Phase diagrams and kinetics of phase transitions in protein solutions

The phase behavior of proteins is of interest for fundamental and practical reasons. The nucleation of new phases is one of the last major unresolved problems of nature. The formation of protein condensed phases (crystals, polymers, and other solid aggregates, as well as dense liquids and gels) underlies pathological conditions, plays a crucial role in the biological function of the respective protein, or is an essential part of laboratory and industrial processes. In this review, we focus on phase transitions of proteins in their properly folded state. We first summarize the recently acquired understanding of physical processes underlying the phase diagrams of the protein solutions and the thermodynamics of protein phase transitions. Then we review recent findings on the kinetics of nucleation of dense liquid droplets and crystals. We explore the transition from nucleation to spinodal decomposition for liquid-liquid separation and introduce the new concept of solution-to-crystal spinodal. We review the two-step mechanism of protein crystal nucleation, in which mesoscopic metastable protein clusters serve as precursors to the ordered crystal nuclei. The concepts and mechanisms reviewed here provide powerful tools for control of the nucleation process by varying the solution thermodynamic parameters.     

Crystallization of a globular protein in lipid cubic phase

We studied the crystallization of a globular protein, lysozyme, in the cubic phase of the lipid mono-olein. The solubility of lysozyme in salt solution decreased by a factor of approximately 4 when confined in cubic phase. Monte Carlo simulations and calculations show that this can be explained by the confinement of lysozyme molecules to the narrow water cells in the cubic phase.     

A change in the physical state of a nonequilibrium blood plasma protein film in patients with carcinoma

Pronounced changes in the physical phase and in the phase transition dynamics of proteins in drying blood plasma are observed. The morphologies (topologies) of these nonequilibrium films in donors and in patients with various types of metastatic carcinoma qualitatively differ by the process of protein film self-assembly and by symmetry type. New types of defects and solid crystals appear, liquid crystals persist for a long time, etc. The microscopic examination of the drying protein plasma topology can be used for diagnosing metastatic carcinoma.     

Protein crystal growth: An approach based on phase diagram determination

There is an increasing need to rationalize protein crystal growth, mostly carried out so far by trial-and-error. The phase rule gives the number of conditions necessary to specify the state of protein crystallization in equilibrium. Unsaturated and supersaturated regions are distinguished in phase diagrams. Examples of such diagrams for protein crystals are tabulated. To determine the phase diagrams reliably, the following items are discussed: (1) proper crystallization methods, (2) the length of time needed for establishing equilibrium, and (3) the importance of specifying the kind of solid state. Various crystallization methods are compared as to their effect on the solution system by using the phase diagrams, and the potential usefulness of the diagrams for crystallizing new proteins is discussed.     

On the existence of regular structures in liquid human blood serum (plasma) and phase transitions in the course of its drying

Experimental data show that regular three-dimensional liquid-crystal protein structures ranging in size from several hundredths to several tenths of a millimeter are present in the whole blood serum (plasma) of patients suffering from various diseases. When a drop of serum dries, some of the structures melt to produce a gel, whereas the rest of them undergo phase transition to form solid crystals. These crystals are shaped like immunoglobulin M molecules enlarged 1000-fold. In the serum (plasma) of ailing people, the amount of the gel formed upon drying increases, breaking the symmetry during the formation of nonequilibrium protein films. It is believed that low-intensity physical factors exert a therapeutic action by changing the phase state of protein in body fluids.     

Viscoelastic phase separation of protein solutions

In addition to the known behavior of normal phase separation and gelation, we report novel phase-separation behavior of protein solutions as their intermediate case. A network structure of the protein-rich phase may be formed even if it is the minority phase, contrary to the conventional wisdom. This behavior is characteristic of viscoelastic phase separation found in polymer solutions. This kinetic pathway may play crucial roles in the complex phase ordering of protein solutions, in particular, protein network formation in biological systems and foods.     

Pressure-induced phase changes of cubic monoolein - Cytochrome C mesophases

Incorporation of the protein cytochrome c (cyt c) into the hydrated bicontinuous Ia3d cubic mesophase of monoolein (MO) was investigated within a wide range of pressures by small-angle X-ray scattering (SAXS). We found that incorporation of cyt c into the cubic phase of MO has a drastic effect on the structure and pressure stability of the system: At high pressures, the lipid systems with less than 0.2 wt.% embedded protein undergo a transition to a fluid lamellar phase with smaller partial molar lipid volume. Incorporation of cyt c at levels above 0.2 wt.% promotes the formation of a new cubic phase, probably a cubic micellar phase of space group P4₃32 (Q²¹²) whose pressure stability rises with increasing protein content.     

Dye-laser threshold in a protein solution

We propose to study the cooperative effects in a system exhibiting a phase transition by measuring the temperature dependence of the threshold pumping power required for laser oscillation from organic dyes in the sample. In the present experiments, we have chosen a protein (lysozyme) as the substance with a phase transition. Comparing measurements of optical absorption, fluorescence and optical rotation spectra, we found two types of temperature-dependent phase transition in lysozyme. The role of the coherent intermolecular interaction is discussed.     

The role of hydrophobic interaction in phase transition and structure formation of lipid membranes and proteins

The hydrophobic interaction arises from the ordered structure of water around nonpolar groups of molecules in an aqueous solvent. Because biological systems are made of various macromolecules and amphiphiles which are suspended in aqueous solution, the hydrophobic interaction plays a very important role in the formation of higher-order structure and phase transitions in biological systems. Considering the hydrophobic interaction, the van der Waals interaction and the entropic effect, an equation of state of a lipid membrane was obtained which was analogous to the van der Waals equation. The characteristics of the lipid bilayer phase transition as well as the phase behaviors of a lipid monolayer were explained by this equation of state. Experimental evidence was obtained from ultrasonic measurements which indicated that its phase transition accompanys significant critical phenomena. Analysis of the hydrophobicity of amino acid sequences revealed that the morphology of the proteins was determined by the hydrophobicity alone. The essential role of the hydrophobic interaction in the morphogenesis of proteins could be confirmed by a denaturation experiment on a soluble protein, carbonic anhydrase B. Fluorescence measurements showed that an intermediate state, the so-called molten globule state, had a quite hydrophobic core, indicating that the globule shape of this protein is stabilized by the hydrophobic interaction.     

蛋白质对测定血清中抗精神病类药物的影响及消除

以人血清白蛋白(HSA)为模型,研究了蛋白质对测定血清样品中抗精神病类药物(APDs：安定、盐酸氯丙嗪及奋乃静)的影响。以分子荧光光谱法详细研究了抗精神病类药物与HSA的相互作用,以瑞利散射光谱研究了不同浓度乙醇对蛋白质变性的影响。研究表明：药物与HSA存在牢固结合作用,体积分数为80%的乙醇水溶液为萃取液提取血清样品中的APDs时,可以使血清中蛋白质缓慢变性,充分释放与其结合的药物。乙醇水溶液萃取液中加入K₂HPO₄无机盐后可形成双水相体系,APDs可进入双水相上相实现药物的均相高效萃取,上相经过滤后可直接进行高效液相色谱(HPLC)检测。该方法用于人血清中三种抗精神病类药物的检测,检出限为18.8～38.4 ng·mL-1,回收率为94.2%～98.7%。方法步骤简单,绿色环保,准确。     

On the nonequilibrium phase transition in protein

Considerations are presented that support the idea that the nonequilibrium phase transition in protein is accompanied by the growth of a colloidal nanocrystal (or the vitrified phase of a liquid crystal). To date, the dynamic transition forms of protein, which are “the source of the catalytic power of enzymes,” have been poorly understood. In our experiments, we observed the dehydration (drying) of the colloid solution of protein in an open (far from thermodynamic equilibrium) one-component protein-water system. The protein in this state is found to acquire properties typical of matter self-organization, including the universal properties of colloidal nanocrystals of different nature, namely, nonequilibrium chaotic dynamics with self-replicability, autocatalysis, coherency, autowave fluctuations, synchronism, fractality, 3D epitaxial growth (stacking) of films, nucleation giving rise to blocks (cells) with shell nuclei, etc. It then follows that our realistic model of the nonequilibrium state of protein during growth of its colloidal nanocrystal provides an opportunity of studying the dynamics of the structural and energy-information features of the transition and solid colloidal nanocrystalline phases of protein. In addition, it allows researchers to gain fundamentally new information about the energy characteristics of protein under abiotic and biotic conditions.     

Evolution of protein synthesis in a lattice model of replicators

The traditional chemical-kinetics approach to the study of prebiotic evolution cannot explain the evolution of protein synthesis in a homogeneous population of self-replicating molecules, because the invasion of a resident population of simpler, template-directed replicators by mutant protein-assisted replicators is deemed impossible. Approaching this problem in a spatial cellular automaton framework, we argue here that in such a setting evolution of protein synthesis is a likely event. In addition, we show that the onset of invasion may be viewed as a nonequilibrium phase transition, that can be characterized quantitatively by a set of critical exponents.     

Landscape of finite-temperature equilibrium behaviour of curvature-inducing proteins on a bilayer membrane explored using a linearized elastic free energy model

Using a recently developed multiscale simulation methodology, we describe the equilibrium behaviour of bilayer membranes under the influence of curvature-inducing proteins using a linearized elastic free energy model. In particular, we describe how the cooperativity associated with a multitude of protein–membrane interactions and protein diffusion on a membrane-mediated energy landscape elicits emergent behaviour in the membrane phase. Based on our model simulations, we predict that, depending on the density of membrane-bound proteins and the degree to which a single protein molecule can induce intrinsic mean curvature in the membrane, a range of membrane phase behaviour can be observed including two different modes of vesicle-bud nucleation and repressed membrane undulations. A state diagram as a function of experimentally tunable parameters to classify the underlying states is proposed.     

Tritium planigraphy and nanosized biological particles

Advances in studies of protein and complex biological systems by tritium planigraphy are considered. Particular attention is paid to detailing the structural organization of the protein components of the influenza virus. The structure of the M1 protein in a solution, virion, and crystal is analyzed. The specific features of this protein identified using tritium planigraphy and computer simulation are indicative of significant changes in its conformation depending on the “phase” state. Models of the spatial structure of the M1 protein in solution and in the virion are proposed. Structural disorder is observed in one of the three domains of this protein. Ideas on the importance of this structure for the multifunctional properties of the protein, such as binding to the membrane and a ribonucleoprotein complex, as well as the transfer of genetic material during viral infection of healthy cells, were suggested.     

Components of released liquid from ultrasonic waste activated sludge disintegration

Ultrasound can be applied as a pretreatment to disintegrate sludge. In this paper, by observing the solution concentration of polysaccharide, protein, DNA, Ca and Mg before and after disintegration, the main components in the released liquid are analyzed. It has been found that the predominant component of the released liquid in this research is protein. Ultrasound can destroy the extracellular polymeric substances (EPS), which is important to the sludge flocs structure. Ca2+ and Mg2+, which play a key role in binding the EPS are released into the aqueous phase. As a result, the sludge flocs are loosened. Under the effect of the hydraulic shear force, the sludge is disintegrated. Then the hydraulic shear forces destroy the cell walls, the substances inside the cells are released into the aqueous phase.     

一级相变时的红外特征辐射–熔融结晶和蒸气冷凝或沉淀

本文报道了一种新的物理现象–一级相变时 (熔融结晶, 蒸气冷凝或沉淀)的红外特征辐射. 实验结果根据相应的理论模型来进行分析. 此理论模型是基于一个论断, 那就是粒子(原子, 分子, 团簇)从高能级亚稳态 (气态或液态) 向低能级稳态 (液态或结晶态) 相变时释放出一个或多个光子. 这些光子的能量取决于相变潜热和新相粒子的结合特性. 对所有研究过的物质来说, 这种能量集中在红外区. 这就是为什么这种辐射被称作红外特征辐射. 在雾和云的形成过程中, 水发生了结晶、冷凝、升华, 从而产生了大量红外辐射留在了大气中. 因而, 该研究的结果必然对大气现象有很重要的影响: 它是地球冷却的因素之一; 冰雹云的形成伴随着强烈的红外辐射, 这种辐射可用来表征高能相转化为低能相的过程, 可以作为一种气象预警. 红外特征辐射似乎可以用来解释木星的呈红色现象. 它可以用于大气储能, 就此, 继风能、水能、太阳能、地热能后, 红外特征辐射成为生态学上第五种纯净的能源. 关键词： 熔体结晶 蒸气结晶 特鲁藤规则 大气蓄能     

朗之万方程及其在蛋白质折叠动力学中的应用

研究了朗之万方程的动力学性质,并用它模拟了蛋白质分子的折叠过程.首先在相空间中对朗之万方程做连续映射,发现做布朗运动的粒子在位置坐标上存在明显的概率分布,这表明蛋白质折叠过程中分子空间构型是非遍历的.此外,本文还通过数值模拟得到了去折叠态蛋白质的紧密度指标,并验证了它与实验结果以及其他理论方法的一致性.本文还提出了一种利用重整化方法研究熔球体状态蛋白质的理论模型,并提供了考虑疏水基影响的蛋白质折叠过程的模拟思路. 关键词： 朗之万方程 蛋白质折叠非遍历性 紧密度指标 重整化     

Simple Model Study of Phase Transition Properties of Isolated and Aggregated Protein

We investigate the phase transition properties of isolated and aggregated protein by exhaustive numerical study in the confined conformation space with maximally compact lattice model. The study within the confined conformation space shows some general folding properties. Various sequences show different folding properties: two-state folding, three-state folding and prion-like folding behavior. We find that the aggregated protein holds a more evident transition than isolated one and the transition temperature is generally lower than that in isolated case.     

Quantum model for understanding protein misfolding behavior——phase diagram and manual intervention

The study of protein folding is fundamental and important in the multidisciplinary field because a diversity of diseases, like Alzheimer's and Parkinson's are relevant to protein misfolding. The current thermodynamic and geometric approaches only phenomenologically describe but do not provide a mechanistic understanding of the competition between correct folding and misfolding. Here we present a model to understand the misfolding behavior. Considering the influence of dissipative strength for all possible sequences and comparing the folding time toward different compact structures, we obtain a phase diagram of the dissipative quantum phase transition that enables us to model the behavior. We also investigate how a perturbation in the Hamiltonian affects the transition point, which motivates us to explore possible manual interventions. Our results indicate that the manual intervention may be effective for some specific sequence but not for everyone. This approach is expected to lay a foundation for further studies on manual intervention in protein misfolding.