Use Of Fluorescence Correlation Spectroscopy Research Articles

Estimating Poly(N-isopropylacrylamide) size in solution below the LCST using fluorescence correlation spectroscopy with non-covalent bound fluorophores

Accurate size measurements of nanosized polymer chains in dilute solutions is important for understanding polymer behavior, however, these measurements can be challenging to implement accurately, and are technique dependent. Here we explore the use of Fluorescence Correlation Spectroscopy (FCS) to determine the size of single polymer chains in dilute solutions (<2 wt%). FCS, a technique with single molecule sensitivity, generally requires the use of covalently labelled polymers which can distort physicochemical behavior. Here, FCS based size measurements were based on the non-covalent interaction of fluorophores (Alexa 405, Atto 390, and Atto 425) with PNIPAm in water at 25 °C (below the Lower Critical Solution Temperature).FCS estimated size (hydrodynamic radius) of three different MW PNIPAm samples in water were: 5.0 ± 1.0 nm (28.5 kDa), 5.0 ± 1.0 nm (38 kDa), 3.3 ± 0.5 nm (55.5 kDa), in reasonable agreement with theoretical calculations. Accuracy was directly related to the fraction of PNIPAm-bound fluorophore, which were, for 1 wt% PNIPAm solutions in water: ∼11.5 % (Atto 390), ∼8.1 % (Atto 425), and 4 % (Alexa 405). This method has several advantages in that it does not require a covalent labelling of PNIPAm, it can be implemented on very small sample volumes, and allows for in-situ measurements.

Fluorescence Correlation Spectroscopy as a Versatile Method to Define Aptamer-Protein Interactions with Single-Molecule Sensitivity.

Aptamers are folded oligonucleotides that selectively recognize and bind a target and are consequently regarded as an emerging alternative to antibodies for sensing and therapeutic applications. The rational development of functional aptamers is strictly related to the accurate definition of molecular binding properties. Nevertheless, most of the methodologies employed to define binding affinities use bulk measurements. Here, we describe the use of fluorescence correlation spectroscopy (FCS) as a method with single-molecule sensitivity that quantitatively defines aptamer-protein binding. First, FCS was used to measure the equilibrium affinity between the CLN3 aptamer, conjugated with a dye, and its target, the c-Met protein. Equilibrium affinity was also determined for other functional aptamers targeting nucleolin and platelet-derived growth factors. Then, association and dissociation rates of CLN3 to/from the target protein were measured using FCS by monitoring the equilibration kinetics of the binding reaction in solution. Finally, FCS was exploited to investigate the behavior of CLN3 exposed to physiological concentrations of the most abundant serum proteins. Under these conditions, the aptamer showed negligible interactions with nontarget serum proteins while preserving its affinity for the c-Met. The presented results introduce FCS as an alternative or complementary analytical tool in aptamer research, particularly well-suited for the characterization of protein-targeting aptamers.

Early Aggregation of Amyloid-β(1-42) Studied by Fluorescence Correlation Spectroscopy.

Alzheimer's disease (AD) is a progressive neurodegenerative disease affecting cognitive and memory abilities and is believed to be linked to the formation and accumulation of neurotoxic aggregates of the Amyloid-β peptide (Aβ). In particular, it is the formation of soluble pre-fibrillar oligomers within the early stage of Aβ aggregation which is thought to represent a key step in the development of AD, thus underlining the interest in characterizing the aggregation process and the nature of these aggregates. In this context, fluorescence correlation spectroscopy (FCS) has emerged as a valuable alternative for the study of these systems in solution. Indeed, the use of FCS to study terminally labelled Aβ provides a means to detect changes in the size and concentration of initially monomeric Aβ samples by monitoring these fluorescently labelled species freely diffusing in solution with single-molecule resolution. Herein, we show how to employ FCS to study the early aggregation process of Aβ(1-42) and how this can be used to estimate the critical concentration for oligomer formation and to characterize the aggregates formed.

Supramolecular association studied by Fluorescence correlation spectroscopy.

A comprehensive description of a supramolecular system involves a full understanding of its thermodynamic and dynamic properties, as well as detailed knowledge of its structure. Fluorescence Correlation Spectroscopy (FCS) constitutes a powerful technique to acquire this information. Fluorescence correlation curves show a characteristic diffusion term that is related to the binding equilibrium constant or other thermodynamic properties of the supramolecular system. The association and dissociation rate constants of the binding process can be determined in FCS when the relaxation time of the binding is faster than the observation time-a regime called fast-exchange dynamics - in opposition to the slow-exchange regime. In all cases, structural information can be inferred from the diffusional properties of the supramolecular complexes. A short overview of the use of FCS for the study of supramolecular systems is given with examples which belong to the fast and slow regime.

The use of fluorescence correlation spectroscopy to characterise the molecular mobility of G protein-coupled receptors in membrane microdomains: an update.

It has become increasingly apparent that some G protein-coupled receptors (GPCRs) are not homogeneously expressed within the plasma membrane but may instead be organised within distinct signalling microdomains. These microdomains localise GPCRs in close proximity with other membrane proteins and intracellular signalling partners and could have profound implications for the spatial and temporal control of downstream signalling. In order to probe the molecular mechanisms that govern GPCR pharmacology within these domains, fluorescence techniques with effective single receptor sensitivity are required. Of these, fluorescence correlation spectroscopy (FCS) is a technique that meets this sensitivity threshold. This short review will provide an update of the recent uses of FCS based techniques in conjunction with GPCR subtype selective fluorescent ligands to characterise dynamic ligand-receptor interactions in whole cells and using purified GPCRs.

Multiple Technique Characterization of the Physical and Chemical Properties of Silica Abrasives Used in Chemical-Mechanical Planarization

The performance of chemical-mechanical planarization (CMP) slurries in the commercial fabrication of integrated circuits depends critically on the physical and chemical properties of abrasive particles used in CMP slurries. A host of analytical methods are used for characterizing the properties of CMP abrasive particles including spectroscopic,1,2 sedimentation,3 viscometric4 and fractionation5 techniques. One particular method, fluorescence correlation spectroscopy (FCS),1 has demonstrated significant versatility in this application due to its ability to simultaneously analyze the particle size distribution and the surface chemistry of abrasive particles in aqueous mixture containing small molecule CMP additives. The need for further characterization of these properties, especially the surface chemistry, is the product of the increasing use of nano-abrasives with reduced hydrodynamic diameters (< 20 nm) in CMP slurries in order to minimize surface defect creation during CMP processes. Incorporation of nano-abrasives in CMP slurries will promote surface chemistry-driven CMP additive adsorption on abrasive particles due to the increase in the total particle surface area as the hydrodynamic diameters of nano-abrasives decrease. The use of FCS in conjunction with the application of complementary techniques, such as dynamic light scattering and fluorescence lifetime analysis, is described in the presentation for the comprehensive characterization of the physical and chemical properties of representative silica abrasives used in CMP slurries.1. Jacobson, L.M.; Turner, D.K.; Wayman, A.; Rawat, A.; Carver, C.T..; Moinpour, M.; Remsen, E.E. “Characterization of Particle Size and Surface Adsorption for SiO2 Abrasives Used in Chemical Mechanical Planarization via Fluorescence Correlation Spectroscopy”, ECS J. Solid State Sci. Tech. 2015, 4, P5053-P5057.2. Marsh, J.L.; Wayman, A.E.; Smiddy, N.M.; Campbell, D.J.; Parker, J.C.; Bosma, W.B.; Remsen, E.E. “Infrared Spectroscopic Analysis of the Adsorption of Pyridine Carboxylic Acids on Colloidal Ceria”, Langmuir 2017, 33 , 13224-13233.3. Kamiti, M.; Boldridge, D.; Ndoping, L.M.; Remsen, E.E. “Simultaneous Absolute Determination of Particle Size and Effective Density of sub-Micron Colloids by Disc Centrifuge Photosedimentometry”, Anal. Chem. 2012, 84, 10526−10530. 4. Boldridge, D.; Kamiti, M.; Remsen, E.E. “Avoiding the Spherical Particle Assumption: Fractal Particle Density, Size and Structure Characterization through Combined Sedimentation and Viscometry Measurements”, Anal. Chem. 2020, 92, 15034-15041.5. Williams, S.K.R.; Park, I.; Remsen, E.E.; Moinpour, M. “New Particle Metrology for CMP Slurries”, Mater. Res. Soc. Symp. Proc. 2007, 991, 249-251.

The use of fluorescence correlation spectroscopy to monitor cell surface β2-adrenoceptors at low expression levels in human embryonic stem cell-derived cardiomyocytes and fibroblasts.

The importance of cell phenotype in determining the molecular mechanisms underlying β2 -adrenoceptor (β2AR) function has been noted previously when comparing responses in primary cells and recombinant model cell lines. Here, we have generated haplotype-specific SNAP-tagged β2AR human embryonic stem (ES) cell lines and applied fluorescence correlation spectroscopy (FCS) to study cell surface receptors in progenitor cells and in differentiated fibroblasts and cardiomyocytes. FCS was able to quantify SNAP-tagged β2AR number and diffusion in both ES-derived cardiomyocytes and CRISPR/Cas9 genome-edited HEK293T cells, where the expression level was too low to detect using standard confocal microscopy. These studies demonstrate the power of FCS in investigating cell surface β2ARs at the very low expression levels often seen in endogenously expressing cells. Furthermore, the use of ES cell technology in combination with FCS allowed us to demonstrate that cell surface β2ARs internalize in response to formoterol-stimulation in ES progenitor cells but not following their differentiation into ES-derived fibroblasts. This indicates that the process of agonist-induced receptor internalization is strongly influenced by cell phenotype and this may have important implications for drug treatment with long-acting β2AR agonists.

Fluorescence Correlation Spectroscopy Reveals Efficient Cytosolic Delivery of Protein Cargo by Cell-Permeant Miniature Proteins.

New methods for delivering proteins into the cytosol of mammalian cells are being reported at a rapid pace. Differentiating between these methods in a quantitative manner is difficult, however, as most assays for evaluating cytosolic protein delivery are qualitative and indirect and thus often misleading. Here we make use of fluorescence correlation spectroscopy (FCS) to determine with precision and accuracy the relative efficiencies with which seven different previously reported “cell-penetrating peptides” (CPPs) transport a model protein cargo—the self-labeling enzyme SNAP-tag—beyond endosomal membranes and into the cytosol. Using FCS, we discovered that the miniature protein ZF5.3 is an exceptional vehicle for delivering SNAP-tag to the cytosol. When delivered by ZF5.3, SNAP-tag can achieve a cytosolic concentration as high as 250 nM, generally at least 2-fold and as much as 6-fold higher than any other CPP evaluated. Additionally, we show that ZF5.3 can be fused to a second enzyme cargo—the engineered peroxidase APEX2—and reliably delivers the active enzyme to the cell interior. As FCS allows one to realistically assess the relative merits of protein transduction domains, we anticipate that it will greatly accelerate the identification, evaluation, and optimization of strategies to deliver large, intact proteins to intracellular locales.

FCS Analysis of Protein Mobility on Lipid Monolayers

In vitro membrane model systems are used to dissect complex biological phenomena under controlled unadulterated conditions. In this context, lipid monolayers are a powerful tool to particularly study the influence of lipid packing on the behavior of membrane proteins. Here, monolayers deposited in miniaturized fixed area-chambers, which require only minute amounts of protein, were used and shown to faithfully reproduce the characteristics of Langmuir monolayers. This assay is ideally suited to be combined with single-molecule sensitive fluorescence correlation spectroscopy (FCS) to characterize diffusion dynamics. Our results confirm the influence of lipid packing on lipid mobility and validate the use of FCS as an alternative to conventional surface pressure measurements for characterizing the monolayer. Furthermore, we demonstrate the effect of lipid density on the diffusional behavior of membrane-bound components. We exploit the sensitivity of FCS to characterize protein interactions with the lipid monolayer in a regime in which the monolayer physical properties are not altered. To demonstrate the potential of our approach, we analyzed the diffusion behavior of objects of different nature, ranging from a small peptide to a large DNA-based nanostructure. Moreover, in this work we quantify the surface viscosity of lipid monolayers. We present a detailed strategy for the conduction of point FCS experiments on lipid monolayers, which is the first step toward extensive studies of protein-monolayer interactions.

Fluorescence-based monitoring of electronic state and ion exchange kinetics with FCS and related techniques: from T-jump measurements to fluorescence fluctuations

In this review, we give a historical view of how our research in the development and use of fluorescence correlation spectroscopy (FCS) and related techniques has its roots and how it originally evolved from the pioneering work of Manfred Eigen, his colleagues, and coworkers. Work on temperature-jump (T-jump) experiments, conducted almost 50 years ago, led on to the development of the FCS technique. The pioneering work in the 1970s, introducing and demonstrating the concept for FCS, in turn formed the basis for the breakthrough use of FCS more than 15 years later. FCS can be used for monitoring reaction kinetics, based on fluctuations at thermodynamic equilibrium, rather than on relaxation measurements following perturbations. In this review, we more specifically discuss FCS measurements on photodynamic, electronic state transitions in fluorophore molecules, and on proton exchange dynamics in solution and on biomembranes. In the latter case, FCS measurements have proven capable of casting new light on the mechanisms of proton exchange at biological membranes, of central importance to bioenergetics and signal transduction. Finally, we describe the transient-state (TRAST) spectroscopy/imaging technique, sharing features with both relaxation (T-jump) and equilibrium fluctuation (FCS) techniques. TRAST is broadly applicable for cellular and molecular studies, and we briefly outline how TRAST can provide unique information from fluorophore blinking kinetics, reflecting e.g., cellular metabolism, rare molecular encounters, and molecular stoichiometries.

In Situ Characterization of Protein Adsorption onto Nanoparticles by Fluorescence Correlation Spectroscopy.

Nanotechnology holds great promise for applications in many fields including biology and medicine. Unfortunately, the processes occurring at the interface between nanomaterials and living systems are exceedingly complex and not yet well understood, which has significantly hampered the realization of many nanobiotechnology applications. Whenever nanoparticles (NPs) are incorporated by a living organism, a protein adsorption layer, also known as the "protein corona", forms on the NP surface. Accordingly, living organisms interact with protein-coated rather than bare NPs, and their biological responses depend on the nature of the protein corona. In recent years, a wide variety of biophysical techniques have been employed to elucidate mechanistic aspects of NP-protein interactions. In most studies, NPs are immersed in protein or biofluid (e.g., blood serum) solutions and then separated from the liquid for analysis. Because this approach may modify the composition and structure of the protein corona, our group has pioneered the use of fluorescence correlation spectroscopy (FCS) as an in situ technique, capable of examining NP-protein interactions while the NPs are suspended in biological fluids. FCS allows us to measure, with subnanometer precision and as a function of protein concentration, the increase in hydrodynamic radius of the NPs due to protein adsorption. This Account aims at reviewing recent progress in the exploration of NP-protein interactions by using FCS. In vitro FCS studies of the adsorption of important serum proteins onto water-solubilized luminescent NPs always showed a stepwise increase of the NP radius upon protein binding in the form of a binding isotherm, regardless of the type of NP and its specific surface functionalization. This observation indicates formation of a protein monolayer on the NP. Structure-based calculations of protein surface potentials revealed that positively charged patches on the proteins interact electrostatically with negatively charged NP surfaces, and the observed protein layer thickness always matched the known molecular dimensions of the proteins binding in certain orientations. Temperature and NP surface functionalization have also been identified as important parameters controlling protein corona formation. Notably, while the corona formed from a single type of serum protein was reversible, protein adsorption from complex biological media such as blood serum was entirely irreversible. These quantitative in vitro studies are of great relevance to the bio-nano community and especially to researchers developing engineered nanomaterials for biological and biomedical applications. Future efforts will be directed toward elucidating kinetic aspects of protein corona formation and the detailed structure of the adsorbed proteins at the molecular level. To better appreciate the biological responses triggered by NP exposure, more efforts will be devoted to the exploration of the biomolecular corona as it forms on NPs in contact with living cells, tissues, and even entire model organisms. These studies are challenging when performed in a well-controlled and quantitative fashion and rely on the availability of sophisticated analytical tools, particularly, quantitative optical imaging techniques including FCS and related fluctuation methods.

Folding Landscape of Mutant Huntingtin Exon1: Diffusible Multimers, Oligomers and Fibrils, and No Detectable Monomer.

Expansion of the polyglutamine (polyQ) track of the Huntingtin (HTT) protein above 36 is associated with a sharply enhanced risk of Huntington’s disease (HD). Although there is general agreement that HTT toxicity resides primarily in N-terminal fragments such as the HTT exon1 protein, there is no consensus on the nature of the physical states of HTT exon1 that are induced by polyQ expansion, nor on which of these states might be responsible for toxicity. One hypothesis is that polyQ expansion induces an alternative, toxic conformation in the HTT exon1 monomer. Alternative hypotheses posit that the toxic species is one of several possible aggregated states. Defining the nature of the toxic species is particularly challenging because of facile interconversion between physical states as well as challenges to identifying these states, especially in vivo. Here we describe the use of fluorescence correlation spectroscopy (FCS) to characterize the detailed time and repeat length dependent self-association of HTT exon1-like fragments both with chemically synthesized peptides in vitro and with cell-produced proteins in extracts and in living cells. We find that, in vitro, mutant HTT exon1 peptides engage in polyQ repeat length dependent dimer and tetramer formation, followed by time dependent formation of diffusible spherical and fibrillar oligomers and finally by larger, sedimentable amyloid fibrils. For expanded polyQ HTT exon1 expressed in PC12 cells, monomers are absent, with tetramers being the smallest molecular form detected, followed in the incubation time course by small, diffusible aggregates at 6–9 hours and larger, sedimentable aggregates that begin to build up at 12 hrs. In these cell cultures, significant nuclear DNA damage appears by 6 hours, followed at later times by caspase 3 induction, mitochondrial dysfunction, and cell death. Our data thus defines limits on the sizes and concentrations of different physical states of HTT exon1 along the reaction profile in the context of emerging cellular distress. The data provide some new candidates for the toxic species and some new reservations about more well-established candidates. Compared to other known markers of HTT toxicity, nuclear DNA damage appears to be a relatively early pathological event.

The use of fluorescence correlation spectroscopy to characterize the molecular mobility of fluorescently labelled G protein-coupled receptors.

The membranes of living cells have been shown to be highly organized into distinct microdomains, which has spatial and temporal consequences for the interaction of membrane bound receptors and their signalling partners as complexes. Fluorescence correlation spectroscopy (FCS) is a technique with single cell sensitivity that sheds light on the molecular dynamics of fluorescently labelled receptors, ligands or signalling complexes within small plasma membrane regions of living cells. This review provides an overview of the use of FCS to probe the real time quantification of the diffusion and concentration of G protein-coupled receptors (GPCRs), primarily to gain insights into ligand–receptor interactions and the molecular composition of signalling complexes. In addition we document the use of photon counting histogram (PCH) analysis to investigate how changes in molecular brightness (ε) can be a sensitive indicator of changes in molecular mass of fluorescently labelled moieties.

Real-Time Monitoring of Alzheimer's-Related Amyloid Aggregation via Probe Enhancement-Fluorescence Correlation Spectroscopy.

This work describes the use of fluorescence correlation spectroscopy (FCS) and a novel amyloid-binding fluorescent probe, ARCAM 1, to monitor the aggregation of the Alzheimer's disease-associated amyloid β-peptide (Aβ). ARCAM 1 exhibits a large increase in fluorescence emission upon binding to Aβ assemblies, making it an excellent candidate for probe enhancement FCS (PE-FCS). ARCAM 1 binding does not change Aβ aggregation kinetics. It also exhibits greater dynamic range as a probe in reporting aggregate size by FCS in Aβ, when compared to thioflavin T (ThT) or an Aβ peptide modified with a fluorophore. Using fluorescent burst analysis (via PE-FCS) to follow aggregation of Aβ, we detected soluble aggregates at significantly earlier time points compared to typical bulk fluorescence measurements. Autocorrelation analysis revealed the size of these early Aβ assemblies. These results indicate that PE-FCS/ARCAM 1 based assays can detect and provide size characterization of small Aβ aggregation intermediates during the assembly process, which could enable monitoring and study of such aggregates that transiently accumulate in biofluids of patients with Alzheimer's and other neurodegenerative diseases.

Use of FCS to Study Protein Denaturation and Aggregation

Fluorescence Correlation Spectroscopy (FCS) is a powerful technique for studying the diffusive dynamics of fluorophore-tagged biomolecules and thereby drawing inferences about their conformational states and transitions. We have employed FCS to investigate the denaturation and aggregation pathways of bovine immunoglobulin (IgG), a multi-subunit all beta-sheet Y-shaped protein prone to aggregation. Each upper arm of the Y consists of a light and a heavy chain connected by cysteine-shy;cysteine disulphide bonds, and the two heavy chains are joined at the bend by more disulphides. While majority of proteins show continuous increase of hydrodynamic radius (Rg) when presented with increasing denaturant concentration, IgG exhibits strikingly different behavior under similar conditions. Its Rg initially decreases with increasing concentration of GdnHCl (from 0 to 3 M) and then goes through two pronounced maxima at 4 M and 7 M [GdnHCl]. We propose that the initial contraction is related to sheet-to-helix transition at the flexible bends of the Y-arms of the protein. The first maximum of Rg is attributed to electrostatic repulsions and their screening by Gdn+ and Cl- ions; the second is shown to arise from disulphide-mediated effects.Previous studies have proposed that IgG aggregation is initiated by intermolecular disulphide bond formation. We studied the aggregation kinetics of IgG by monitoring the time evolution of characteristic diffusion time (τD) under two different conditions: with the cysteine residues free to interact or blocked. The progress curves (τD vs.elapsed time) were sigmoidal in both cases, but the delay time was almost an order of magnitude longer, and the apparent polymerization rate half as fast, for the case where the cysteines were blocked than when they were free. This result indicates that intermolecular disulphide bond formation is a key factor driving the aggregation process of IgG.

The Bacterial Membrane Insertase YidC Is a Functional Monomer and Binds Ribosomes in a Nascent Chain-Dependent Manner

Thesynthesisandfoldingofproteinsoften occursin kinetically coupled processes in order to avoidthe accumulation of aggregation-prone unfoldedpolypeptide chains. Chaperones are specificallyrecruited to the ribosomal tunnel exit where theywait for the appearance of appropriate binding siteson the emerging polypeptide chains in order toensureearlybindingoffoldingfactors.Foranumberof chaperones and nascent chain modifying en-zymes, the binding sites on the ribosome weremapped to high molecular resolution and theirrelevance for cotranslational protein folding hasbeen studied in detail[1].In contrast, our understanding of how ribosomesassociate with the components that mediate proteininsertionintolipidbilayersisstillrelativelyscarce.Dueto their hydrophobic nature, nascent membraneproteins have a much higher risk to form insolubleaggregates. Hence, a tight coupling of the synthesisand insertion of membrane proteins appears crucial.Two evolutionary conserved systems mediate themembrane integration of proteins: the Sec61/SecYtranslocon (Sec complex) and protein insertases ofthe YidC/Oxa1/Alb3 protein family. The Sec complexforms a translocation pore that allows the completetranslocation of secretory proteins through the mem-brane, as well as the insertion of membrane proteinsviaalateralgate[2].Ribosomescandirectlydockontothe Sec complex so that the ribosomal polypeptidetunnel and the translocation pore in the membraneform one continuous channel [3,4]. The targeting ofribosomes to the Sec complex is thereby initiated bythe signal recognition particle in conjunction with itsreceptor [5].Members of the YidC/Oxa1/Alb3 protein familymediate the insertion of proteins into membranes ofbacteria, mitochondria and chloroplasts, respectively.The precise mode of action is not known, but theirsubstrates typically lack larger soluble domains onthe trans side of the membrane, suggesting thattheir major activity is the integration and folding oftransmembrane segments in the lipid bilayer ratherthanthe translocationof hydrophilic segmentsacrossthe membrane. Nevertheless, the Oxa1 protein of themitochondrialinnermembrane,incooperationwithitshomolog Cox18, was shown to facilitate membranetranslocation of even long hydrophilic domains [6,7]and electrophysiological measurements with purifiedOxa1showedanabilitytoformsubstrate-gatedpores[8].The membrane-embedded insertase domain ofbacterial YidC (Fig. 1a), mitochondrial Oxa1 andchloroplast Alb3 are closely related and functionallyinterchangeable[9–11],butOxa1,incontrasttoYidC,contains a C-terminal ribosome binding domain(Fig. 1b) that connects the protein permanently tomitochondrial ribosomes [12,13]. The permanenttetheringofmitochondrialribosomestothemembraneis presumably a consequence of the evolution of themitochondrial genome during which genes coding forhydrophilic proteins were lost.The bacterial YidC protein lacks a C-terminalribosome binding domain and only exposes a veryshortC-terminalstretchof13residuesintothecytosol.Cross-linking studies indicated that, during the syn-thesis of membrane proteins, YidC and bacterialribosomesareinclosecontact[14,15]butitremainedunclear whether YidC binds bacterial ribosomesdirectly. This important issue was now carefullyaddressed by the group of Arnold Driessen in astudy published in this issue of Journal of MolecularBiology [16]. By use of fluorescence correlationspectroscopy, the authors showed that YidC doesnot associate with bacterial ribosomes (Fig. 1b).Interestingly, the addition of just six histidine residuesto the C-terminus of YidC is sufficient to bind YidC toribosomesparticularlyuponlowpHwheremoreofthehistidineresiduesarepositivelycharged.Thisisavery

The use of fluorescence correlation spectroscopy (FCS) as an alternative biomarker detection technique: a preliminary study

Biomarkers are essential part of daily medical practice. Currently, biomarkers are being used both for diagnostic and prognostic purposes. There are many approaches e.g. ELISA by which biomarker levels are detected from patient samples. However, all these approaches are laborious, time consuming and expensive. There is therefore a general need for exploring new technique which can overcome these drawbacks. Here, we present a preliminary study for detection of serum biomarkers by fluorescence correlation spectroscopy (FCS) based diagnostic technique. FCS is a technique basically used for spatial and temporal analysis of molecular interactions of extremely low-concentration biomolecules in solution. FCS is able to measure diffusion time of the fluorescent molecules passing through the open detection volume and it can also measure the average number of fluorescent molecules passing through the detection volume. Because diffusion speed is correlated with shape and molecular mass of the fluorescent molecule, this property makes it possible to study the complex formation between a small fluorescently labelled and a large unlabelled molecule. In this preliminary study, we utilize this FCS property for detection of serum biomarker. Further studies on various pathological serum samples are warranted to explore further aspects of this technique.

Relaxation Kinetics by Fluorescence Correlation Spectroscopy: Determination of Kinetic Parameters in the Presence of Fluorescent Impurities.

The use of Fluorescence Correlation Spectroscopy (FCS) in combination with Förster Resonance Energy Transfer (FRET) is gaining popularity as a tool to investigate kinetics in equilibrium conditions. The technique is based on the study of fluorescence fluctuations in small numbers of molecules, and is particularly well-suited to investigate conformational dynamics in biopolymers. In practice, its applicability is often hindered by the presence of certain impurities such as partially labeled biomolecules, excess of free fluorophore, or partially dissociated multi-subunit complexes. Here, we show that the simultaneous measurement of the fluctuations in the donor and acceptor intensities allows the determination of the kinetic relaxation time of the reaction in the presence of donor-only particles when cross-talk is negligible, or in cases where all species have the same diffusion coefficient. Theoretical predictions are supported with the results of Monte Carlo simulations, and demonstrate that the applicability of the technique is more general than previously thought.

FRET Fluctuation Spectroscopy of Diffusing Biopolymers: Contributions of Conformational Dynamics and Translational Diffusion

The use of fluorescence correlation spectroscopy (FCS) to study conformational dynamics in diffusing biopolymers requires that the contributions to the signal due to translational diffusion are separated from those due to conformational dynamics. A simple approach that has been proposed to achieve this goal involves the analysis of fluctuations in fluorescence resonance energy transfer (FRET) efficiency. In this work, we investigate the applicability of this methodology by combining Monte Carlo simulations and experiments. Results show that diffusion does not contribute to the measured fluctuations in FRET efficiency in conditions where the relaxation time of the kinetic process is much shorter than the mean transit time of the molecules in the optical observation volume. However, in contrast to what has been suggested in previous work, the contributions of diffusion are otherwise significant. Neglecting the contributions of diffusion can potentially lead to an erroneous interpretation of the kinetic mechanisms. As an example, we demonstrate that the analysis of FRET fluctuations in terms of a purely kinetic model would generally lead to the conclusion that the system presents complex kinetic behavior even for an idealized two-state system.

Direct measurement of diffusing proteins in nanochannels using fluorescence correlation spectroscopy

Transport of proteins in liquid-filled nanometer-sized apertures with charged surfaces based on both diffusion and convective flow is investigated with the use of fluorescence correlation spectroscopy. The measured fluctuations of fluorescence allow the evaluation of the concentration of molecules directly inside the nanochannel. Here, we supply theoretical models describing the concentration profile of proteins inside nano-confinements, taking into account the steric exclusion, the presence of reversibly adsorbed molecules on surfaces and the exclusion-enrichment effect due to interaction with the electrical double layer. Therefore, this study presents a new step for a deeper understanding of the transport of molecules in nanofluidics.