DNA binding activity of Anabaena sensory rhodopsin transducer probed by fluorescence correlation spectroscopy

Anabaena sensory rhodopsin transducer (ASRT) is believed to be a major player in the photo-signal transduction cascade, which is triggered by Anabaena sensory rhodopsin. Here, we characterized DNA binding activity of ASRT probed by using fluorescence correlation spectroscopy. We observed clear decrease of diffusion coefficient of DNA upon binding of ASRT. The dissociation constant, K_D, of ASRT to 20?bp-long DNA fragments lied in micro-molar range and varied moderately with DNA sequence. Our results suggest that ASRT may interact with several different regions of DNA with different binding affinity for global regulation of several genes that need to be activated depending on the light illumination.     

Studies on the formation and stability of triplex DNA using fluorescence correlation spectroscopy

Triplex DNA has become one of the most useful recognition motifs in the design of new molecular biology tools, therapeutic agents and sophisticated DNA‐based nanomaterials because of its direct recognition of natural double‐stranded DNA. In this paper, we developed a sensitive and microscale method to study the formation and stability characterization of triplex DNA using fluorescence correlation spectroscopy (FCS). The principle of this method is mainly based on the excellent capacity of FCS for sensitively distinguishing between free single‐strand DNA (ssDNA) fluorescent probes and fluorescent probe–double‐strand DNA (dsDNA) hybridized complexes. First, we systematically investigated the experimental conditions of triplex DNA formation. Then, we evaluated the equilibrium association constants (K_a) under different ssDNA probe lengths, composition and pH. Finally, we used FCS to measure the hybridization fraction of a 20‐mer perfectly matched ssDNA probe and three single‐base mismatched ssDNA probes with 146‐mer dsDNA. Our data illustrated that FCS is a useful tool for the direct determination of the thermodynamic parameters of triplex DNA formation and discrimination of a single‐base mismatch of triplex DNA without denaturation. Compared with current methods, our method is characterized by high sensitivity, good universality and small sample and reagent requirements. More importantly, our method has the potential to become a platform for triplex DNA research in vitro. Copyright © 2015 John Wiley & Sons, Ltd.     

Characterizing specific phage-protein interactions by fluorescence correlation spectroscopy

The interactions of several affinity reagent displayed T7 and M13 phage particles with their corresponding target molecules were examined using Fluorescence Correlation Spectroscopy (FCS). Diffusion times, relative fractions of each component in the recognition reactions at the equilibrium state, and ultimately the dissociation constants were deduced from analyzing the fluorescence autocorrelation curves. Although the sample preparation and FCS characterization of icosahedral T7-related systems were relatively straight forward, procedures with filamentous M13-related systems were complicated by the physical size of M13 and its aggregate formation. Methods that accommodate the FCS measurement of the M13 phage via changing confocal optics, fitting procedures, and aggregate discrimination are presented and discussed.     

Fluorescence correlation spectroscopy in the nanosecond time range: Photon antibunching in dye fluorescence

Possibilities for the use of fluorescence correlation spectroscopy in the nanosecond time range are demonstrated. The experiment is based on a cw argon ion laser, a microfluorimeter, two photon detectors, and a time-to-analog converting system. Experiments using solutions of rhodamine 6G and pyronine G in water at concentrations of about 20 molecules per sample volume are reported. The photon anticorrelation component decaying with a time constant close to the excited state lifetime was observed.     

Study of protein aggregation using two-dimensional correlation infrared spectroscopy and spectral simulations

Two-dimensional (2D) correlation spectroscopy establishes correlations between intensity variations in a series of spectra obtained by the application of an external perturbation. However, spectral effects (wavenumber shift or bandwidth change) are known to generate apparent asynchronisms in 2D maps. Surprisingly, spectral effects are often neglected in the literature when interpreting experimental maps, which can lead to erroneous conclusions. In an attempt to evaluate the contribution of these effects and that of true asynchronisms on 2D maps, the heat-induced aggregation of glutamyl-tRNA synthetase (GluRS) was studied as a typical example of the application of Fourier transform infrared (FTIR) spectroscopy in the amide I region. The data were compared with those obtained from a mutant protein that differs by one amino acid. To determine whether the aggregation mechanisms are identical for both proteins, the experimental 2D maps were compared to simulations based on curve fitting of the initial and final spectra of the series, which allows change in position and bandwidth of the components to be taken into account. Intermediate spectra were generated using a convenient function that mimics the spectral evolution. The speed and the delay of each component were controlled. Apart from the appearance of turns that occur for the mutant and not for GluRS, the aggregation mechanisms of both proteins seems to be essentially identical. In particular, the loss of alpha-helices seems to be concomitant with the formation of intermolecular beta-sheets, whereas the loss of intramolecular beta-sheets is delayed. Since the experimental maps are satisfactorily simulated when almost all the components are in phase, it appears that many of the asynchronous features are mainly due to spectral effects. Thus, one has to be aware that true asynchronisms are not necessarily at the origin of peaks observed in asynchronous maps.     

High‐throughput screening of optimal solution conditions for structural biological studies by fluorescence correlation spectroscopy

Protein aggregation is an essential molecular event in a wide variety of biological situations, and is a causal factor in several degenerative diseases. The aggregation of proteins also frequently hampers structural biological analyses, such as solution NMR studies. Therefore, precise detection and characterization of protein aggregation are of crucial importance for various research fields. In this study, we demonstrate that fluorescence correlation spectroscopy (FCS) using a single‐molecule fluorescence detection system enables the detection of otherwise invisible aggregation of proteins at higher protein concentrations, which are suitable for structural biological experiments, and consumes relatively small amounts of protein over a short measurement time. Furthermore, utilizing FCS, we established a method for high‐throughput screening of protein aggregation and optimal solution conditions for structural biological experiments.     

Investigation on the effect of fluorescence quenching of bovine serum albumin by cefoxitin sodium using fluorescence spectroscopy and synchronous fluorescence spectroscopy

The reaction mechanism of cefoxitin sodium with bovine serum albumin was investigated using fluorescence spectroscopy and synchronous fluorescence spectroscopy at different temperatures. The results showed that the change of binding constant of the synchronous fluorescence method with increasing temperature could be used to estimate the types of quenching mechanisms of drugs with protein and was consistent with one of fluorescence quenching method. In addition, the number of binding sites, type of interaction force, cooperativity between drug and protein and energy‐transfer parameters of cefoxitin sodium and bovine serum albumin obtained from two methods using the same equation were consistent. Electrostatic force played a major role in the conjugation reaction between bovine serum albumin and cefoxitin sodium, and the type of quenching was static quenching. The primary binding site for cefoxitin sodium was sub‐hydrophobic domain IIA, and the number of binding sites was 1. The value of Hill's coefficients (n_H) was approximately equal to 1, which suggested no cooperativity in the bovine serum albumin–cefoxitin sodium system. The donor‐to‐acceptor distance r < 7 nm indicated that static fluorescence quenching of bovine serum albumin by cefoxitin sodium was also a non‐radiation energy‐transfer process. The results indicated that synchronous fluorescence spectrometry could be used to study the reaction mechanism between drug and protein, and was a useful supplement to the conventional method. Copyright © 2015 John Wiley & Sons, Ltd.     

Dynamics of unfolded polypeptide chains in crowded environment studied by fluorescence correlation spectroscopy

Proteins have evolved to fold and function within a cellular environment that is characterized by high macromolecular content. The earliest step of protein folding represents intrachain contact formation of amino acid residues within an unfolded polypeptide chain. It has been proposed that macromolecular crowding can have significant effects on rates and equilibria of biomolecular processes. However, the kinetic consequences on intrachain diffusion of polypeptides have not been tested experimentally, yet. Here, we demonstrate that selective fluorescence quenching of the oxazine fluorophore MR121 by the amino acid tryptophan (Trp) in combination with fast fluorescence correlation spectroscopy (FCS) can be used to monitor end-to-end contact formation rates of unfolded polypeptide chains. MR121 and Trp were incorporated at the terminal ends of polypeptides consisting of repetitive units of glycine (G) and serine (S) residues. End-to-end contact formation and dissociation result in "off" and "on" switching of MR121 fluorescence and underlying kinetics can be revealed in FCS experiments with nanosecond time resolution. We revisit previous experimental studies concerning the dependence of end-to-end contact formation rates on polypeptide chain length, showing that kinetics can be described by Gaussian chain theory. We further investigate effects of solvent viscosity and temperature on contact formation rates demonstrating that intrachain diffusion represents a purely diffusive, entropy-controlled process. Finally, we study the influence of macromolecular crowding on polypeptide chain dynamics. The data presented demonstrate that intrachain diffusion is fast in spite of hindered diffusion caused by repulsive interactions with macromolecules. Findings can be explained by effects of excluded volume reducing chain entropy and therefore accelerating the loop search process. Our results suggest that within a cellular environment the early formation of structural elements in unfolded proteins can still proceed quite efficiently in spite of hindered diffusion caused by high macromolecular content.     

Fluorescence correlation spectroscopy and quantitative cell biology

Fluorescence correlation spectroscopy (FCS) analyzes fluctuations in fluorescence within a small observation volume. Autocorrelation analysis of FCS fluctuation data can be used to measure concentrations, diffusion properties, and kinetic constants for individual fluorescent molecules. Photon count histogram analysis of fluorescence fluctuation data can be used to study oligomerization of individual fluorescent molecules. If the FCS observation volume is positioned inside a living cell, these parameters can be measured in vivo. FCS can provide the requisite quantitative data for analysis of molecular interaction networks underlying complex cell biological processes.     

Investigation on the interaction of cefpirome sulfate with lysozyme by fluorescence quenching spectroscopy and synchronous fluorescence spectroscopy

The reaction mechanism of cefpirome sulfate with lysozyme at different temperatures (298, 310 and 318 K) was investigated using fluorescence quenching and synchronous fluorescence spectroscopy under simulated physiological conditions. The results clearly demonstrated that cefpirome sulfate caused strong quenching of the fluorescence of lysozyme by a static quenching mechanism. The binding constants obtained using the above methods were of the same order of magnitude and very similar. Static electric forces played a key role in the interaction between cefpirome sulfate and lysozyme, and the number of binding sites in the interaction was close to 1. The values of Hill's coefficients were > 1, indicating that drugs or proteins showed a very weakly positive cooperativity in the system. In addition, the conclusions obtained from the two methods using the same equation were consistent. The results indicated that synchronous fluorescence spectrometry could be used to study the binding mechanism between drug and protein, and was a useful supplement to the fluorescence quenching method. In addition, the effect of cefpirome sulfate on the secondary structure of lysozyme was analyzed using circular dichroism spectroscopy. Copyright © 2015 John Wiley & Sons, Ltd.     

Protein aggregation processes: In search of the mechanism

Amyloid formation typically follows a time course in which there is a long lag period followed by a rapid formation of fibrils. In this review, I show that the standard mechanisms of polymerization need to be expanded to consider that the monomeric proteins/peptides involved in amyloid formation are intrinsically disordered and exist as an ensemble of disordered-collapsed states. The review focuses primarily on events which occur in the long lag period defining these as protein folding issues, coupled with formation of oligomers. Experimental methods to explore folding and oligomerization issues over a wide range of protein concentrations using primarily fluorescence and 19F-NMR methods are discussed.     

Self‐assembly of lipid rafts revealed by fluorescence correlation spectroscopy in living breast cancer cells

Lipids and proteins in the plasma membrane are laterally heterogeneous and formalised as lipid rafts featuring unique biophysical properties. However, the self‐assembly mechanism of lipid raft cannot be revealed even its physical properties and components were determined in specific physiological processes. In this study, two‐photon generalised polarisation imaging and fluorescence correlation spectroscopy were used to study the fusion of lipid rafts through the membrane phase and the lateral diffusion of lipids in living breast cancer cells. A self‐assembly model of lipid rafts associated with lipid diffusion and membrane phase was proposed to demonstrate the lipid sorting ability of lipid rafts in the plasma membrane. The results showed that the increased proportion of slow subdiffusion of G_M1‐binding cholera toxin B‐subunit (CT‐B) was accompanied with an increased liquid‐ordered domain during the β‐estradiol‐induced fusion of lipid rafts. And slow subdiffusion of CT‐B was vanished with the depletion of lipid rafts. Whereas the dialkylindocarbocyanine (DiIC₁₈) diffusion was not specifically regulated by lipid rafts. This study will open up a new insight for uncovering the self‐assembly of lipid rafts in specific pathophysiological processes.     

Probing receptor-translocator interactions in the oligopeptide ABC transporter by fluorescence correlation spectroscopy

The oligopeptide transporter Opp is a five-component ABC uptake system. The extracytoplasmic lipid-anchored substrate-binding protein (or receptor) OppA delivers peptides to an integral membrane complex OppBCDF (or translocator), where, on ATP binding and hydrolysis, translocation across the membrane takes place. OppA and OppBCDF were labeled with fluorescent probes, reconstituted into giant unilamellar vesicles, and the receptor-translocator interactions were investigated by fluorescence correlation spectroscopy. Lateral mobility of OppA was reduced on incorporation of OppBCDF into giant unilamellar vesicles, and decreased even further on the addition of peptide. Fluorescence cross-correlation measurements revealed that OppBCDF distinguished liganded from unliganded OppA, binding only the former. Addition of ATP or its nonhydrolyzable analog AMP-PNP resulted in release of OppA from OppBCDF. In vanadate-trapped “transition state” conditions, OppA also was not bound by OppBCDF. A model is presented in which ATP-binding to OppDF results in donation of peptide to OppBC and simultaneous release of OppA. ATP-hydrolysis would complete the peptide translocation and reset the transporter for another catalytic cycle. Implications in terms of a general transport mechanism for ABC importers and exporters are discussed.     

Fluorescence correlation spectroscopy in the nanosecond time range: rotational diffusion of bovine carbonic anhydrase B

A fluorescence correlation experiment for measurement of rotational diffusion in the nanosecond time scale is described. Using this method, the rotational diffusion coefficient of bovine carbonic anhydrase B labelled with tetramethylrhodamine isothiocyanate was estimated to be D _r=(1.14±0.15)×10⁷ s^-1 at 22°C. The experiment is based on a cw argon ion laser, a microfluorimeter with local solution flow inside the sample cell, and two photon detectors. The fluorescence intensity autocorrelation function in the nanosecond time range is computed with the help of a time-to-amplitude converter and a multichannel pulse-amplitude analyser.     

Counting nucleosomes in living cells with a combination of fluorescence correlation spectroscopy and confocal imaging

Although methods for light microscopy of chromatin are well established, there are no quantitative data for nucleosome concentrations in vivo. To establish such a method we used a HeLa clone expressing the core histone H2B fused to the enhanced yellow fluorescent protein (H2B-EYFP). Quantitative gel electrophoresis and fluorescence correlation spectroscopy (FCS) of isolated oligonucleosomes show that 5% of the total H2Bs carry the fluorescent tag and an increased nucleosome repeat length of 204 bp for the fluorescent cells. In vivo, the mobility and distribution of H2B-EYFP were studied with a combination of FCS and confocal imaging. With FCS, concentration and brightness of nascent molecules were measured in the cytoplasm, while in the nucleoplasm a background of mobile fluorescent histones was determined by continuous photobleaching. Combining these results allows converting confocal fluorescence images of nuclei into calibrated nucleosome density maps. Absolute nucleosome concentrations in interphase amount up to 250 microM locally, with mean values of 140(+/-28)microM, suggesting that a condensation-controlled regulation of site accessibility takes place at length scales well below 200 nm.     

Characterizing the inhibition of α‐synuclein oligomerization by a pharmacological chaperone that prevents prion formation by the protein PrP

Aggregation of the disordered protein α‐synuclein into amyloid fibrils is a central feature of synucleinopathies, neurodegenerative disorders that include Parkinson's disease. Small, pre‐fibrillar oligomers of misfolded α‐synuclein are thought to be the key toxic entities, and α‐synuclein misfolding can propagate in a prion‐like way. We explored whether a compound with anti‐prion activity that can bind to unfolded parts of the protein PrP, the cyclic tetrapyrrole Fe‐TMPyP, was also active against α‐synuclein aggregation. Observing the initial stages of aggregation via fluorescence cross‐correlation spectroscopy, we found that Fe‐TMPyP inhibited small oligomer formation in a dose‐dependent manner. Fe‐TMPyP also inhibited the formation of mature amyloid fibrils in vitro, as detected by thioflavin T fluorescence. Isothermal titration calorimetry indicated Fe‐TMPyP bound to monomeric α‐synuclein with a stoichiometry of 2, and two‐dimensional heteronuclear single quantum coherence NMR spectra revealed significant interactions between Fe‐TMPyP and the C‐terminus of the protein. These results suggest commonalities among aggregation mechanisms for α‐synuclein and the prion protein may exist that can be exploited as therapeutic targets.