber evolution in solution and solid-state, including stimuli-responsive reversible interconversion among diverse nanoarchitectures, was probed through a combined spectroscopic and microscopic approach.

G protein-coupled receptors (GPCRs) are the largest class of transmembrane proteins, making them an important target for therapeutics. Activation of these receptors is modulated by orthosteric ligands, which stabilize one or several states within a complex conformational ensemble. The intra- and inter-state dynamics, however, is not well documented. Here, we used single-molecule fluorescence to measure ligand-modulated conformational dynamics of the adenosine A2A receptor (A2AR) on nanosecond to millisecond timescales. Experiments were performed on detergent-purified A2R in either the ligand-free (apo) state, or when bound to an inverse, partial or full agonist ligand. Single-molecule Förster resonance energy transfer (smFRET) was performed on detergent-solubilized A2AR to resolve active and inactive states via the separation between transmembrane (TM) helices 4 and 6. The ligand-dependent changes of the smFRET distributions are consistent with conformational selection and with inter-state exchange lifetimes ≥ 3 ms. Local conformational dynamics around residue 2296.31 on TM6 was measured using fluorescence correlation spectroscopy (FCS), which captures dynamic quenching due to photoinduced electron transfer (PET) between a covalently-attached dye and proximal aromatic residues. Global analysis of PET-FCS data revealed fast (150–350 ns), intermediate (50–60 μs) and slow (200–300 μs) conformational dynamics in A2AR, with lifetimes and amplitudes modulated by ligands and a G-protein mimetic (mini-Gs). Most notably, the agonist binding and the coupling to mini-Gs accelerates and increases the relative contribution of the sub-microsecond phase. Molecular dynamics simulations identified three tyrosine residues (Y112, Y2887.53, and Y2907.55) as being responsible for the dynamic quenching observed by PET-FCS and revealed associated helical motions around residue 2296.31 on TM6. This study provides a quantitative description of conformational dynamics in A2AR and supports the idea that ligands bias not only GPCR conformations but also the dynamics within and between distinct conformational states of the receptor.

Super-resolution microscopy techniques have pushed the limit of optical imaging to unprecedented spatial resolutions. However, one of the frontiers in nanoscopy is its application to intact living organisms. Here we describe the implementation and application of super-resolution single-particle tracking photoactivated localization microscopy (sptPALM) to probe single-molecule dynamics of membrane proteins in live roots of the model plant Arabidopsis thaliana. We first discuss the advantages and limitations of sptPALM for studying the diffusion properties of membrane proteins and compare this to fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS). We describe the technical details for handling and imaging the samples for sptPALM, with a particular emphasis on the specificity of imaging plant cells, such as their thick cell walls or high degree of autofluorescence. We then provide a practical guide from data collection to image analyses. In particular, we introduce our sptPALM_viewer software and describe how to install and use it for analyzing sptPALM experiments. Finally, we report an R statistical analysis pipeline to analyze and compare sptPALM experiments. Altogether, this protocol should enable plant researchers to perform sptPALM using a benchmarked reproducible protocol. Routinely, the procedure takes 3–4 h of imaging followed by 3–4 d of image processing and data analysis. This protocol describes how to perform single-particle tracking photoactivated localization microscopy (sptPALM) of membrane proteins in living plant tissues. The procedure covers all stages, from sample preparation to data analysis.

Molecular chaperone Hsp70 plays important roles in the pathology of amyloid diseases by inhibiting aberrant aggregation of proteins. However, the biophysical mechanism of the interaction of Hsp70 with the intrinsically disordered proteins (IDPs) is unclear. Here, we report that Hsp70 inhibits aggregation of islet amyloid polypeptide (IAPP) at substoichiometric concentrations under diverse solution conditions, including in the absence of ATP. The inhibitory effect is strongest if Hsp70 is added in the beginning of aggregation but progressively less if added later, indicating a role for Hsp70 in preventing nucleation of IAPP. However, ensemble measurement of the binding affinity suggests poor interactions between Hsp70 and IAPP. Therefore, we hypothesize that the interaction must involve a rare species (e.g., the oligomeric intermediates of IAPP). Size exclusion chromatography and field flow fractionation are then used to fractionate the constituent species. Multiangle light scattering and fluorescence correlation spectroscopy measurements indicate that the dominant fraction in size exclusion chromatography contains a few nanomolar Hsp70-IAPP complexes amid several μmoles of free Hsp70. Using single-particle two-color coincidence detection measurements, we detected a minor fraction that exhibits fluorescence bursts arising from heterogeneous oligomeric complexes of IAPP and Hsp70. Taken together, our results indicate that Hsp70 interacts poorly with the monomers but strongly with oligomers of IAPP. This is likely a generic feature of the interactions of Hsp70 chaperones with the amyloidogenic IDPs. Whereas high-affinity interactions with the oligomers prevent aberrant aggregation, poor interaction with the monomers averts interference with the physiological functions of the IDPs.

Understanding of prion aggregation in a membrane environment may help to ameliorate neurodegenerative complications caused by the amyloid forms of prions. Here, we investigated the membrane binding-induced aggregation of yeast prion protein Sup35. Using the combination of fluorescence correlation spectroscopy (FCS) at single molecule resolution and other biophysical studies, we establish that lipid composition and lipid/protein ratio are key modulators of the aggregation kinetics of Sup35. In the presence of a zwitterionic membrane (DMPC), Sup35 exhibited novel biphasic aggregation kinetics at lipid/protein ratios ranging between 20 : 1 and 70 : 1 (termed here as the optimum lipid concentration, OLC). In ratios below (low lipid concentration, LLC) and above (ELC, excess lipid concentration) that range, the aggregation was found to be monophasic. In contrast, in the presence of negatively charged membranes, we did not observe any bi-phasic aggregation kinetics in the entire range of protein to lipid ratios. Our results provide a mechanistic description of the role that membrane concentration/composition-modulated aggregation may play in neurodegenerative diseases., This study provides a mechanistic description of how the membrane composition and lipid to protein ratio modulate amyloid kinetics of yeast prion protein.

Protein oligomerization is a commonly encountered strategy by which the functional repertoire of proteins is increased. This, however, is a double-edged sword strategy because protein oligomerization is notoriously difficult to control. Living organisms have therefore developed a number of chaperones that prevent protein aggregation. The small ATP-independent molecular chaperone domain proSP-C BRICHOS, which is mainly trimeric, specifically inhibits fibril surface-catalyzed nucleation reactions that give rise to toxic oligomers during the aggregation of the Alzheimer’s disease-related amyloid-β peptide (Aβ42). Here, we have created a stable proSP-C BRICHOS monomer mutant and show that it does not bind to monomeric Aβ42 but has a high affinity for Aβ42 fibrils, using surface plasmon resonance. Kinetic analysis of Aβ42 aggregation profiles, measured by thioflavin T fluorescence, reveals that the proSP-C BRICHOS monomer mutant strongly inhibits secondary nucleation reactions and thereby reduces the level of catalytic formation of toxic Aβ42 oligomers. To study binding between the proSP-C BRICHOS monomer mutant and small soluble Aβ42 aggregates, we analyzed fluorescence cross-correlation spectroscopy measurements with the maximum entropy method for fluorescence correlation spectroscopy. We found that the proSP-C BRICHOS monomer mutant binds to the smallest emerging Aβ42 aggregates that are comprised of eight or fewer Aβ42 molecules, which are already secondary nucleation competent. Our approach can be used to provide molecular-level insights into the mechanisms of action of substances that interfere with protein aggregation.

Transactive response element DNA/RNA-binding protein 43 kDa (TDP-43) is the causative protein of amyotrophic lateral sclerosis (ALS); several ALS-associated mutants of TDP-43 have been identified. TDP-43 has several domains: an N-terminal domain, two RNA/DNA-recognition motifs, and a C-terminal intrinsically disordered region (IDR). Its structures have been partially determined, but the whole structure remains elusive. In this study, we investigate the possible end-to-end distance between the N- and C-termini of TDP-43, its alterations due to ALS-associated mutations in the IDR, and its apparent molecular shape in live cells using Förster resonance energy transfer (FRET) and fluorescence correlation spectroscopy (FCS). Further-more, the interaction between ALS-associated TDP-43 and heteronuclear ribonucleoprotein A1 (hnRNP A1) is slightly stronger than that of wild-type TDP-43. Our findings provide insights into the structure of wild-type and ALS-associated mutants of TDP-43 in a cell.

With an aim to understand the interaction mechanism of bovine serum albumin (BSA) with copper nanoclusters (CuNCs), three different types CuNCs having chemically different surface ligands, namely, tannic acid (TA), chitosan, and cysteine (Cys), have been fabricated, and investigations are carried out in the absence and presence of protein (BSA) at ensemble-averaged and single-molecule levels. The CuNCs, capped with different surface ligands, are consciously chosen so that the role of surface ligands in the overall protein-NCs interactions is clearly understood, but, more importantly, to find whether these CuNCs can interact with protein in a new pathway without forming the "protein corona", which otherwise has been observed in relatively larger nanoparticles when they are exposed to biological fluids. Analysis of the data obtained from fluorescence, ζ-potential, and ITC measurements has clearly indicated that the BSA protein in the presence of CuNCs does not attain the binding stoichiometry (BSA/CuNCs > 1) that is required for the formation of "protein corona". This conclusion is further substantiated by the outcome of the fluorescence correlation spectroscopy (FCS) study. Further analysis of data and thermodynamic calculations have revealed that the surface ligands of the CuNCs play an important role in the protein-NCs binding events, and they can alter the mode and thermodynamics of the process. Specifically, the data have demonstrated that the binding of BSA with TA-CuNCs and Chitosan-CuNCs follows two types of binding modes; however, the same with Cys-CuNCs goes through only one type of binding mode. Circular dichroism (CD) measurements have indicated that the basic structure of BSA remains almost unaltered in the presence of CuNCs. The outcome of the present study is expected to encourage and enable better application of NCs in biological applications.

Lateral compartmentalization of the plasma membrane is a prominent feature present at multiple spatiotemporal scales that regulates key cellular functions. The extracellular glycocalyx matrix has recently emerged as an important player that modulates the organization of specific receptors and patterns the lipid bilayer itself. However, experimental limitations in investigating its impact on the membrane nanoscale dynamics have hampered detailed studies. Here, we used photonic nanoantenna arrays combined with fluorescence correlation spectroscopy to investigate the influence of hyaluronic acid (HA), a prominent glycosaminoglycan, on the nanoscale organization of mimetic lipid bilayers. Using atomic force microscopy and force spectroscopy, we further correlated our dynamic measurements with the morphology and mechanical properties of bilayers at the nanoscale. Overall, we find that HA has a profound effect on the dynamics, nanoscale organization, and mechanical properties of lipid bilayers that are enriched in sphingolipids and/or cholesterol, such as those present in living cells.

Protein phosphorylation is a very important mechanism for regulating and controlling the activity and function of proteins, and is closely associated with signal transduction, gene expression, cell cycle and other life activities in organisms. In this paper, we proposed a new strategy for studying protein phosphorylation in living cells by combining fluorescence resonance energy transfer (FRET) with a small molecule adenosine 5'-triphosphate (ATP) analogue. We synthesized a new ATP analogue functionalized by norbornene (ATP-NB), and a tetrazine modified fluorescent probe Cyanine3 (TZ-Cy3). Based on the inverse electron demand Diels-Alder (D-A) reaction, ATP-NB phosphorylated proteins in solution and in living cells were in situ labelled with TZ-Cy3. By combining FRET with fluorescence correlation spectroscopy (FRET-FCS) and imaging technology, we established an efficient method for studying the phosphorylation of proteins in solution and in living cells using an ATP analogue instead of natural ATP. We studied the effects of phosphatase inhibitors on the phosphorylation of proteins in living cells. Our results documented that ATP-NB is a small molecule ATP analogue with hydrophobicity, which can penetrate cells and efficiently phosphorylate proteins in living cells. This strategy is well suitable for in situ study of protein phosphorylation in living cells.

Gold nanoclusters (AuNCs) synthesized within a protein (Human Serum Albumin, HSA) template exhibited intense red luminescence accompanied by a quantum yield >10% and remarkable photo and cluster-core stability for a prolonged period (more than a year). These photoluminescent nanoclusters (NCs) were resistant to chemical and thermal perturbations but break down selectively and highly sensitively in the presence of mercury, Hg(ii), ions. The AuNCs were efficient in quantifying Hg(ii) ions in solution as well as bound to the hormone insulin. By exploiting the auto-fluorescence of these AuNCs, we demonstrated that our AuNCs were able to sense Hg(ii) ions at single-molecule sensitivity using Fluorescence Correlation Spectroscopy (FCS), highlighting a detection limit in the sub-nanomolar regime. The translational diffusion time of the AuNCs decreased significantly upon the interaction with Hg(ii) ions and resulted in the formation of smaller sized clusters. A cell viability study reveals the non-toxic nature of these nano-probes, which thus can be used for cell imaging. Interestingly, a cell line-based study reveals that the fluorescence intensity of AuNCs could be detected in cancerous MDA-MB-231 cells but not in non-cancerous breast-derived MCF10A cells. Further, time lapse fixed cell imaging by confocal microscopy revealed that the fluorescence of AuNCs could be quenched by Hg(ii) ions inside the MDA-MB-231 cells. Thus the objective of our study is to appraise the sensitive in vivo as well as in vitro detection of Hg(ii) ions using AuNCs as a probe.

Protein expression is closely related to many biological processes including cell growth, differentiation and signaling. It is a challenge to selectively monitor newly synthesized proteins under both physiological and pathological conditions due to shortage of efficient analytical methods. Here, we proposed a new strategy to selectively monitor newly synthesized proteins in cells by combining fluorescence correlation spectroscopy (FCS) with bioorthogonal noncanonical amino acid tagging (BONCAT) technique. Firstly, homopropargylglycine (HPG), an alkyne surrogate of methionine, was metabolically incorporated into newly synthesized proteins in living cells, and the proteins containing the alkyne functional group were subsequently labeled with chemoselective fluorescence reporters using the Cu(i)-catalyzed azide-alkyne cycloaddition (CuAAC) reaction. Then, FCS was used to analyze the newly synthesized proteins based on the difference in the characteristic diffusion times of labeled proteins and free fluorescent dyes. We optimized the conditions of HPG metabolic incorporation and the CuAAC click reaction and applied this new method to study autophagic protein degradation and in situ monitor secreted proteins in cells. Compared to current methods, our method is simple, fast, and without separation, and it may become a promising approach for in situ studying protein expression in living cells.

Sliding clamps are oligomeric ring-shaped proteins that increase the efficiency of DNA replication. The stability of the Escherichia coli β-clamp, a homodimer, is particularly remarkable. The dissociation equilibrium constant of the β-clamp is of the order of 10 pM in buffers of moderate ionic strength. Coulombic electrostatic interactions have been shown to contribute to this remarkable stability. Increasing NaCl concentration in the assay buffer results in decreased dimer stability and faster subunit dissociation kinetics in a way consistent with simple charge-screening models. Here, we examine non-Coulombic ionic effects on the oligomerization properties of sliding clamps. We determined relative diffusion coefficients of two sliding clamps using fluorescence correlation spectroscopy. Replacing NaCl by KGlu, the primary cytoplasmic salt in E. coli, results in a decrease of the diffusion coefficient of these proteins consistent with the formation of protein assemblies. The UV-vis spectrum of the β-clamp labeled with tetramethylrhodamine shows the characteristic absorption band of dimers of rhodamine when KGlu is present in the buffer. This suggests that KGlu induces the formation of assemblies that involve two or more rings stacked face-to-face. Results can be quantitatively explained on the basis of unfavorable interactions between KGlu and the functional groups on the protein surface, which drive biomolecular processes that bury exposed surface. Similar results were obtained with the Saccharomyces cerevisiae PCNA sliding clamp, suggesting that KGlu effects are not specific to the β-clamp. Clamp association is also promoted by glycine betaine, a zwitterionic compound that accumulates intracellularly when E. coli is exposed to high concentrations of extracellular solute. Possible biological implications are discussed.

DNA methylation is a critical part of epigenetics and plays a vital role in maintaining normal cell function, genetic imprinting, and human tumorigenesis. Thus, it is important to develop a sensitive method for the determination of DNA methyltransferase (MTase) activity. Here, we present a simple and sensitive method based on single molecule fluorescence correlation spectroscopy (FCS) and polystyrene polymer dots (PS Pdots) for the quantitative detection of DNA adenine methylation (Dam) MTase activity and its inhibitor screening in homogeneous solution without separation. Its principle is based on the measurement of the characteristic diffusion time (τD) of unmethylated and methylated DNA-fluorescent probes by FCS. A hairpin DNA probe including the 5'-GATC-3' sequence is used by doubly labelling fluorophore Alexa Fluor 488 (Alexa 488) and biotin at the 5'- and 3'-terminus, respectively. Dam MTase catalyzed the methylation of the sequence of 5'-GATC-3', and DpnI cleaved the sequence of 5'-G-Am-TC-3'. Streptavidin conjugated PS Pdots were used to react with DNA probes without methylation to further increase the difference in τD values between methylated and unmethylated DNA-Alexa 488 probes. We used the FCS method to measure the τD values of DNA-Alexa 488 probes and further obtained the activity of Dam MTase. It is found that the τD value of the methylated DNA probe is negatively correlated with the logarithm of Dam MTase concentration in the range from 0.025 U mL-1 to 3 U mL-1. The detection limit is as low as 0.025 U mL-1. Furthermore, we evaluated the inhibition effect of drug-related DNA methylation and the half-maximal inhibitory concentration (IC50) value is consistent with a previous study. The results demonstrated that our proposed method will become a promising platform for the determination of Dam MTase activity and inhibitor screening.

Fluorescence correlation spectroscopy based on upconverting nanoparticles (UCNPs) can detect single particles in strongly autofluorescent samples, such as plasma. Nanoparticle aggregation, however,...

Cell membranes are fluidic, and the fluidic properties give unique biological functions to the membranes. Here, we review the application of fluorescence lifetime correlation analyses to elucidate ...

Enzymatic proteolysis or protein digestion is the fragmentation of protein into smaller peptide units under the action of peptidase enzymes. In this contribution, the directionality of proteolysis has been studied using fluorescence correlation spectroscopy (FCS), taking human serum albumin (HSA) as the model protein and papain, chymotrypsin and trypsin as the model enzymes. Domain-I of HSA has been tagged with tetramethylrhodamine-5-maleimide (TMR) and domain-III with p-nitrophenylcoumarin ester (NPCE) separately and subjected to proteolysis. Following the change in hydrodynamic radius, as monitored by FCS, it has been confirmed that under similar experimental conditions the order of efficiency of digestion is papain > trypsin > chymotrypsin. More interestingly, a faster decrease of hydrodynamic radius was observed when the fluorescence from domain-I was monitored in FCS, compared to that of domain-III. This observation clearly indicates that all these enzymes prefer to start cleaving HSA from domain-I. We assign this preference to the hydrophilic natures of the enzyme active site and domain-I surface. The dependence of the proteolysis on temperature and enzyme concentration has also been studied for papain using the same approach. Reverse-phase HPLC results are found to be in line with the FCS results and validates the applicability of our proposed method.

RNA and DNA play distinct roles in biological systems. However, the underlying physicochemical difference has been poorly understood, in particular, that in dynamical aspects. In this paper, we report on a comparative study of the formation-dissociation dynamics of a hairpin structure of RNA and DNA with development of two-color two-dimensional fluorescence lifetime correlation spectroscopy (two-color 2D FLCS). In this extension of 2D FLCS, we newly introduce the two-color detection scheme to analyze not only donor fluorescence photons but also acceptor fluorescence photons from a doubly labeled Förster resonance energy transfer (FRET) pair. This new 2D FLCS is utilized to resolve multiple species present in an equilibrated condition with a microsecond time resolution and enhanced sensitivity, and the combined use with the filtered fluorescence correlation spectroscopy (FCS) method enables a quantitative discussion on microsecond structural dynamics occurring in the equilibrium. This integrated approach is applied to FRET-labeled RNA/DNA oligonucleotides having analogous hairpin-forming sequences, and it was revealed that the hairpin dissociation rate of RNA is an order of magnitude slower than that of DNA while their hairpin-forming rates are comparable. This marked difference is attributable to the distinct duplex structure of RNA and DNA. The present study demonstrates that the integrated approach combining two-color 2D FLCS and filtered FCS has a high potential for quantifying microsecond kinetics at the single-molecule level, which allows us to experimentally construct a free energy landscape.

Here, we studied the interaction of Cys-substituted (G56C) cytochrome c labeled with sulfocyanin-3 fluorescent dye (fCyt) with artificial and natural lipid membranes by using fluorescence correlation spectroscopy (FCS). Compared to mitochondria, mitoplasts were shown to have more fCyt binding sites with a lower affinity for this protein. The fCyt affinity for cardiolipin-containing liposomes depended on the content of cardiolipin in liposomes and decreased upon raising the ionic strength of the solution. A high value of the constant of the fCyt binding with mitochondria could be explained by the presence of specific binding sites for this protein on the mitochondrial outer membrane surface. This explanation is confirmed by observation of more efficient displacement of fluorescent cytochrome c by the unlabeled mutant variant K8T than by the WT protein, whereas in mitoplasts and liposomes the WT protein displaced fCyt more efficiently than K8T.

Correlation has a variety of applications that require signal processing. However, it is computationally intensive, and software correlators require high-performance processors for real-time data analysis. This is a challenge for embedded devices because of the limitation of computing resources. Hardware correlators that use Field Programmable Gate Array (FPGA) technology can significantly boost computational power and bridge the gap between the need for high-performance computing and the limited processing power available in embedded devices. This paper presents a detailed FPGA-based correlator design at the register level along with the open-source Very High-Speed Integrated Circuit Hardware Description Language (VHDL) code. It includes base modules for linear and multi-tau correlators of varying sizes. Every module implements a simple and unified data interface for easy integration with standard and publicly available FPGA modules. Eighty-lag linear and multi-tau correlators were built for validation of the design. Three input data sets—constant signal, pulse signal, and sine signal—were used to test the accuracy of the correlators. The results from the FPGA correlators were compared against the outputs of equivalent software correlators and validated with the corresponding theoretical values. The FPGA correlators returned results identical to those from the software references for all tested data sets and were proven to be equivalent to their software counterparts. Their computation speed is at least 85,000 times faster than the software correlators running on a Xilinx MicroBlaze processor. The FPGA correlator can be easily implemented, especially on System on a Chip (SoC) integrated circuits that have processor cores and FPGA fabric. It is the ideal component for device-on-chip solutions in biosensing.

Voltage-gated Kv1.3 potassium channels are essential for maintaining negative membrane potential during T-cell activation. They interact with membrane-associated guanylate kinases (MAGUK-s) via their C-terminus and with TCR/CD3, leading to enrichment at the immunological synapse (IS). Molecular interactions and mobility may impact each other and the function of these proteins. We aimed to identify molecular determinants of Kv1.3 mobility, applying fluorescence correlation spectroscopy on human Jurkat T-cells expressing WT, C-terminally truncated (ΔC), and non-conducting mutants of mGFP-Kv1.3. ΔC cannot interact with MAGUK-s and is not enriched at the IS, whereas cells expressing the non-conducting mutant are depolarized. Here, we found that in standalone cells, mobility of ΔC increased relative to the WT, likely due to abrogation of interactions, whereas mobility of the non-conducting mutant decreased, similar to our previous observations on other membrane proteins in depolarized cells. At the IS formed with Raji B-cells, mobility of WT and non-conducting channels, unlike ΔC, was lower than outside the IS. The Kv1.3 variants possessing an intact C-terminus had lower mobility in standalone cells than in IS-engaged cells. This may be related to the observed segregation of F-actin into a ring-like structure at the periphery of the IS, leaving much of the cell almost void of F-actin. Upon depolarizing treatment, mobility of WT and ΔC channels decreased both in standalone and IS-engaged cells, contrary to non-conducting channels, which themselves caused depolarization. Our results support that Kv1.3 is enriched at the IS via its C-terminal region regardless of conductivity, and that depolarization decreases channel mobility.

Fluorescentna korelacijska spektroskopija je etablirana tehnika spektroskopije na razini jedne molekule kojom se promatraju vremenske fluktuacije intenziteta fluorescencije biomolekula markiranih nekom fluorescentnom bojom. Fluktuacije se vremenski autokoreliraju, što potom omogućuje daljnju analizu i mjerenje parametara biomolekule od interesa. Parametri mogu biti koncentracija, difuzijska konstanta i sl. U ovom radu je kao izvor fluorescencije korištena boja Alexa647 u nevezanom obliku. Korištenjem aproksimativne teorijske autokorelacijske funkcije, koja uključuje difuzijsku i tripletnu dinamiku molekule, izračunate su dimenzije detekcijskog volumena u konfokalnom mikroskopu. Zatim su izračunati parametri fiksirani u teorijskom modelu ˇsto je omogućilo određivanje vremena difuzije i difuzijske konstante promatrane molekule. Kao alternativna metoda obrade autokorelacijskih krivulja korišteno je strojno učenje. Točnije, korištena je jednostavna neuronska mreža, trenirana na skupu uzoraka poznatih parametara. Potom su provedena predviđanja difuzijske konstante na podskupovima mjerenja koji nisu bili dio treninga modela. Strojno učenje je korišteno kao pokušaj da se odziv optičkog postava odredi izravno iz podataka. Na taj način su se pokusale izbjeći nepouzdanosti mjerenja koje dolaze od optičkih artefakata, a koji nisu predvedeni teorijskim modelima. Vrijednosti dobivene neuronskom mrežom su uspoređeni s vrijednostima dobivenim klasičnim pristupom gdje se parametri sustava dobivaju prilagodbom teorijske funkcije na podatke. Rezultati ukazuju da je pristup korištenjem strojnog učenja i s tako malim brojem podataka koji su se koristili za trening modela, barem usporediv po preciznosti s predviđanjima koje daje klasičan pristup. Fluorescence correlation spectroscopy is an established technique in single-molecule spectroscopy used for observing temporal fluctuations of the fluorescence intensity of marked biomolecules. Fluctuations are temporally autocorrelated which enables further analysis and measurement of relevant system parameters. Examples of observables are molecule concentrations, diffusion coefficients, etc. In this master’s thesis an unbound Alexa647 dye molecule was used as source of fluorescence. By using an approximate theoretical function, which incorporates diffusion dynamics and triplet dynamics of the dye, dimensions of the observational volume of the confocal microscope were calculated. The latter parameters are then fixed in the theoretical model which enabled determination of diffusion time and diffusion constant of the dye. As an alternative method, machine learning was used to process autocorrelation curves. Specifically, a neural network was trained on a dataset that included data of known parameters. After the model was trained, it was used to predict diffusion coefficients of the data in the test set which was not used during the model training. System parameters were measured directly from the data. Doing it that way enables us to potentially avoid systematic errors which come from optical artifacts of the experimental apparatus because approximate theoretical functions do not take optical artifacts into account. The results of the model are compared with the results obtained in the theoretical function fitting procedure. Obtained results signalled that even though the training set used for model training was small, the machine learning model was at least as successful in predicting diffusion coefficients as was the theoretical function fitting procedure.

A vaccine antigen, when launched as DNA or RNA, can be presented in various forms, including intracellular, secreted, membrane-bound, or on extracellular vesicles (EVs). Whether an antigen in one or more of these forms is superior in immune induction remains unclear. In this study, we used GFP as a model antigen and first compared the EV-loading efficiency of transmembrane domains (TMs) from various viral glycoproteins, and then investigated whether EV-bound GFP (EV-GFP) would enhance immune induction. Our data showed that GFP fused to viral TMs was successfully loaded onto the surface of EVs. In addition, GFP-bound EVs were predominantly associated with the exosome marker CD81. Immunogenicity study with EV-GFP-producing plasmids in mice demonstrated that antigen-specific IgG and IgA were significantly increased in EV-GFP groups, compared to soluble and intracellular GFP groups. Similarly, GFP-specific T cell response-related cytokines produced by antigen-stimulated splenocytes were also enhanced in mice immunized with EV-GFP constructs. Immunogenicity study with purified soluble GFP and GFP EVs further confirmed the immune enhancement property of EV-GFP in mice. In vitro uptake assays indicated that EV-GFP was more efficiently taken up than soluble GFP by mouse splenocytes and such uptake was B cell preferential. Taken together, our data indicate that viral TMs can efficiently load antigens onto the EV surface, and that EV-bound antigen enhances both humoral and cell-mediated antigen-specific responses.

Significance Liquid–liquid phase separation causes formation of membraneless condensates containing concentrated biomolecules, which play essential roles in diverse cellular processes. Currently, micrometer-scale phase-separated condensates are examined routinely to elucidate their functions and mechanisms in details. However, limited by current commonly used methods, the transition process from miscible individual molecules to micrometer-scale condensates is mostly unknown. Herein, we captured formation of nanoscale condensates, whose size, growth rate, molecular stoichiometry, and binding affinity to recruit client molecules were all quantified. To achieve this, we developed a dual-color fluorescence cross-correlation spectroscopy (dcFCCS) method, and we expect that dcFCCS can be widely applied to investigate phase separation of other biomolecules at the nanoscale., Liquid–liquid phase separation, driven by multivalent macromolecular interactions, causes formation of membraneless compartments, which are biomolecular condensates containing concentrated macromolecules. These condensates are essential in diverse cellular processes. Formation and dynamics of micrometer-scale phase-separated condensates are examined routinely. However, limited by commonly used methods which cannot capture small-sized free-diffusing condensates, the transition process from miscible individual molecules to micrometer-scale condensates is mostly unknown. Herein, with a dual-color fluorescence cross-correlation spectroscopy (dcFCCS) method, we captured formation of nanoscale condensates beyond the detection limit of conventional fluorescence microscopy. In addition, dcFCCS is able to quantify size and growth rate of condensates as well as molecular stoichiometry and binding affinity of client molecules within condensates. The critical concentration to form nanoscale condensates, identified by our experimental measurements and Monte Carlo simulations, is at least several fold lower than the detection limit of conventional fluorescence microscopy. Our results emphasize that, in addition to micrometer-scale condensates, nanoscale condensates are likely to play important roles in various cellular processes and dcFCCS is a simple and powerful quantitative tool to examine them in detail.

Cell behaviour and function is determined through the interactions of a multitude of molecules working in concert. To observe these molecular dynamics, biophysical studies have been developed that track single interactions. Fluorescence correlation spectroscopy (FCS) is an optical biophysical technique that non-invasively resolves single molecules through recording the signal intensity at the femtolitre scale. However, recording the behaviour of these biomolecules using in vitro-based assays often fails to recapitulate the full range of variables in vivo that directly confer dynamics. Therefore, there has been an increasing interest in observing the state of these biomolecules within living organisms such as the zebrafish Danio rerio. In this review, we explore the advancements of FCS within the zebrafish and compare and contrast these findings to those found in vitro.

The composition, stoichiometry and interactions of supramolecular protein complexes are a critical determinant of biological function. Several techniques have been developed to study molecular interactions and quantify subunit stoichiometry at the single molecule level; however, these typically require artificially low expression levels to achieve the low fluorophore concentration required for single molecule imaging, or use of detergent isolation of complexes that may perturb native subunit interactions. Here we present an alternative approach where protein complexes are assembled at physiological concentrations and subsequently diluted in situ for single-molecule level observations while preserving them in a near-native cellular environment. We show that coupling this in situ dilution strategy with single molecule techniques such as in vivo Fluorescence Correlation Spectroscopy (FCS), bleach step counting for quantifying protein complex stoichiometry, and two-color single molecule colocalization, improves the quality of data obtained using these single molecule fluorescence methods. Single Protein Recovery After Dilution (SPReAD) is a simple and versatile means of extending the concentration range of single molecule measurements into the cellular regime while minimizing potential artifacts and perturbations of protein complex stoichiometry.SIGNIFICANCE STATEMENTQuantifying the composition and stoichiometry of protein complexes in live cells is critical to understanding mechanisms involved in their function. Here we detail a method in which protein complexes are assembled intracellularly at physiological concentrations, but then diluted to levels suitable for single-molecule fluorescence observations while still within a cellular environment. The technique permits the use of common single molecule analysis techniques such as stepwise photobleaching quantification and fluorescence correlation spectroscopy to determine stoichiometry and functional interactions while avoiding artifacts that may occur from the use of detergent isolation methods or from the artificially low expression levels sometimes used to attain single molecule observation levels.

Owing to their potential applications in the extraction, purification, and preservation of biomolecules and biocatalysis, ionic liquids (ILs) have gained great attention in biotechnology. Although it is known that the structure and dynamics of proteins in ILs depend on the nature of both proteins and ILs, the biophysical mechanism governing the protein-IL interaction, which determines the stability of proteins or the activity of an enzyme in these nonconventional media, is yet to be understood clearly. Herein, we study the effect of two ammonium ILs, triethylammonium dihydrogen phosphate (TEAP) and tributylammonium dihydrogen phosphate (TBAP), on the stability and conformational dynamics of cytochrome c (Cyt c) in its native and unfolded states, employing primarily the single molecule-based fluorescence correlation spectroscopy (FCS) technique. The results show that the native structure of Cyt c is not significantly altered by TEAP, but the tertiary structure is perturbed to a great extent by TBAP, which comprises a longer alkyl chain. Fluctuations of the fluorescence intensity of Alexa488 dye-labeled Cyt c in FCS measurements reveal conformational dynamics (67 ± 10 μs) in the native state of Cyt c that is accelerated in the presence of both ILs but not affected when Cyt c is in its unfolded state. The present findings demonstrate how the stability of this protein can be modulated by using ammonium ILs of different alkyl chain lengths.