Fluorescence Correlation Spectroscopy Studies Research Articles

Spectroscopy and Fluorescence Lifetime Imaging Microscopy To Probe the Interaction of Bovine Serum Albumin with Graphene Oxide.

The interaction of graphene oxide (GO) with bovine serum albumin (BSA) in aqueous buffer solution has been investigated with various spectroscopic and imaging techniques. At single molecular resolution this interaction has been performed using fluorescence correlation spectroscopy (FCS) and fluorescence lifetime imaging microscopy (FLIM) techniques. The conformational dynamics of BSA on GO's influence have been explored by FCS and circular dichroism (CD) spectroscopy. For the FCS studies BSA was labeled covalently by a fluorophore, Alexa Fluor 488. On the addition of GO in phosphate buffer of 10 mM at pH 7.4 the diffusion time (τD) and the hydrodynamic radius (Rh) of BSA increase due to adsorption of BSA. Conformational relaxation time components of native BSA drastically vary with the addition of GO, signifying the change of conformational dynamics of BSA after addition of GO. The adsorption isotherm also indicates significant adsorption of BSA on the GO surface. Adsorption of BSA on the GO surface has shown in direct images of atomic force microscopy (AFM) and FLIM. Fluorescence quenching study of BSA with addition of GO also indicates that there is strong interaction between BSA and GO.

Investigation of GPCR allosterism and dimerization in single living cells using fluorescent ligands.

Previous work, using fluorescent adenosine receptor agonists and antagonists, has provided novel insights into the allosteric regulation of adenosine A3 (A3AR) and A1 (A1AR) receptors by allosteric ligands and receptor dimerization in single living cells [1, 2]. We have also used a fluorescent analogue of {type:entrez-protein,attrs:{text:CGP12177,term_id:877152897,term_text:CGP12177}}CGP12177 to investigate ligand binding to the human β1-adrenoceptor. This work has demonstrated that there is negative cooperativity between the two different ligand-binding conformations of the β1-adrenoceptor activated by catecholamines and {type:entrez-protein,attrs:{text:CGP12177,term_id:877152897,term_text:CGP12177}}CGP12177 respectively [3]. Finally, we have used fluorescence correlation spectroscopy (FCS) to investigate ligand binding to A1AR and A3AR in small 0.2 µm2 microdomains of single living cells [4]. FCS studies with a fluorescent A3-agonist have enabled high affinity labeling of the active conformation (R*) of the receptor [4]. We have also used a fluorescent adenosine A3-antagonist ({type:entrez-nucleotide,attrs:{text:CA200645,term_id:35234116,term_text:CA200645}}CA200645) to study the binding characteristics of antagonist-occupied receptor conformations (R) in membrane microdomains of single cells [5]. Investigation of the dissociation kinetics of {type:entrez-nucleotide,attrs:{text:CA200645,term_id:35234116,term_text:CA200645}}CA200645 provided further support for allosteric regulation of this receptor by homodimerization [5].

Probing the Role of an Intermediate State in TDP‐43: A Gateway to Developing New Biomarkers and Therapies for Neurodegenerative Disease

Pathological alteration of TDP-43 is a hallmark feature of multiple neurodegenerative diseases, including amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Fragments, comprised of the second RNA recognition motif (RRM2) and the disordered C-terminus, have been observed in cytoplasmic inclusions, suggesting that conformational changes within these regions play a role in aggregation and toxicity. Circular dichroism and fluorescence spectroscopies reveal a three-state equilibrium unfolding model for RRM2 with a partially-folded intermediate state. This state likely plays a biological role by enhancing access to the nuclear export signal (NES) contained within RRM2; however, it may also serve as a molecular hazard linking productive folding and function with pathological misfolding and aggregation. Structural insights using nuclear magnetic resonance, small-angle X-ray scattering and mutational analysis have shown the intermediate is unfolded-like with potential residual structure in the N-terminus of RRM2. These results suggest that the NES may be unfolded in this state for recognition by the export machinery. Ongoing fluorescence correlation spectroscopy and cellular-based studies will investigate how this intermediate state, in the context of the disordered C-terminus, may disrupt normal folding and function to enhance misfolding and toxicity. These insights will provide essential information for designing therapeutic strategies targeting the RRM2 intermediate state for preventing TDP-43 dysfunction and aggregation in a set of neurodegenerative diseases.

Actin cytoskeleton differentially alters the dynamics of lamin A, HP1α and H2B core histone proteins to remodel chromatin condensation state in living cells.

Cells in physical microenvironments regulate their functioning and geometry in response to mechanical stimuli. Recent studies have demonstrated the influence of the integrated actin cytoskeleton on nuclear integrity and chromatin organization. However, the mechanisms underlying the mechanotransduction of their physical coupling to nuclear protein dynamics are not well understood. In this study, we take advantage of micropatterned geometric substrates in NIH3T3 mouse fibroblasts to probe the functional influence of actin organization on nuclear lamina and chromatin assembly. Fluorescence correlation spectroscopy studies demonstrate that stabilization of perinuclear actin strengthens the transient interactions of lamin A with the chromatin. Correspondingly, fluorescence recovery after photobleaching studies reveal enhanced mobility of these nuclear lamina proteins when actin organization is perturbed. Combining these fluorescence dynamics assays, we also demonstrate an actin-driven differential modulation of core histone H2B and heterochromatin HP1α protein dynamics with chromatin. These altered dynamics are reflected structurally by concomitant changes in the architecture of the heterochromatin foci as seen by immunofluorescence assays. Taken together, our study provides a demonstration of the differential mechanical control of perinuclear actin on the dynamics of the nuclear lamina, euchromatin and heterochromatin regimes of the nucleus, and suggests an actin-mediated route to spatially and structurally tune chromatin organization and dynamics.

ATP and Magnesium Promote Cotton Short-Form Ribulose-1,5-bisphosphate Carboxylase/Oxygenase (Rubisco) Activase Hexamer Formation at Low Micromolar Concentrations

We report a fluorescence correlation spectroscopy (FCS) study of the assembly pathway of the AAA+ protein ribulose-1,5-bisphosphate carboxylase/oxygenase (Rubisco) activase (Rca), a ring-forming ATPase responsible for activation of inhibited Rubisco complexes for biological carbon fixation. A thermodynamic characterization of simultaneously populated oligomeric states appears critical in understanding Rca structure and function. Using cotton β-Rca, we demonstrate that apparent diffusion coefficients vary as a function of concentration, nucleotide, and cation. Using manual fitting procedures, we provide estimates for the equilibrium constants for the stepwise assembly and find that in the presence of ATPγS, the Kd for hexamerization is 10-fold lower than with ADP (∼0.1 vs ∼1 μM). Hexamer fractions peak at 30 μM and dominate at 8-70 μM Rca, where they comprise 60-80% of subunits with ATPγS, compared with just 30-40% with ADP. Dimer fractions peak at 1-4 μM Rca, where they comprise 15-18% with ATPγS and 26-28% with ADP. At 30 μM Rca, large aggregates begin to form that comprise ∼10% of total protein with ATPγS and ∼25% with ADP. FCS data collected on the catalytically impaired WalkerB-D173N variant in the presence of ATP provided strong support for these results. Titration with free magnesium ions lead to the disaggregation of larger complexes in favor of hexameric forms, suggesting that a second magnesium binding site with a Kd value of 1-3 mM mediates critical subunit contacts. We propose that closed-ring toroidal hexameric forms are stabilized by binding of Mg·ATP plus Mg2+, whereas Mg·ADP promotes continuous assembly to supramolecular aggregates such as spirals.

Effects of surface functionalization on the adsorption of human serum albumin onto nanoparticles - a fluorescence correlation spectroscopy study.

By using fluorescence correlation spectroscopy (FCS), we have studied the adsorption of human serum albumin (HSA) onto Fe–Pt nanoparticles (NPs, 6 nm radius), CdSe/ZnS quantum dots (QDs, 5 nm radius) and Au and Ag nanoclusters (1–4 nm radius), which are enshrouded by various water-solubilizing surface layers exposing different chemical functional groups (carboxyl, amino and both), thereby endowing the NPs with different surface charges. We have also measured the effects of modified surface functionalizations on the protein via succinylation and amination. A step-wise increase in hydrodynamic radius with protein concentration was always observed, revealing formation of protein monolayers coating the NPs, independent of their surface charge. The differences in the thickness of the protein corona were rationalized in terms of the different orientations in which HSA adsorbs onto the NPs. The midpoints of the binding transition, which quantifies the affinity of HSA toward the NP, were observed to differ by almost four orders of magnitude. These variations can be understood in terms of specific Coulombic interactions between the proteins and the NP surfaces.

Effect of ethanol-water mixture on the structure and dynamics of lysozyme: A fluorescence correlation spectroscopy study

Effect of ethanol-water mixture on the hydrodynamic radius (r(H)) and conformational dynamics of lysozyme has been studied by circular dichroism, emission spectra, and fluorescence correlation spectroscopy. For this purpose, the protein lysozyme is covalently labeled near the active site with a fluorescent probe, alexa 488. The ethanol molecules are sequestered near the hydrophobic tryptophan residues as indicated by the blue shift of the emission maximum of tryptophan. It is observed that both size (r(H)) and time constant of conformational relaxation (τ(R)) of lysozyme oscillate with increase in ethanol concentration. The r(H) of the protein fluctuates from 19 Å in the native state, to a minimum of 13 Å, and a maximum of 29 Å. It is proposed that the oscillating behavior arises from competition between mutual interaction among protein, ethanol, and water. The fluorescence intensity fluctuates because of quenching of the fluorescence of the probe (alexa) by the free amino group of certain residues (e.g., tryptophan). Rate of inter-conversion (folding dynamics) between the open (fluorescent) and closed (non-fluorescent) form has been determined and is found to exhibit similar oscillation with variation in ethanol content.

What Kind of Microviscosity Does a Molecule Experience During its Rotational and Translational Diffusion in Crowded Environments?

Living cells are known to be crowded with organelles, biomembranes, and macromolecules such as proteins, DNA, RNA, and actin filaments. In such crowded environments it is reasonable to believe that cellular viscosity is heterogeneous, which is likely to influence biomolecular diffusion, protein-protein interactions, protein-substrate interaction, and protein folding. In this contribution, we investigate the difference between bulk viscosity and microviscosity in crowded environments and their effects on both rotational (ps-ns) and translational (ms-s) diffusion of rhodamine green (as a probe) using time-resolved fluorescence anisotropy (TRFA) and fluorescence correlation spectroscopy (FCS), respectively. For biomimetic crowding, Ficoll-70, BSA and ovalbumin were used as crowding agents and compared with glycerol-rich solutions as a homogeneous environment. Assuming a Stockes-Einstein model, the microviscosity was calculated using TRFA and FCS, assuming no binding, and the results are compared with the bulk viscosity, which was measured using a conventional viscometer. Our results indicate that the micro- and bulk viscosities in a homogeneous environment like glycerol-rich solutions are similar over the 1-20 cP range. In Ficoll-70, BSA and ovalbumin-crowded environments, the microviscosity differs from the corresponding bulk viscosity, depending on the nature of crowding agents (i.e., proteins versus polymers) and the concentration of crowding agents. These results are discussed in terms of both non-specific binding and heterogeneous viscosity in crowded solutions, which in return provide an apparent deviation from the Stokes-Einstein model (i.e., Brownian diffusion). Our findings provide a foundation for FCS and TRFA-based studies of diffusion and binding of biomolecules in the crowded milieu of living cells.

FCS Study of the Structural Stability of Lysozyme in the Presence of Morpholinium Salts

Ability of the ionic liquids to alter the structural stability of proteins in aqueous solution is a topic of considerable interest in modern bioscientific research because of possible applications of these substances in protein purification and as refolding agents. A few early studies involving the imidazolium ionic liquids have demonstrated their role as both denaturants and refolding agents. As the influence of an ionic liquid on a given protein depends on the identity of both species, it is necessary to extend the studies to a wider number of ionic liquids and proteins to obtain insight into the mechanism of interaction between the two and to arrive at a comprehensive picture. It is in this context that we have studied the effect of two morpholinium salts, [Mor1,2][Br] and [Mor1,4][Br], differing in the alkyl chain length of cation, on chicken egg white lysozyme in its native and chemically denatured states employing primarily the fluorescence correlation spectroscopy (FCS) technique. Fluorescence signal of Alexa488-labeled lysozyme (A488-Lysz) has been used to determine the changes in hydrodynamic radius of protein in the presence of additives. The results reveal a conformational dynamics of lysozyme with a time constant of 56 ± 10 μs in its native state. It is observed, when in its native state, both the morpholinium salts induce structural changes of lysozyme. However, when in its unfolded state, [Mor1,4][Br] at low concentration compacts the protein, but at higher concentration, it stabilizes the unfolded state, unlike [Mor1,2][Br], which compacts lysozyme at both low and high concentrations. A comparison of the effect of these salts and arginine, a protein stabilizer, on lysozyme indicates that [Mor1,2][Br] is a superior compacting agent for the unfolded state of the protein compared to arginine.

High-throughput fluorescence assay for membrane-protein interaction

Membrane-protein interaction plays key roles in a wide variety of biological processes. Although various methods have been employed to measure membrane binding of soluble proteins, a robust high-throughput assay that is universally applicable to all proteins is lacking at present. Here we report a new fluorescence quenching assay utilizing enhanced green fluorescence protein (EGFP)-fusion proteins and a lipid containing a dark quencher, N-dimethylaminoazobenzenesulfonyl-phosphatidylethanolamine (dabsyl-PE). The EGFP fluorescence emission intensity showed a large decrease (i.e., >50%) when EGFP-fusion proteins bound the vesicles containing 5 mol% dabsyl-PE. This simple assay, which can be performed using either a cuvette-based spectrofluorometer or a fluorescence plate reader, allowed rapid, sensitive, and accurate determination of lipid specificity and affinity for various lipid binding domains, including two pleckstrin homology domains, an epsin N-terminal homology domain, and a phox homology domain. The assay can also be applied to high-throughput screening of small molecules that modulate membrane binding of proteins.

Fluorescence Correlation Spectroscopy Study on the Effects of the Shape and Size of a Protein on Its Diffusion Inside a Crowded Environment

Fluorescence correlation spectroscopy (FCS) has been commonly used to study the diffusional and conformational fluctuations of labeled molecules at single-molecule resolution. Here, we explored the applications of FCS inside a polyacrylamide gel to study the effects of molecular weight and molecular shape in a crowded environment. To understand the effect of molecular weight, we carried out FCS experiments with four model systems of different molecular weights in the presence of varying concentrations of acrylamide. The correlation curves were fit adequately using a model containing two diffusing components: one representing unhindered diffusion and one representing slow hindered diffusion in the gel phase. A large number of measurements carried out at different randomly chosen spots on a gel were used to determine the most probable diffusion time values using Gaussian distribution analysis. The variation of the diffusivity with the molecular weight of the model systems could be represented well using the effective medium model. This model assumes a combination of hydrodynamic and steric effects on solute diffusivity. To study the effects of solute shape, FCS experiments were carried inside a urea gradient gel to probe the urea-induced unfolding transition of Alexa488Maleimide-labeled bovine serum albumin. We showed that the scaling behavior, relating the hydrodynamic radius and the number of amino acids, changes inside an acrylamide gel for both folded and unfolded proteins. We showed further that crowding induced by a polyacrylamide gel increases the resolution of measuring the difference in hydrodynamic radii between the unfolded and folded states.

A Fluorescence Correlation Spectroscopy, Steady-State, and Time-Resolved Fluorescence Study of the Modulation of Photophysical Properties of Mercaptopropionic Acid Capped CdTe Quantum Dots upon Exposure to Light

Light-induced modulation of the fluorescence behavior of mercaptopropionic acid (MPA) capped CdTe quantum dots (QDs) in aqueous solution is studied by a combination of fluorescence correlation spectroscopy (FCS) and steady-state and time-resolved fluorescence techniques. These investigations reveal a dramatic variation in the fluorescence properties of the QDs under exposure to light. In the FCS measurement, a large decrease in amplitude and change in shape of the correlation curves are observed with increasing excitation power. The change in the shape of the correlation curves, particularly at short lag time, e.g., a faster relaxation at high excitation power, is attributed to the increasing contribution of the off state of the QDs. Interestingly, despite this increasing contribution of the off state, which reduces the effective number of emitters in the observation volume and hence should increase the amplitude of the correlation curve, the latter actually decreases at high excitation power. This appare...

Structural Transformation of Bovine Serum Albumin Induced by Dimethyl Sulfoxide and Probed by Fluorescence Correlation Spectroscopy and Additional Methods

Determining the structure of a protein and its transformation under different conditions is key to understanding its activity. The structural stability and activity of proteins in aqueous-organic solvent mixtures, which is an intriguing topic of research in biochemistry, is dependent on the nature of the protein and the properties of the medium. Herein, the effect of a commonly used cosolvent, dimethyl sulfoxide (DMSO), on the structure and conformational dynamics of bovine serum albumin (BSA) protein is studied by fluorescence correlation spectroscopy (FCS) measurements on fluorescein isothiocyanate (FITC)-labeled BSA. The FCS study reveals a change of the hydrodynamic radius of BSA from 3.7 nm in the native state to 7.0 nm in the presence of 40% DMSO, which suggests complete unfolding of the protein under these conditions. Fluorescence self-quenching of FITC has been exploited to understand the conformational dynamics of BSA. The time constant of the conformational dynamics of BSA is found to change from 35 μs in its native state to 50 μs as the protein unfolds with increasing DMSO concentration. The FCS results are corroborated by the near-UV circular dichroism spectra of the protein, which suggest a loss of its tertiary structure with increasing concentration of DMSO. The intrinsic fluorescence of BSA and the fluorescence response of 1-anilinonaphthalene-8-sulfonic acid, used as a probe molecule, provide information that is consistent with the FCS measurements, except that aggregation of BSA is observed in the presence of 40% DMSO in the ensemble measurements.

Discrepancy between fluorescence correlation spectroscopy and fluorescence recovery after photobleaching diffusion measurements of G-protein-coupled receptors

Fluorescence recovery after photobleaching (FRAP) and fluorescence correlation spectroscopy (FCS) are the two most direct methods to measure the diffusion of molecules in intact living cells. Ideally, these methods should produce similar results for an identical system. We have used these methods to monitor the diffusion of two G-protein-coupled receptors and their associated proteins in the plasma membranes of cells that do not or do contain invaginated protein domains called caveolae. FRAP studies show that caveolae domains increase the immobile fraction of receptors without significantly changing their mobility. On the other hand, FCS studies show an unexpected increase the mobility of caveolae-associated proteins. Our data suggest that the geometry of caveolae domains gives rise to a confined diffusion of its attached proteins, resulting in an apparent increase in mobility.

In what time scale proton transfer takes place in a live CHO cell?

Excited state proton transfer (ESPT) of pyranine (8-hydroxypyrene-1,3,6-trisulfonate, HPTS) in a live Chinese hamster ovary (CHO) cell is studied by time resolved confocal microscopy. The cytoplasm region of the cell is stained by a photoacid, HPTS (HA). The time constant of initial proton transfer (τ(PT)) in the cell is found to be ~10 times longer than that in bulk water, while the time constants of recombination (τ(rec)) and dissociation (τ(diss)) in the cell are ~3 times and ~2 times longer, respectively. The slower rate of proton transfer (~10 times) inside the CHO cell compared to that in bulk water is ascribed to slower solvation dynamics, lower availability of free water molecules, and disruption of hydrogen-bond network inside the cell. Translational and rotational diffusion of HPTS inside a single CHO cell have been investigated by fluorescence correlation spectroscopy (FCS) and picosecond anisotropy measurement, respectively. Both the translational and rotational diffusion slow down inside the live cell. FCS studies indicate that HPTS remains tightly bound to a macromolecule inside the cell.

Exploring the role of internal friction in the dynamics of unfolded proteins using simple polymer models

Recent experiments showed that the reconfiguration dynamics of unfolded proteins are often adequately described by simple polymer models. In particular, the Rouse model with internal friction (RIF) captures internal friction effects as observed in single-molecule fluorescence correlation spectroscopy (FCS) studies of a number of proteins. Here we use RIF, and its non-free draining analog, Zimm model with internal friction, to explore the effect of internal friction on the rate with which intramolecular contacts can be formed within the unfolded chain. Unlike the reconfiguration times inferred from FCS experiments, which depend linearly on the solvent viscosity, the first passage times to form intramolecular contacts are shown to display a more complex viscosity dependence. We further describe scaling relationships obeyed by contact formation times in the limits of high and low internal friction. Our findings provide experimentally testable predictions that can serve as a framework for the analysis of future studies of contact formation in proteins.

Combined analytical ultracentrifugation, light scattering, and fluorescence correlation spectroscopy studies on the associations and assembly of the Escherichia coli cell division FtsZ protein

This work was supported by the Spanish government through grants BIO2008-04478-C03 and BIO2011-28941-C03-03 to GR, BFU2010-14910 and BIO2011-28941-C03-02 to SZ; by the European Commission through contract HEALTH-F3-2009-223432, by Human Frontiers Science Program through grant RGP0050/2010-C102, and Comunidad de Madrid through grant S-BIO-0260/2006 to GR; and by the CSIC through grants 200980I186 and 201020I001 to SZ and CA, respectively

Fluorescence Studies of the Bradykinin 2 and µ-Opioid Receptors Suggest that Caveolae Localization of GPCRs is Mediated by their Attached G Proteins

Caveolae are 50-100 nm plasma membrane invaginations that have several proposed functions. Some studies suggest that caveolae may influence cell signaling by sequestering certain signaling proteins such as G-protein coupled receptors. We have previously found that Caveolin-1, the main structural protein of caveolae specifically binds to Gαq and that their binding is strengthened upon Gαq activation. Here, we have used fluorescence methods to determine the effect of caveolae on the functional properties and localization of two G-protein coupled receptors: the bradykinin receptor type 2 (B2R), which is coupled to Gαq, and the µ opioid receptor (µOR), which is coupled to Gαi. While caveolae do not affect cAMP signals mediated by µOR, they prolong Ca2+ signals mediated by B2R. In A10 cells, down-regulation of Caveolin-1 ablates the prolonged calcium signal in accord with idea that caveolae binds to Gαq. Immunofluorescence and FRET studies show that a significant fraction of B2R resides at or close to caveolae domains while none or very little µOR resides in caveolae domains. FRET between B2R and caveolae is reduced by down-regulation of Gαq or by addition of a peptide that interferes with Gαq/Caveolin-1 interactions suggesting that Gαq promotes localization of B2R to caveolae domains. Fluorescence Correlation Spectroscopy studies on live cells show that the presence of caveolae changes the distribution of the apparent diffusion coefficients of B2R and Gαq but not µOR and Gαi. Our results suggest that Gαq can localize its associated receptors to caveolae domains to enhance their signals.

Loop Length and Counter Ion Dependent Folding Kinetics and Thermodynamic Stability of Nucleic Acid Hairpin

Stem-loop structured hairpins represent major structural elements in nucleic acid. An intra-molecular double helix (stem) defines a DNA hairpin, usually stabilized by canonical base pairs or mis-pairs and capped by number of unpaired and paired nucleosides (loop). It is well known that nucleic acid hairpins play a pivotal role in DNA recombination, gene expression, and DNA transposition. A thorough investigation of kinetics and thermodynamic stability is the key to understand the role of DNA hairpin formation. It is well known that the structure and thermodynamic stability of DNA hairpin depend on stem size, loop size, stem and loop composition, base stacking, base-pairing, hydrogen bonds on the loop and closing base-pair of the loop. Here we use rapid-mixing stopped-flow kinetics and Fluorescence Correlation Spectroscopy (FCS) techniques to probe the loop length dependent folding kinetics and thermodynamic stability of DNA hairpins. Each hairpin is labeled with fluorophore TAMRA and quencher DABCYL at the 5' and 3' end respectively. FCS is a single molecule technique to measure reaction kinetics in the microsecond timescale where as stopped flow kinetics is a rapid mixing technique to monitor the reaction kinetics on the millisecond timescale. The reactions were observed to occur on a time scale of milliseconds by stopped-flow kinetics, considerably longer than tens of microseconds time scale suggested by previous kinetic studies of similar sized hairpins. Stopped flow kinetics and FCS studies show that nucleic acid folding and unfolding reactions occur over a broad range of time scales from the microseconds to milliseconds range. FCS can probe intermediate reaction and complete folding reaction can be probed by Stopped-flow kinetics. The combination of these two complementary techniques can probe the complete folding trajectory of the DNA hairpin.

Evaluation of the inhibitory effect of docosahexaenoic acid and arachidonic acid on the initial stage of amyloid β1-42 polymerization by fluorescence correlation spectroscopy

Amyloid β(Aβ)1-42 fibrillation is a crucial step in the development of pathological hallmarks, such as neuritic plaques and neurofibrillary tangles, of Alzheimer’s disease (AD). In this study, we evaluated the effects of free docosahexaenoic acid (DHA), an essential brain polyunsaturated fatty acid (PUFA), on the inhibition of Aβ1-42 fibrillation by fluorescence correlation spectroscopy (FCS), a technique capable of detecting molecular movements and interactions in solution. We also examined whether free arachidonic acid (AA), eicosapentaenoic acid (EPA), and metabolites of DHA, including neuroprotectin D1 (NPD1, 10S, 17S-dihydroxy-DHA), resolvin D1 (RvD1, 7S, 8R, 17S-trihydroxy-DHA), and didocosahexaenoyl glycerol (diDHA), affect Aβ1-42 polymerization. The results of the FCS study reveal that DHA and AA significantly reduced the diffusion time of TAMRA (5-carboxytetramethylrhoda-mine)-Aβ1-42 by 28% and 31%, respectively, while EPA, NPD1, RvD1, and diDHA had no effects on diffusion time. These results indicate that DHA and AA inhibited Aβ1-42 polymerization and that their inhibitory effects occurred at the initial stage of Aβ1-42 polymerization. This study will advance the research on PUFAs in preventing AD progression.