蛋白质多态解折叠行为与稳定性Ⅱ:酶的多态解折叠行为

基于结构基元模型,进一步假设,由n个结构基元组成的蛋白酶,其活性中心由na(na?n)个结构基元组成,酶活性仅与组成活性中心的结构基元相关.由此,推导出适合于蛋白酶解折叠研究的变性曲线、解折叠结构基元平均自由能、物种分布等表达式.本文以盐酸胍诱导的卵清溶菌酶解折叠为例,通过荧光方法测定的溶菌酶解折叠曲线,得出卵清溶菌酶由2个结构基元组成,结构基元平均自由能?G0element(H2O)为48.47 kJ/mol.物种分布表明,酶活性随盐酸胍浓度的变化仅仅反映的是结构基元1(?-片结构域)的解折叠,而结构基元2(?-螺旋结构域)的解折叠反映在3.8~5.0 mol/L盐酸胍浓度范围内.结构基元模型既可描述蛋白酶多态解折叠的谱学行为,又可解释蛋白酶活性的两态性质.     

Temperature-induced unfolding of ribonuclease A embedded in spherical polyelectrolyte brushes

We use Fourier Transform infrared spectroscopy (FT-IR) spectroscopy to study the thermal unfolding and refolding behavior of ribonuclease (RNase A) adsorbed to spherical polyelectrolyte brushes (SPB). The SPB consist of a solid poly(styrene) core of ca. 100 nm diameter onto which long chains of poly(styrene sulfonic acid), PSS have been densely attached. The particles bearing the adsorbed protein are dispersed in aqueous buffer solution at a pH close to the isoelectric point (9.6) of the protein. The secondary structure of the protein was analyzed by FT-IR spectroscopy and compared to the structure of the native protein before adsorption. The unfolding of the free RNase A in solution was found to be fully reversible with an unfolding temperature of 65 degrees C, in accordance to previous studies. However, after adsorption to the SPB, the unfolding temperature of the protein molecule is lowered by 10 degrees C and the Van't Hoff enthalpy of the unfolding process is significantly reduced. Moreover the unfolding of the adsorbed protein is irreversible. The phenomenon may be explained by an increase in binding sites due to unfolding of the globular structure. Protein adsorption to a spherical polyelectrolyte brush.     

Assessing the Functional and Structural Stability of the Met80Ala Mutant of Cytochrome c in Dimethylsulfoxide

The Met80Ala variant of yeast cytochrome c is known to possess electrocatalytic properties that are absent in the wild type form and that make it a promising candidate for biocatalysis and biosensing. The versatility of an enzyme is enhanced by the stability in mixed aqueous/organic solvents that would allow poorly water-soluble substrates to be targeted. In this work, we have evaluated the effect of dimethylsulfoxide (DMSO) on the functionality of the Met80Ala cytochrome c mutant, by investigating the thermodynamics and kinetics of electron transfer in mixed water/DMSO solutions up to 50% DMSO v/v. In parallel, we have monitored spectroscopically the retention of the main structural features in the same medium, focusing on both the overall protein structure and the heme center. We found that the organic solvent exerts only minor effects on the redox and structural properties of the mutant mostly as a result of the modification of the dielectric constant of the solvent. This would warrant proper functionality of this variant also under these potentially hostile experimental conditions, that differ from the physiological milieu of cytochrome c.     

白藜芦醇与CopC的相互作用研究

白藜芦醇是一种天然的植物抗毒素,存在于花生、葡萄和多种浆果之中。CopC是一种由β-折叠片组成的铜伴侣蛋白,含有102个氨基酸,Cu~+结合在C端,Cu~(2+)结合在N端,其作为氧化还原开关参与铜的调控,它与小分子的作用影响其氧化还原开关功能。本研究主要利用红外光谱(FTIR)、圆二色谱(CD)、荧光寿命、三维荧光光谱、荧光光谱和分子对接等方法研究白藜芦醇与CopC的相互作用。通过FTIR、CD、化学变性和荧光寿命测定表明,白藜芦醇的结合使得CopC的稳定性降低,可能源于CopC的β-折叠片含量减少,自由卷曲含量增加。此外,三维荧光光谱和荧光光谱表明,白藜芦醇与CopC结合后,其荧光光谱出现蓝移,表明白藜芦醇可能结合在CopC的疏水区域;并且,可得白藜芦醇与CopC的结合比为1∶1,结合常数为(6.76±0.17)×10~5 L·mol~(-1),疏水作用力是维持两者之间形成复合物的主要作用力。通过F9rster能量转移和分子对接模拟确定CopC中白藜芦醇和色氨酸之间的平均结合距离,模拟的结果和实验结果具有较好的一致性。本文结果将为阐明CopC的铜调控机理提供帮助。     

Stability of S-layer proteins for electrochemical nanofabrication

Crystalline cell surface layer proteins (S-layers) can be used in electrochemical fabrication to create nanoscale arrays of metals and oxides on surfaces so long as the proteins maintain their long-range order during processing. We have explored the stability of the HPI layer protein (the S-layer protein from the microorganism Deinococcus radiodurans) adsorbed onto platinum surfaces after immersion in sulfuric acid or sodium hydroxide electrolytes ranging in pH from 0 to 14 over time periods ranging from 1 to 1000 s. Topographic data obtained by atomic force microscopy (AFM) was used to characterize the protein stability, judged by its retention of long-range order after immersion. The compiled data revealed that, under these solution conditions and in this environment, the HPI layer protein has a dose-dependent structural stability “envelope” in the acidic range from 1 < pH < 4. The protein retains its long-range order up to 1000 s from pH 4 to 11, and has a sharp stability edge between pH 12 and 13. Interestingly, the more stringent requirement of stability (i.e., retention of long-range order) defined in the context of electrochemical fabrication for this protein narrowed the window of stability in pH and time when compared to previous stability studies reported for this protein.     

Monitoring of unfolding of metallo-proteins by electrospray ionization mass spectrometry

An electrospray ionisation (ESI) mass spectrometric method for the determination of the equilibrium constant and free energy (DeltaG) of protein unfolding was used to monitor the denaturation process at different pH of three metallo-proteins, i.e. wild-type copper azurin, zinc azurin and wild-type amicyanin. The time course of the unfolding process was followed by dissolving the proteins under denaturing conditions (methanol-water (1 : 1, v/v)) at different pH (2.5, 3.0, 3.5) and recording ESI spectra at time intervals. The spectra showed two series of peaks, corresponding to the native holo-protein and the unfolded apo-protein. From the intensity ratio of these two series of peaks at increasing time and at equilibrium, the equilibrium constants for the unfolding process for the three proteins could be determined. From these equilibrium constants a DeltaG degrees derivation was attempted. The DeltaG degrees values obtained decrease with decrease in pH, in agreement with the expected reduction of conformational stability of proteins at lower pH. The results obtained confirm that ESI-MS can be used for monitoring of unfolding process and to derive quantitative thermodynamic data.     

Electron transfer and interfacial behavior of redox proteins

This paper reviews the recent progress in the electron transfer and interfacial behavior of redox proteins. Significant achievements in the relevant fields are summarized including the direct electron transfer between proteins and electrodes, the thermodynamic and kinetic properties, catalytic activities and activity regulation of the redox proteins. It has been demonstrated that the electrochemical technique is an effective tool for protein studies, especially for probing into the electron transfer and int...     

A convenient tool for studying the stability of proteins and nucleic acids

The aim of this work is to discuss the thermodynamic properties, obtained by differential scanning calorimetry (DSC), of the thermal transition of proteins and nucleic acids and to analyze these data using statistical thermodynamic relations. The denaturation of the ordered, specific structures of biological macromolecules is a cooperative process and in many cases the macromolecules undergo a two-state transition. Differential scanning calorimetry, giving direct thermodynamic information, has proved to be very useful in clarifying the energetics of macromolecule transitions and in characterizing their thermal stability. Here, various examples are discussed: i) the equilibrium thermal denaturation of ribonuclease A, a model for the use of DSC by following the temperature-unfolding of the proteins, a monomolecular transition; ii) the equilibrium thermal dissociation of a DNA double helix in two strands, an example of how DSC is used to follow a bimolecular process; iii) an example of the use of DSC for studying the melting of unimolecular and tetramolecular DNA quadruple-helices.     

Site-selective probe for investigating the asynchronous unfolding of domains in bovine serum albumin

A convenient method is proposed for precise investigation of the asynchronous structural transition of the domains in bovine serum albumin (BSA) during unfolding process. The method is based on a site-selective probe, alizarin red S (ARS), which has a high affinity to the subdomain IIA of BSA. BSA-ARS complex was formed and gradually unfolded by urea from 0 to 8.0 M. The unfolding occurred in different domains of BSA resulted in distinct alterations of the microenvironment of the bound ARS. The spectral response of BSA-ARS complex, including the color, the UV absorption at 530 and 432 nm, and the intrinsic fluorescence at 342 and 310 nm with the excitation wavelength of 280 nm, showed slight changes in the urea concentration from 0 to 4.5 M, drastic changes from 4.5 to 6.0 M, and almost no changes from 6.0 to 8.0 M. The redox behavior of bound ARS between 0.3 and 0.8 V also showed the same trend. Consequently, a two-step, three-state transition process was monitored by naked eyes, UV-vis spectroscopy and electrochemistry. It is the first report to realize the indicator of the intermediate state during the unfolding process of BSA through convenient methods instead of expensive approaches. The work provides a facile method for the investigation of the unfolding process of multidomain proteins.     

Computational modeling of acrylodan-labeled cAMP dependent protein kinase catalytic subunit unfolding

Structure of the cAMP-dependent protein kinase catalytic subunit, where the asparagine residue 326 was replaced with acrylodan-cystein conjugate to implement this fluorescence reporter group into the enzyme, was modeled by molecular dynamics (MD) method and the positioning of the dye molecule in protein structure was characterized at temperatures 300 K, 500 K and 700 K. It was found that the acrylodan moiety, which fluorescence is very sensitive to solvating properties of its microenvironment, was located on the surface of the native protein at 300 K that enabled its partial solvation with water. At high temperatures the protein structure significantly changed, as the secondary and tertiary structure elements were unfolded and these changes were sensitively reflected in positioning of the dye molecule. At 700 K complete unfolding of the protein occurred and the reporter group was entirely expelled into water. However, at 500 K an intermediate of the protein unfolding process was formed, where the fluorescence reporter group was directed towards the protein interior and buried in the core of the formed molten globule state. This different positioning of the reporter group was in agreement with the two different shifts of emission spectrum of the covalently bound acrylodan, observed in the unfolding process of the protein.     

Interactions of proteins with uniform colloidal hematite and chromium hydroxide particles. II. Stability and mobility

Stability of hematite and chromium hydroxide particles covered by ovalbumin, -globulin, and lysozyme, respectively, and mobilities of the same coated particles in aqueous media were investigated as a function of the pH, ionic strenght [NaNO₃, Mg(NO₃)₂], and the amount of added proteins. It was found that ovalbumin causes electrosteric stabilization of dispersions at pH values other than 5, while flocculation occurred at pH 5 (which was the i.e.p. of the coated particles). Mobility curves of ovalbumin covered particles resembled those of the pure protein. Dispersions with -globulin flocculated at and around the i.e.p. of the macromolecule (pH7). The mobility curves of -gobulin-coated sols were intermediate between those of the protein and of the cores, and were dependent upon the amount of adsorbed -globulin. Heterocoagulation was observed for both hematite and chromium hydroxide dispersions with lysozyme. Mobilities of lysozyme covered particles were between those of the cores and of the protein, but were not dependent upon the quantity of adsorbed polymer.Supported by the NSF Grant CHE-9108420Part of a Ph.D. Thesis     

Influence of Interfacial Engineering on Stability of Emulsions Stabilized with Soy Protein Isolate

The objectives of this study were to examine the influence interfacial composition on environmental stresses stability of oil in water emulsions. An electrostatic layer-by-layer deposition method was used to create the multilayered interfacial membranes with different compositions: (i) primary emulsion (Soy protein Isolate); (ii) secondary emulsion (Soy protein Isolate – OSA-starch); (iii) tertiary emulsion (Soy protein isolate – OSA-starch – chitosan). Fourier transform-infrared (FTIR) and scanning electron microscopy (SEM) results confirmed the adsorption of charged polyelectrolyte onto oppositely charge polyelectrolyte-coated oil droplets. The stability of primary, secondary, and tertiary emulsions to thermal treatment (30 min at 30–90°C), pH (3–7) and NaCl (0–500 mM) were determined using ζ-potential, particle diameter, and microstructure analysis. Primary emulsions were unstable at pH 4–7, salt concentrations, and thermal treatments. Secondary emulsions were stable to creaming and droplet aggregation at pH 3–5, at ≤50 mM NaCl, and unstable at thermal treatments, whereas tertiary emulsions were stable at all salt concentrations, thermal treatments, and at pH 3–6. These results demonstrate that these polymers can be used to engineer oil in water emulsion systems and improve the emulsion stability to environmental stresses.     

Influence of the sample-solvent on protein retention,mass transfer and unfolding kinetics in hydrophobic interaction chromatography

Typical mobile phase employed in hydrophobic interaction chromatography contains cosmotropic salts, which promote retention and simultaneously reduce the protein solubility in the mobile phase. To increase mass overloading in the separation process the protein can be dissolved in a sample-solvent with concentration of salt lower than that in the mobile phase or in salt free solutions. However, this methodology may cause band splitting and band deformation, which results in yield losses. In this study, these phenomena were analyzed based on the retention behavior of two model proteins, i.e., lysozyme and bovine serum albumin. Retention of these proteins was accompanied by strong band broadening originated from slow rates of mass transfer and/or of adsorption–desorption process involving the protein conformational changes. The mass transport resistances and unfolding kinetics were found to contribute to the sample-solvent effects. To avoid band deformations the process variables such as the salt concentration and temperature were adjusted in such a way that complete resolution between band profile of the sample-solvent and the protein was achieved. For the process simulation a dynamic model, which accounted for underlying kinetics was used. General guidelines of the process design were developed.     

Stability of bovine serum albumin at different pH

Summary The effect of pH on the thermal denaturation of BSA containing fatty acids was studied by use of differential scanning calorimetry (DSC). Thermal scanning of BSA aqueous solutions gave various types of DSC curves depending on the protein concentration and on the pH. The broad bimodal endothermic transition was suggested to be connected with loose protein structure in contradistinction to single peak for compact molecule structure. The propensity toward precipitation at pH conditions ranging from 3.8 to 5 was observed. A scan-rate independent and partly reversible behavior of the thermal heating of BSA was found. Deconvolution of DSC traces in non-two-state model with assumption of two- or three-component transition allowed to study the effect of pH on different parts of BSA molecule.     

Protein Stability in Reverse Micelles

The N‐terminal SH3 domain of the Drosophila signal transduction protein drk was encapsulated in reverse micelles. Both the temperature of maximum stability and the melting temperature decreased on encapsulation. Dissecting the temperature‐dependent stability into enthalpic and entropic contributions reveals a stabilizing enthalpic and a destabilizing entropic contribution. These results do not match the expectations of hard‐core excluded volume theory, nor can they be wholly explained by interactions between the head groups in the reverse micelle and the test protein. We suggest that geometric constraints imposed by the reverse micelles need to be considered.     

血红蛋白和细胞色素c构象去折叠行为研究

利用紫外-可见吸收和荧光光谱法研究了血红蛋白(Hb)与细胞色素c(Cytc)两种血红素蛋白的去折叠行为。采用化学变性剂盐酸胍(GdHCl)和尿素(Urea)诱导两种蛋白构象去折叠,阐述了两种蛋白的去折叠机理。Hb的血红素(Heme)辅基通过与卟啉铁原子和组氨酸配位,与肽链键合的稳定性较差,在3.0 mol/L的盐酸胍作用下即发生解离。而Cyt c的Heme辅基通过卟啉与半胱氨酸形成二硫键呈现较强的稳定性,盐酸胍浓度达到6.0 mol/L也难使其发生解离。该研究为阐释蛋白构象与功能之间的关系提供了重要依据。     

Elasticity of the Transition State Leading to an Unexpected Mechanical Stabilization of Titin Immunoglobulin Domains

The giant protein titin plays a critical role in regulating the passive elasticity of muscles, mainly through the stochastic unfolding and refolding of its numerous immunoglobulin domains in the I‐band of sarcomeres. The unfolding dynamics of titin immunoglobulin domains at a force range greater than 100 pN has been studied by atomic force microscopy, while that at smaller physiological forces has not been measured before. By using magnetic tweezers, it is found that the titin I27 domain unfolds in a surprising non‐monotonic force‐dependent manner at forces smaller than 100 pN, with the slowest unfolding rate occurring around 22 pN. We further demonstrate that a model with single unfolding pathway taking into account the elasticity of the transition state can reproduce the experimental results. These results provide important novel insights into the regulation mechanism of the passive elasticity of muscle tissues.