Sensing Tryptophan Microenvironment of Amyloid Protein Utilizing Wavelength-Selective Fluorescence Approach

Structural transition among various forms of proteins involves subtle interplay between structure and dynamics and is crucial in human diseases. Red edge excitation shift (REES) represents a suitable approach to explore the environmental organization and dynamics surrounding tryptophan residues in proteins. Although REES from tryptophan residues has been reported for native, molten globule and denatured states of proteins, such data on the amyloid form of proteins is lacking. κ-casein is one of the most important constituents of casein micelles in milk and has a tendency to form amyloid fibril. We report here REES of the sole tryptophan residue for native, acid-denatured and urea-denatured forms of κ-casein. More importantly, we show that the amyloid form of κ-casein displays REES of 4 nm. We analyze these results in terms of tryptophan microenvironment in various forms of κ-casein, particularly the amyloid form. We conclude that REES is a sensitive tool to monitor structural plasticity in proteins.     

Origin of Tryptophan Fluorescence Lifetimes. Part 2: Fluorescence Lifetimes Origin of Tryptophan in Proteins

Fluorescence intensity decays of L-tryptophan in proteins dissolved in pH 7 buffer, in ethanol and in 6 M guanidine pH 7.8 and in lyophilized proteins were measured. In all protein conditions, three lifetimes were obtained along the emission spectrum (310–410 nm). The two shortest lifetimes are in the same range of those obtained for L-Trp in water or in ethanol. Thus, these two lifetimes originate from specific two sub-structures existing in the excited state and are inherent to the tryptophan structure independently of the surrounding environment (amino acids residues, solvent, etc.) In proteins, the third lifetime originates from the interactions that are occurring between tryptophan residues and neighboring amino acids. Populations of these lifetimes are independent of the excitation wavelength and thus originate from pre-defined sub structures existing in the excited state and put into evidence after tryptophan excitation. Fluorescence decay studies of different tripeptides having a tryptophan residue in second position show that the best analysis is obtained with two fluorescence lifetimes. Consequently, this result seems to exclude the possibility that peptide bond induces the third fluorescence lifetimes. Indole dissolved in water and/or in ethanol emits with two fluorescence lifetimes that are completely different from those observed for L-Trp. Absence of the third lifetime in ethanol demonstrates that indole behaves differently when compared to tryptophan. Thus, it seems not adequate to attribute fluorescence lifetime or fluorescence properties of tryptophan to indole ring and to compare tryptophan fluorescence properties in proteins to molecules having close structures such as NATA which fluoresces with one lifetime.     

Origin of Tryptophan Fluorescence Lifetimes Part 1. Fluorescence Lifetimes Origin of Tryptophan Free in Solution

Fluorescence intensity decays of L-tryptophan free in polar, hydrophobic and mixture of polar-hydrophobic solvents were recorded along the emission spectrum (310–410 nm). Analysis of the data show that emission of tryptophan occurs with two lifetimes in 100 % polar and hydrophobic environments. The values of the two lifetimes are not the same in both environments while their populations (pre-exponentials values) are identical. Fluorescence lifetimes and pre-exponentials values do not change with the excitation wavelength and thus are independent of excitation energy. Our results indicate that tryptophan emission occurs from two specific sub-structures existing in the excited state. These sub-structures differ from those present in the ground states and characterize an internal property and/or organization of the tryptophan structure in the excited state. By sub-substructure, we mean here tryptophan backbone and its electronic cloud. In ethanol, three fluorescence lifetimes were measured; two lifetimes are very close to those observed in water (0.4–0.5 ns and 2–4 ns). Presence of a third lifetime for tryptophan in ethanol results from the interaction of both hydrophobic and hydrophilic dipoles or chemical functions of ethanol with the fluorophore.     

Multi-Photon Fluorescence Correlation Spectroscopy: a Quantification of Tryptophan Methylester Solutions by Visible Emission

A multi-photon excitation fluorescence correlation system has been developed. The emission from tryptophan methylester solution was observed by this system and analyzed by the intensity correlation function of the visible emission, which originates from the two-photon excitation of photo products generated through a five-photon process. The intensity and the product concentration were proportional to the concentration of tryptophan methylester at a lower concentration range and thus the generation process is a single molecular reaction. The correlation analysis determined the concentration of tryptophan methylester down to 5 μM. The photo product generation from tryptophan solution was enhanced by a potassium iodide addition. These results suggest a new quantification method of tryptophan derivatives.     

Thermal Effects of Interfacial Dynamics

Dynamical Ginzburg-Landau theory is applied to the study of thermal effects of motion of interfaces that appear after different phase transitions. These effects stem from the existence of the surface internal energy, entropy and temperature gradients in the interfacial transition region. Evolution equations for the interfacial motion are derived. For the experimental verification of the thermal effects the expression is derived for the amplitude of temperature waves during continuous ordering.     

New Insights in the Interpretation of Tryptophan Fluorescence

Origin of tryptophan fluorescence is still up to these days a quiz which is not completely solved. Fluorescence emission properties of tryptophan within proteins are in general considered as the result of fluorophore interaction within its environment. For example, a low fluorescence quantum yield is supposed to be the consequence of an important fluorophore–environment interaction. However, are we sure that the fluorophore has been excited upon light absorption? What if fluorophore excitation did not occur as the result of internal conformation specific to the fluorophore environment? Are we sure that all absorbed energy is used for the excitation process? Fluorescence lifetimes of Trp residues are considered to originate from rotamers or conformers resulting from the rotation of the indole ring within the peptide bonds. However, how can we explain the fact that in most of the proteins, the two lifetimes 0.5 and 3 ns, attributed to the conformers, are also observed for free tryptophan in solution? The present work, performed on free tryptophan and tyrosine in solution and on different proteins, shows that absorption and excitation spectra overlap but their intensities at the different excitation wavelengths are not necessarily equal. Also, we found that fluorescence emission intensities recorded at different excitation wavelengths depend on the intensities at these excitation wavelengths and not on the optical densities. Thus, excitation is not equal to absorption. In our interpretation of the data, we consider that absorbed photons are not necessary used only for the excitation, part of them are used to reorganize fluorophore molecules in a new state (excited structure) and another part is used for the excitation process. A new parameter that characterizes the ratio of the number of emitted photons over the real number of photons used to excite the fluorophore can be defined. We call this parameter, the emission to excitation ratio. Since our results were observed for fluorophores free in solution and present within proteins, structural reorganization does not depend on the protein backbone. Thus, fluorescence lifetimes (0.5 and 3 ns) observed for tryptophan molecules result from the new structures obtained in the excited state. Our theory allows opening a new way in the understanding of the origin of protein fluorescence and fluorescence of aromatic amino acids.     

Constrained Markovian Dynamics of Random Graphs

We introduce a statistical mechanics formalism for the study of constrained graph evolution as a Markovian stochastic process, in analogy with that available for spin systems, deriving its basic properties and highlighting the role of the ‘mobility’ (the number of allowed moves for any given graph). As an application of the general theory we analyze the properties of degree-preserving Markov chains based on elementary edge switchings. We give an exact yet simple formula for the mobility in terms of the graph’s adjacency matrix and its spectrum. This formula allows us to define acceptance probabilities for edge switchings, such that the Markov chains become controlled Glauber-type detailed balance processes, designed to evolve to any required invariant measure (representing the asymptotic frequencies with which the allowed graphs are visited during the process). As a corollary we also derive a condition in terms of simple degree statistics, sufficient to guarantee that, in the limit where the number of nodes diverges, even for state-independent acceptance probabilities of proposed moves the invariant measure of the process will be uniform. We test our theory on synthetic graphs and on realistic larger graphs as studied in cellular biology, showing explicitly that, for instances where the simple edge swap dynamics fails to converge to the uniform measure, a suitably modified Markov chain instead generates the correct phase space sampling.     

Tryptophan Fluorescence Yields and Lifetimes as a Probe of Conformational Changes in Human Glucokinase

Five variants of glucokinase (ATP-D-hexose-6-phosphotransferase, EC 2.7.1.1) including wild type and single Trp mutants with the Trp residue at positions 65, 99, 167 and 257 were prepared. The fluorescence of Trp in all locations studied showed intensity changes when glucose bound, indicating that conformational change occurs globally over the entire protein. While the fluorescence quantum yield changes upon glucose binding, the enzyme’s absorption spectra, emission spectra and fluorescence lifetimes change very little. These results are consistent with the existence of a dark complex for excited state Trp. Addition of glycerol, L-glucose, sucrose, or trehalose increases the binding affinity of glucose to the enzyme and increases fluorescence intensity. The effect of these osmolytes is thought to shift the protein conformation to a condensed, high affinity form. Based upon these results, we consider the nature of quenching of the Trp excited state. Amide groups are known to quench indole fluorescence and amides of the polypeptide chain make interact with excited state Trp in the relatively unstructured, glucose-free enzyme. Also, removal of water around the aromatic ring by addition of glucose substrate or osmolyte may reduce the quenching.     

基于色氨酸本征荧光测量的生物气溶胶检测技术研究

生物气溶胶与环境质量和人类健康密切相关。基于光学测量的气溶胶检测技术具有快速、无损和灵敏的优点,是目前的研究主流。而对检测系统的标定技术及其评价方法尚无国家标准可依。基于本征荧光测量技术,研制完成了一套针对包括病毒气溶胶在内的生物气溶胶检测装置。并在此基础上,以色氨酸气溶胶作为被检物,提出了一种生物气溶胶检测与标定方法。实验结果表明,研制的检测装置对色氨酸气溶胶具良好的线性响应特性,线性相关系数R2≥0.99,灵敏度达到4000L-1。证明了通过测量气溶胶中色氨酸含量检测生物气溶胶技术的可行性与可靠性。还探讨了利用多通道本征荧光检测技术实现对生物粒子种类进行预判别的可行性。     

一种新型卟啉侧链聚合物的飞秒荧光动力学

研究了一种新型的卟啉侧链聚合物丙烯腈丙烯酸卟啉酯共聚物{poly[porphyrin acrylate-acrylonitrile](p[(por)A-AN]}的链间和链内的卟啉分子的相互作用对聚合物薄膜发光性质的影响。通过采用飞秒荧光光谱技术测量了p[(por)A-AN]薄膜的荧光动力学过程。测量结果表明:纯p[(por)A-AN]薄膜(~450ps)显示出了比混合物薄膜p[(por)A-AN]/polystyrene(PSE)(~1.3 ns)快得多的荧光弛豫过程。而p[(por)A-AN]/PSE混合物薄膜显示出较纯p[(por)A-AN]薄膜增强的荧光效率。增加p[(por)A-AN]分子内卟啉侧链基团的浓度导致纯p[(por)A-AN]薄膜和p[(por)A-AN]/PSE混合物薄膜的荧光效率的增强和寿命(由近26~36 ps)的增加。分子间和分子内卟啉侧链基团之间的无辐射能量转移和分子内卟啉侧链基团的旋转运动在p[(por)A-AN]的荧光动力学过程中起着重要的作用。     

色氨酸和酪氨酸的三维荧光光谱特征参量提取

氨基酸是维持生命活动的重要物质,而色氨酸和酪氨酸又是天然氨基酸中重要的发光组分,应用荧光光谱法对其进行测量和分辨具有重要的意义。文章用美国Pekin-Elmer LS55型荧光分光光度计,对色氨酸和酪氨酸的三维荧光光谱进行了测量。将测量的数据用激发-发射-荧光强度的三维坐标表示,得到三维荧光谱图,但色氨酸和酪氨酸存在共性峰,通过波峰位置简单地来辨别两种混叠的物质很有难度。以数理统计概念为基础,提取该三维荧光光谱的特征参数,得到两种物质荧光光谱中最相关的信息,可以解决两种物质光谱混叠的分辨问题。结果表明,色氨酸和酪氨酸的三维荧光光谱平均值、标准差、原点矩、混合中心矩等参数差值百分比分别为330.37%, 102.86%, 329.16%, 329.63%,区别较大;而边际分布、相关系数值差值百分比仅为10.61%和2.40%。因而平均值、标准差、原点矩、混合中心矩可作为敏感特征参数,用其分辨谱图混叠的色氨酸和酪氨酸是可行的。这种“数学预提取”的三维光谱分析法可以找出组分之间的敏感特征参量,能够取代传统的三维荧光光谱分析法。     

Fluorescence Dynamics of Three UV-B Sunscreens

Polarity of the surrounding medium affects the excited states of UV-B sunscreens. Therefore understanding excited state processes in a mixed polarity model system similar to skin is essential. We report the excited state lifetimes, quantum yields, radiative and non-radiative rates of three sunscreens. Among the three UV-B sunscreens studied, octyl salicylate emits from a single excited state, while padimate O and octyl methoxy cinnamate show multiple states. The radiative rates of salicylate and cinnamate are approximately constant, while that of padimate O depends strongly on solvent. The non-radiative rates of all sunscreens vary with solvent polarity. Compared to salicylate and cinnamate, padimate O is complex to analyze because of its two emission peaks and one peak’s strong dependence on the dielectric constant. High absorbance, broad absorption peak with small fluorescence quantum yield, and low radiative rate make octyl methoxy cinnamate a superior UV-B sunscreen ingredient. The complexity in excited-state analysis shows that the lifetimes of the sunscreens are critical parameters, in addition to absorbance and quantum yield. Fluorescence lifetime substantiates the use of polystyrene nanospheres as a model host to study the photo-physical properties of sunscreen in a heterogeneous environment.     

Fluorescence and Rotational Dynamics of Dityrosine

We have examined the lifetimes and rotational correlation times of dityrosine emission by time-correlated single-photon counting. We first noticed dityrosine fluorescence in samples of tyrosine and tyrosine dipeptides by its characteristic red-shifted emission at 400 to 430 nm. The longer rotational correlation time relative to tyrosine proved that this fluorescence emanated from a distinct species. Comparison with the fluorescence properties of synthesized dityrosine established the identity of the emitting species. Fluorescence intensity decays of dityrosine are generally characterized by two decay components, one with a lifetime in the range of 150 to 800 ps and another between 2.5 and 4.5 ns. We found no evidence for an excited-state reaction, since a rising phase (negative-amplitude component) was not observed. In the pH range from 4 to 10, two ground-state species exist in equilibrium with pK _a 7. Both species exhibit two fluorescence decays. The average fluorescence lifetime increases gradually with pH over the pH range from 4 to 10 and decreases at pH 2. Anisotropy decays were measured for dityrosine and the alanine–dityrosine–alanine and leucine–dityrosine–leucine dipeptides. The rotational correlation times of dityrosine and dityrosine dipeptides increase linearly with van der Waals volumes. The slope indicates a stronger solute–solvent interaction than predicted with stick boundary conditions. It is suggested that these interactions result from the presence of two zwitterionic pairs.     

Molecular Dynamics of Guest Molecules in Micelles: Models Based on Fluorescence Polarization Dynamics

Time-resolved fluorescence polarization (anisotropy) of a probe (guest molecule) in a micelle is used for testing different models of molecular dynamics. The experimental studies so far support the model that includes wobbling motion and translational diffusion for the guest molecule in the micelle.     

同型二聚体肽模型的二维红外光谱

利用一个同型二聚体模型,甲酰胺二聚体,模拟一对肽基团.用量子化学从头计算考察了二聚体中酰胺-I带振动模式之间的振动耦合及其在空间的行为. 研究发现C=O伸缩振动耦合以静电作用为主,耦合有效距离能超过10 oA. 一维和二维红外光谱的激子模拟计算表明,耦合常数的空间依赖性能够清楚地表现在光谱特征中. 这些结果意味着多肽中C=O伸缩振动模式能够在很远的距离相互耦合并产生振动态的离域化.     

A Note on the Quantization of Constrained Generalized Dynamics

It is proved that the canonical quatization starting from a singular first-order Lagrangian yields the same physical results as a second-order Lagrangian which differs from the original singular Lagrangian by a total-time derivative term for a system with finite degrees of freedom. A typical example is given. For the case in the field theories a brief discussion is given and an example is illustrated.     

Entanglement Dynamics of a Generic-Spin Model

We investigate the entanglement dynamics of a generic-spin model with weak external field. We first derive the time-dependent solutions of angular momentum operators with short-time approximation and then numerically calculate the entangled witness. It’s shown that one can dynamically generate quantum entanglement by adjusting coupling strength.     

含单个色氨酸的多肽分子的荧光特性研究:pH及金属离子的响应

由于金属离子包括铜离子对人体的重要性,报道了一种含单个色氨酸的新型多肽分子(WDAHSS),证明利用这种分子可以通过荧光光谱测量的方法实现铜离子灵敏检测。WDAHSS的荧光由其包含的色氨酸残基的本征荧光贡献,易被铜离子猝灭。通过分析铜离子在不同pH值条件下与WDAHSS作用的荧光光谱并与单个色氨酸分子的光谱相比较,详细研究了铜离子猝灭WDAHSS荧光的机理。研究表明,WDAHSS结构中的组氨酸通过金属配位与铜离子作用,并联合肽键形成稳固的四方形平面结构,螯合铜离子,致使色氨酸残基发生荧光猝灭。同时详细讨论了不同pH值环境对WDAHSS荧光光谱的影响。通过荧光光谱测量和数值拟合,推测了WDAHSS和铜离子的结合常数。为了增强WDAHSS抗pH干扰的能力,特意对其氨基端乙酰化,在生理pH值范围内稳定了其荧光发射。此外,WDAHSS也采用了一些特殊设计的结构,很好地增强了它对铜离子灵敏探测的特异选择性和生物相容性。对WDAHSS的进一步研究有望用于生物体内或细胞内荧光成像检测。     

The Asymmetric One-Dimensional Constrained Ising Model: Rigorous Results

We study a one-dimensional spin (interacting particle) system, with product Bernoulli (p) stationary distribution, in which a site can flip only when its left neighbor is in state +1. Such models have been studied in physics as simple exemplars of systems exhibiting slow relaxation. In our East model the natural conjecture is that the relaxation time (p), that is 1/(spectral gap), satisfies log (p) as p0. We prove this up to a factor of 2. The upper bound uses the Poincaré comparison argument applied to a wave (long-range) comparison process, which we analyze by probabilistic techniques. Such comparison arguments go back to Holley (1984, 1985). The lower bound, which atypically is not easy, involves construction and analysis of a certain coalescing random jumps process.