Photodynamics of Red Fluorescent Proteins Studied by Fluorescence Correlation Spectroscopy

Red fluorescent proteins are important tools in fluorescence-based life science research. Recently, we have introduced eqFP611, a red fluorescent protein with advantageous properties from the sea anemone Entacmaea quadricolor. Here, we have studied the submillisecond light-driven intramolecular dynamics between bright and dark states of eqFP611 and, for comparison, drFP583 (DsRed) by using fluorescence correlation spectroscopy on protein solutions. A three-state model with one dark and two fluorescent states describes the power-dependence of the flickering dynamics of both proteins at different excitation wavelengths. It involves two light-driven conformational transitions. We have also studied the photodynamics of individual (monomeric) eqFP611 molecules immobilized on surfaces. The flickering rates and dark state fractions of eqFP611 bound to polyethylene glycol-covered glass surfaces were identical to those measured in solution, showing that the bound FPs behaved identically. A second, much slower flickering process was observed on the 10-ms timescale. Deposition of eqFP611 molecules on bare glass surfaces yielded bright fluorescence without any detectable flickering and a >10-fold decreased photobleaching yield. These observations underscore the intimate connection between protein motions and photophysical processes in fluorescent proteins.     

Hydrodynamic Properties of Human Adhesion/Growth-Regulatory Galectins Studied by Fluorescence Correlation Spectroscopy

Fluorescence correlation spectroscopy is applied on homologous human lectins (i.e., adhesion/growth-regulatory galectins) to detect influence of ligand binding and presence of the linker peptide in tandem-repeat-type proteins on hydrodynamic properties. Among five tested proteins, lactose binding increased the diffusion constant only in the cases of homodimeric galectin-1 and the linkerless variant of tandem-repeat-type galectin-4. To our knowledge, the close structural similarity among galectins does not translate into identical response to ligand binding. Kinetic measurements show association and dissociation rate constants in the order of 1 to 10³ M⁻¹ s⁻¹ and 10⁻⁴ s⁻¹, respectively. Presence of the linker peptide in tandem-repeat-type protein leads to anomalous scaling with molecular mass. These results provide what we believe to be new insights into lectin responses to glycan binding, detectable so far only by small angle neutron scattering, and the structural relevance of the linker peptide. Methodologically, fluorescence correlation spectroscopy is shown to be a rather simple technical tool to characterize hydrodynamic properties of these proteins at a high level of sensitivity.     

Single-Molecule Analysis of Restriction DNA Fragments Using Fluorescence Correlation Spectroscopy

The cleavage of fluorescence-labeled M13DNA (7250 bp) usingHaeIII,HgaI,BsmAI, andBspMI was analyzed by fluorescence correlation spectroscopy (FCS) in a small volume (1.5 × 10⁻¹⁵liters). The digestion process can be monitored by the decrease in amplitude of the fluorescence correlation function while the original DNA molecule is divided into several fragments by the enzymes. To analyze this reaction by FCS, we derived a practical equation for estimating the number of molecules in the FCS measurements. Under standard enzymatic conditions,HaeIII andBsmAI digested fluorescence-labeled DNA to completion in the range of 8 h, whereasHgaI andBspMI digested the DNA after 40 h. The comparison of recognition sequences suggested that some tagged nucleotides could be inserted between the recognition site and the cleavage site of the slow enzyme group. The decrease in amplitude in the fluorescence correlation function quantitatively monitors the hydrolysis of DNA during the digestion process.     

Suppression of Exonucleolytic Degradation of Double-Stranded DNA and Inhibition of Exonuclease III by PNA

Abstract

Degradation of double-stranded DNA by Exonuclease III in the presence of complementary anti-parallel PNA was studied. It was found for the first time that the PNA suppresses the degradation of dsDNA in a sequence-specific manner as well as inhibits the activity of Exonuclease III in a non-specific way.     

荧光相关谱技术及其应用

基于对处于平衡态少量荧光分子集合的强度涨落进行时间平均的技术,荧光相关谱fluoreswceance correlation spectroscopy,FCS)技术最近已经应用于细胞环境过程的研究。FCS优秀的灵敏特性为我们实时测量许多参数提供了途径,而且具有快速的时间特性和高空间分辨率。测量的参数包括扩散速率、局部浓度、聚合状态和分子间的相互作用。荧光互相关谱(fluorescence cross-correlation spectroscopy,FCCS)进一步扩展了FCS技术的应用,包括在活细胞中的广泛应用。本文介绍了FCS技术的原理、实验装置及其应用。     

Tilt and Rotation Angles of a Transmembrane Model Peptide as Studied by Fluorescence Spectroscopy

In this study the membrane orientation of a tryptophan-flanked model peptide, WALP23, was determined by using peptides that were labeled at different positions along the sequence with the environmentally sensitive fluorescent label BADAN. The fluorescence properties, reflecting the local polarity, were used to determine the tilt and rotation angles of the peptide based on an ideal α-helix model. For WALP23 inserted in dioleoylphosphatidylcholine (DOPC), an estimated tilt angle of the helix with respect to the bilayer normal of 24° ± 5° was obtained. When the peptides were inserted into bilayers with different acyl chain lengths or containing different concentrations of cholesterol, small changes in tilt angle were observed as response to hydrophobic mismatch, whereas the rotation angle appeared to be independent of lipid composition. In all cases, the tilt angles were significantly larger than those previously determined from ²H NMR experiments, supporting recent suggestions that the relatively long timescale of ²H NMR measurements may result in an underestimation of tilt angles due to partial motional averaging. It is concluded that although the fluorescence technique has a rather low resolution and limited accuracy, it can be used to resolve the discrepancies observed between previous ²H NMR experiments and molecular-dynamics simulations.     

抗癌药物ADM与DNA相互作用的紫外共振拉曼光谱的研究

用紫外共振拉曼光谱研究了ＡＤＭ与小牛胸腺ＤＮＡ相互作用,分析表明：ＡＤＭ插入ＤＮＡ的ＧＣ－ＣＧ位置;ＡＤＭ与ＤＮＡ之间的主要相互作用是蒽环π电子与碱基Ｇ和Ｃ的π电子形成的π－π电子相互作用,并通过碱基Ｇ、Ｃ的ＮＨ＿２的氨的孤对Ｐ电子与ＡＤＭ的π电子形成的π－Ｐ电子相互作用以及ＡＤＭ和ＤＮＡ碱基Ｇ、Ｃ和ＰＯ＿２之间形成的氢键使相互作用加强;ＡＤＭ插入ＤＮＡ使其构象产生一定变化,但未破坏ＤＮＡ碱基对间的氢键。     

基于双光子激发的荧光相关谱测量

本文利用多光子激发激光扫描显微镜的部分光路和探测器．建立了双光子荧光相关谱系统(Two-Photo Fluorescence Correlation Spectroscopy．简称TP-FCS)。利用TP-FCS系统观察到了“光子爆发”现象．实现了染料分子的双光子激发,测量出若丹明B染料分子在蔗糖溶液中的扩散系数。实验证明该系统具有操作简便、可靠性高,费用低廉等等点,可实现单分子检测。     

荧光相关光谱在生物化学领域中的应用

荧光相关光谱(fluorescence correlation spectroscopy,FCS)是一种通过监测荧光涨落从而获得单分子水平的分子扩散行为信息的技术。FCS高灵敏度的优点使得它已发展成为一种可以在活体外与活体内检测分子浓度、扩散系数、结合和解离常数等参数的有力工具。荧光互相关光谱(fluorescence cross-correlation spectroscopy,FCCS)是FCS技术的进一步发展,其大大扩展了FCS技术的应用范围。本文介绍了FCS及其衍生技术的原理及其在生物化学领域的应用。     

Fluorescence Correlation Spectroscopy: The Case of Subdiffusion

The theory of fluorescence correlation spectroscopy is revisited here for the case of subdiffusing molecules. Subdiffusion is assumed to stem from a continuous-time random walk process with a fat-tailed distribution of waiting times and can therefore be formulated in terms of a fractional diffusion equation (FDE). The FDE plays the central role in developing the fluorescence correlation spectroscopy expressions, analogous to the role played by the simple diffusion equation for regular systems. Due to the nonstationary nature of the continuous-time random walk/FDE, some interesting properties emerge that are amenable to experimental verification and may help in discriminating among subdiffusion mechanisms. In particular, the current approach predicts 1), a strong dependence of correlation functions on the initial time (aging); 2), sensitivity of correlation functions to the averaging procedure, ensemble versus time averaging (ergodicity breaking); and 3), that the basic mean-squared displacement observable depends on how the mean is taken.     

Pin-Hole Array Correlation Imaging: Highly Parallel Fluorescence Correlation Spectroscopy

In this work, we describe pin-hole array correlation imaging, a multipoint version of fluorescence correlation spectroscopy, based upon a stationary Nipkow disk and a high-speed electron multiplying charged coupled detector. We characterize the system and test its performance on a variety of samples, including 40 nm colloids, a fluorescent protein complex, a membrane dye, and a fluorescence fusion protein. Our results demonstrate that pin-hole array correlation imaging is capable of simultaneously performing tens or hundreds of fluorescence correlation spectroscopy-style measurements in cells, with sufficient sensitivity and temporal resolution to study the behaviors of membrane-bound and soluble molecules labeled with conventional chemical dyes or fluorescent proteins.     

Exonuclease proofreading by human mitochondrial DNA polymerase

We have examined the ability of the human mitochondrial DNA polymerase to correct errors in DNA sequence using single turnover kinetic methods. The rate of excision of single-stranded DNA ranged from 0.07 to 0.17 x s(-1), depending on the identity of the 3'-base. Excision of the 3'-terminal base from correctly base paired DNA occurred at a rate of 0.05 x s(-1), indicating that the cost of proofreading is minimal, as defined by the ratio of the k(exo) for correctly base-paired DNA divided by the rate of forward polymerization (0.05/37 = 0.14%). Excision of duplex DNA containing 1-7 mismatches was biphasic, and the rate and amplitude of the fast phase increased with the number of mismatches, reaching a maximum of 9 x s(-1). We showed that transfer of DNA from the polymerase to the exonuclease active site and back again occurs through an intramolecular reaction, allowing for a complete cycle of reactions for error correction. For DNA containing a buried mismatch (T:T followed by C:G base pairs), the 3' base was removed at a rate of 3 x s(-1). The addition of nucleotide to the reaction that is identical to the 3' base increased the rate of excision 7-fold to 21 x s(-1). We propose that the free nucleotide enhances the rate of transfer of the DNA to the exonuclease active site by interrupting the correct 3' base pair through interaction with the template base. The exonuclease contribution to fidelity is minimal if the calculation is based on hydrolysis of a single mismatch: (k(exo) + k(pol,over))/(k(pol,over)) = 10, but this value increases to approximately 200 when examining error correction in the presence of nucleotides.     

Direct Observation of T4 Lysozyme Hinge-Bending Motion by Fluorescence Correlation Spectroscopy

Bacteriophage T4 Lysozyme (T4L) catalyzes the hydrolysis of the peptidoglycan layer of the bacterial cell wall late in the infection cycle. It has long been postulated that equilibrium dynamics enable substrate access to the active site located at the interface between the N- and C-terminal domains. Crystal structures of WT-T4L and point mutants captured a range of conformations that differ by the hinge-bending angle between the two domains. Evidence of equilibrium between open and closed conformations in solution was gleaned from distance measurements between the two domains but the nature of the equilibrium and the timescale of the underlying motion have not been investigated. Here, we used fluorescence fluctuation spectroscopy to directly detect T4L equilibrium conformational fluctuations in solution. For this purpose, Tetramethylrhodamine probes were introduced at pairs of cysteines in regions of the molecule that undergo relative displacement upon transition from open to closed conformations. Correlation analysis of Tetramethylrhodamine intensity fluctuations reveals hinge-bending motion that changes the relative distance and orientation of the N- and C-terminal domains with ≅15 μs relaxation time. That this motion involves interconversion between open and closed conformations was further confirmed by the dampening of its amplitude upon covalent substrate trapping. In contrast to the prevalent two-state model of T4L equilibrium, molecular brightness and number of particles obtained from cumulant analysis suggest that T4L populates multiple intermediate states, consistent with the wide range of hinge-bending angles trapped in the crystal structure of T4L mutants.     

Anomalous Diffusion in Inverted Variable-Lengthscale Fluorescence Correlation Spectroscopy

Using fluorescence correlation spectroscopy (FCS) to distinguish between different types of diffusion processes is often a perilous undertaking because the analysis of the resulting autocorrelation data is model dependant. Two recently introduced strategies, however, can help move toward a model-independent interpretation of FCS experiments: 1) the obtention of correlation data at different length scales and 2) their inversion to retrieve the mean-squared displacement associated with the process under study. We use computer simulations to examine the signature of several biologically relevant diffusion processes (simple diffusion, continuous-time random walk, caged diffusion, obstructed diffusion, two-state diffusion, and diffusing diffusivity) in variable-length-scale FCS. We show that, when used in concert, length-scale variation and data inversion permit us to identify non-Gaussian processes and, regardless of Gaussianity, to retrieve their mean-squared displacement over several orders of magnitude in time. This makes unbiased discrimination between different classes of diffusion models possible.     

Fluorescence Correlation Spectroscopy Measures Molecular Transport in Cells

Fluorescence correlation spectroscopy (FCS) can measure dynamics of fluorescent molecules in cells. FCS measures the fluctuations in the number of fluorescent molecules in a small volume illuminated by a thin beam of excitation light. These fluctuations are processed statistically to yield an autocorrelation function from which rates of diffusion, convection, chemical reaction, and other processes can be extracted. The advantages of this approach include the ability to measure the mobility of a very small number of molecules, even down to the single molecule level, over a wide range of rates in very small regions of a cell. In addition to rates of diffusion and convection, FCS also provides unique information about the local concentration, states of aggregation and molecular interaction using fluctuation amplitude and cross-correlation methods. Recent advances in technology have rendered these once difficult measurements accessible to routine use in cell biology and biochemistry. This review provides a summary of the FCS method and describes current areas in which the FCS approach is being extended beyond its original scope.     

荧光相关谱测量技术研究

荧光相关谱(fluorescence correlation spectroscopy,FCS)是对处于热平衡态条件下的荧光分子发出的荧光强度涨落进行时间相关处理的一种单分子检测方法,能够直接测量分子在溶液里的扩散系数和浓度.影响FCS测量扩散系数准确性的因素有分子量子效率,测量时间,样本折射率和温度偏差等.用FCS分别测量溶有荧光染料罗丹明6G(rhodamine 6G,Rh.6G)和青色素Cy5甘油水溶液的粘滞系数,实验结果表明:荧光分子的量子效率是影响测量准确性的重要因素,要求其每秒发射的光子数目(photon counts per second,cps)至少达到1 000(photons/s).     

Sucrose monoester micelles size determined by Fluorescence Correlation Spectroscopy (FCS)

One of the several uses of sucrose detergents, as well as other micelle forming detergents, is the solubilization of different membrane proteins. Accurate knowledge of the micelle properties, including size and shape, are needed to optimize the surfactant conditions for protein purification and membrane characterization. We synthesized sucrose esters having different numbers of methylene subunits on the substituent to correlate the number of methylene groups with the size of the corresponding micelles. We used Fluorescence Correlation Spectroscopy (FCS) and two photon excitation to determine the translational D of the micelles and calculate their corresponding hydrodynamic radius, R(h). As a fluorescent probe we used LAURDAN (6-dodecanoyl-2-dimethylaminonaphthalene), a dye highly fluorescent when integrated in the micelle and non-fluorescent in aqueous media. We found a linear correlation between the size of the tail and the hydrodynamic radius of the micelle for the series of detergents measured.