偏最小二乘红外光谱法及其对三种过氧化氢酶二级结构的测定

以九种蛋白质及其晶体结构数据为校准，建立了蛋白质二级结构测定的偏最小二乘红外光谱定量法，并分析了三种过氧化氢酶在水溶液中的二级结构，结果表明本法对于测定水溶液中二级结构是一种简便，有效，准确和可靠的方法。     

傅里叶变换红外光谱对阴离子表面活性剂SDS与牛血清白蛋白相互作用的研究

对牛血清白蛋白BSA与阴离子表面活性剂SDS在水溶液中不同浓度和不同相互作用时间的红外光谱进行了研究。研究表明,短时间和低浓度时SDS使BSA的α-螺旋结构增加,无规结构降低,没有破坏蛋白质的二级结构;当SDS浓度很大或者SDS与BSA的作用时间长时,蛋白质的二级结构遭到破坏,BSA的α-螺旋结构降低,无规结构增加。     

Micro－FTIR法研究肺癌细胞组织内蛋白质二级结构

采用Ｍｉｃｒｏ－ＦＴＩＲ法研究了不同程度的肺癌细胞组织内蛋白质二级结构的变化，获得了肺癌的恶性程度与表征蛋白质二级结构的α螺旋与β折叠两者之比值的关系。     

Micro—FTIR法研究肺癌细胞组织内蛋白质二级结构

采用Ｍｉｃｒｏ－ＦＴＩＲ法研究了不同程度的肺癌细胞组织内蛋白质二级结构的变化，获得了肺癌的恶性程度与表征蛋白质二级结构的ａ螺旋与β折叠两者之比值的关系。     

应用FTIR法研究磷脂酶A2改性蛋黄粉乳化性的构效关系

乳化性能是磷脂酶A2改性蛋黄粉的重要指标.将磷脂酶A2改性蛋黄液分别在150,170及190℃条件下喷雾干燥制成蛋黄粉,运用FTIR技术并结合去卷积、二阶导数、曲线拟合方法对蛋白质二级结构进行分析.结果表明,随喷雾干燥温度的升高,蛋黄粉的乳化性能及α-螺旋含量均呈现先升高后下降的趋势,蛋黄粉乳化液的热稳定性不受喷雾干燥温度的影响,但二级结构会呈现出从α-螺旋向β-折叠转变的趋势,而代表蛋白质分子内部紧密程度的β+α结构总含量相对稳定.     

超高压处理优化藜麦蛋白的乳化性能

利用超高压处理藜麦蛋白,研究超高压保压压力、超高压保压时间及蛋白质量分数对藜麦蛋白乳化性的影响。采用响应面法优化超高压处理条件,得到最佳工艺条件,并利用傅里叶红外光谱、粒度仪、X射线衍射(XRD)等表征方法分析乳液蛋白质的表面性质及结构特征。结果表明:保压压力为235 MPa、保压时间为5.2 min、蛋白质量分数为0.34%时,乳化指数为119 m~2/g。同时,由傅里叶红外光谱分析蛋白二级结构可知,变性后藜麦蛋白的α-螺旋结构含量降低,β-转角结构含量增加,分子无序性增加,蛋白乳化性提高。XRD分析发现,改性后蛋白在2θ=10°附近的峰强度明显减小,说明α-螺旋结构含量降低。改性后乳液蛋白粒度减小,其乳化性提升。因此,适当的超高压处理可以改善藜麦蛋白的乳化性。     

Molecular Dynamics Simulation on Stability of Insulin on Graphene

通过分子动力学模拟研究了胰岛素与不同尺寸石墨烯的相互作用,并比较了固定的石墨烯与不固定的石墨烯对蛋白质吸附行为及动力学的影响. 通过分析胰岛素蛋白在吸附过程中构象与取向的变化和吸附过程的动力学行为证实了石墨烯对胰岛素蛋白的有效吸附. 通过对蛋白质结构的分析发现蛋白质的三级结构在所有的体系中均有不同程度的破坏,而蛋白质二级结构的稳定性则与体系有关,其中非固定的石墨烯因其具有较大柔性而表现出对蛋白质二级结构的破坏具有一定选择性. 同时还发现石墨烯的尺寸对于胰岛素的吸附动力学过程也有较大影响.     

FTIR光学信息研究PLD蛋白分子冻干胁迫下的二级结构变化

通过FTIR光学信息研究液态和固态磷脂酶D的二级结构,结果表明FTIR光学信息是一种有效的蛋白质分子二级结构研究方法。FTIR的光学信息分析表明冻干胁迫下,磷脂酶D的FTIR光学信息及二级结构发生较大变化。在磷脂酶D活性构象时的pH冻干使磷脂酶Dα螺旋大大下降,无规卷曲大大上升,β折叠几乎没有变化,说明PLD蛋白质分子空间有序构象解体严重。功能性试验表明,磷脂酶D二级结构的变化直接导致了其活性发生很大变化。     

抑癌基因p53的单细胞傅里叶变换显微红外光谱

为探索单细胞红外光谱技术对单基因差异的鉴别能力,利用同步辐射傅里叶变换红外显微光谱技术采集含抑癌基因p53(野生型)和敲除抑癌基因p53(敲除型)结肠癌细胞的单细胞显微红外光谱。研究分析光谱发现,二者在脂质、蛋白质以及核酸吸收谱带峰强度和位置都有明显的差异。敲除p53后脂质、核酸以及蛋白特征吸收峰均减弱,且几乎所有的吸收峰都向高波数位移。分析了酰胺I带与酰胺II带的相对吸收强度比,发现敲除型比值明显变大;酰胺I带拟合结果表明野生型细胞中蛋白质二级结构的α螺旋和无规则卷曲含量明显低于敲除型,转角和非典型螺旋的含量则高于敲除型,而β折叠的含量无明显变化。研究表明,同步辐射单细胞红外光谱可以在分子水平上鉴别因p53基因差异而产生的细胞代谢变化。     

基于动态光散射研究pH值对茄红素胶囊粒径的影响

用微乳化技术结合复合团聚法制备了纳米及亚微米茄红素胶囊,基于动态光散射测量分析了环境p H值对胶囊的粒径大小及其分布的影响.结果表明,对纳米茄红素胶囊,当p H值为3.5、6.0及6.8时能稳定的分散,而p H值为7.4时则会出现明显的聚集现象;对亚微米茄红素胶囊,当p H值为3.5、6.0、6.8及7.4时其粒径分布均为双峰状态,其中粒径较大的为亚微米茄红素胶囊,粒径较小的为包覆不良的颗粒,且p H=7.4时的粒径明显小于其它三种p H值时的粒径.虽然亚微米胶囊在p H=7.4时粒径明显变小的结果与纳米胶囊相同,但亚微米胶囊并不像纳米胶囊那样出现聚集现象.     

COSY with in-phase cross peaks

We have designed two pulse sequences that give rise to COSY-type spectra with in-phase multiplet structure. In these spectra, the cross peaks are absorptive and in-phase along both dimensions. Such 2D spectra are useful for quantitative measurements, such as measurement of the extent of H/D exchange in proteins and measurement of the concentrations of individual components in a series of mixtures. These spectra can also be used to emphasize cross peaks between weakly coupled protons.     

德国小蠊变应原Bla g 2蛋白变复性的荧光光谱研究

包涵体中的重组蛋白经抽提后可以在变性状态下纯化,而纯化后的复性过程是基因工程下游处理的重要环节。通过对变应原Bla g 2蛋白复性前后荧光光谱的比较和分析、变性剂(尿素和SDS)对复性后Bla g 2蛋白的荧光滴定实验、以及复性后Bla g 2在不同pH下的荧光光谱分析,推断出Bla g 2蛋白分子在不同环境下构象的变化及其光谱学特征,初步建立了一种新的检测重组变应原蛋白变复性的光谱实验方法。     

Electronic and vibrational spectra of a gel of single-wall carbon nanotubes in an ionic liquid

A gel of single-wall carbon nanotubes in an ionic liquid has been prepared using the technique of mixing nanotubes with ionic liquids. The gel obtained has a high concentration of nanotubes (～1%). The absorption spectra of the gel and its individual components have been measured. The absorption spectrum of the gel exhibits reconstruction of the electronic and vibrational spectra of the gel with respect to its individual components. This reconstruction is explained by the interaction of nanotubes with the ionic liquid in the gel structure. Reconstruction of the electronic subsystem of nanotubes has also been observed in a suspension of nanotubes in an aqueous solution of a surfactant.     

傅里叶变换红外光谱法和紫外-可见谱线组法分析广西特产罗汉果

采用红外光谱和紫外-可见谱线组法全面分析了广西特产罗汉果,并将紫外-可见谱线组法延伸至红外光谱,采集了4种浸泡液的红外谱图。罗汉果红外光谱中出现了羟基、亚甲基、链状羧酸酯羰基、酰胺和苷键的特征吸收峰,表明其中主要含有油脂、蛋白质和甜甙等成分。4种不同极性溶剂水、乙醇、氯仿和石油醚浸泡液的紫外-可见谱线中吸收峰个数、峰形和峰位都存在很大差别,全面反映出罗汉果所含成分的整体效应。4种浸泡液的红外谱图中分别出现了不同成分的特征峰,说明水浸泡液中含有蛋白质和甜甙等极性分子,乙醇浸泡液中既含有蛋白质、甜甙,也含有油脂等脂溶性成分,氯仿浸泡液中除了含有大量油脂,还含有少量蛋白质等成分,而石油醚浸泡液中只含有油脂等脂溶性成分。     

'q-Titration' of long-chain and short-chain lipids differentiates between structured and mobile residues of membrane proteins studied in bicelles by solution NMR spectroscopy

'q-Titration' refers to the systematic comparison of signal intensities in solution NMR spectra of uniformly (15)N labeled membrane proteins solubilized in micelles and isotropic bicelles as a function of the molar ratios (q) of the long-chain lipids (typically DMPC) to short-chain lipids (typically DHPC). In general, as q increases, the protein resonances broaden and correspondingly have reduced intensities due to the overall slowing of protein reorientation. Since the protein backbone signals do not broaden uniformly, the differences in line widths (and intensities) enable the narrower (more intense) signals associated with mobile residues to be differentiated from the broader (less intense) signals associated with "structured" residues. For membrane proteins with between one and seven trans-membrane helices in isotropic bicelles, we have been able to find a value of q between 0.1 and 1.0 where only signals from mobile residues are observed in the spectra. The signals from the structured residues are broadened so much that they cannot be observed under standard solution NMR conditions. This q value corresponds to the ratio of DMPC:DHPC where the signals from the structured residues are "titrated out" of the spectrum. This q value is unique for each protein. In magnetically aligned bilayers (q>2.5) no signals are observed in solution NMR spectra of membrane proteins because the polypeptides are "immobilized" by their interactions with the phospholipid bilayers on the relevant NMR timescale (～10(5)Hz). No signals are observed from proteins in liposomes (only long-chain lipids) either. We show that it is feasible to obtain complementary solution NMR and solid-state NMR spectra of the same membrane protein, where signals from the mobile residues are present in the solution NMR spectra, and signals from the structured residues are present in the solid-state NMR spectra. With assigned backbone amide resonances, these data are sufficient to describe major features of the secondary structure and basic topology of the protein. Even in the absence of assignments, this information can be used to help establish optimal experimental conditions.     

Analysis of nitroxide spin label motion in a protein-protein complex using multiple frequency EPR spectroscopy

X- and W-band EPR spectra, at room and low temperatures, are reported for nitroxide spin labels attached to cysteine residues selectively introduced into two proteins, the DNase domain of colicin-E9 and its immunity protein, Im9. The dynamics of each site of attachment on the individual proteins and in the tight DNase-Im9 complex have been analysed by computer simulations of the spectra using a model of Brownian dynamics trajectories for the spin label and protein. Ordering potentials have been introduced to describe mobility of labels restricted by the protein domain. Label mobility varies with position from completely immobilised, to motionally restricted and to freely rotating. Bi-modal dynamics of the spin label have been observed for several sites. We show that W-band spectra are particularly useful for detection of anisotropy of spin label motion. On complex formation significant changes are observed in the dynamics of labels at the binding interface region. This work reveals multi-frequency EPR as a sensitive and valuable tool for detecting conformational changes in protein structure and dynamics especially in protein-protein complexes.     

High flow-resolution for mobility estimation in 2D-ENMR of proteins using maximum entropy method (MEM-ENMR)

Multidimensional electrophoretic NMR (nD-ENMR) is a potentially powerful tool for structural characterization of co-existing proteins and protein conformations. By applying a DC electric field pulse, the electrophoretic migration rates of different proteins were detected experimentally in a new dimension of electrophoretic flow. The electrophoretic mobilities were employed to differentiate protein signals. In U-shaped ENMR sample chambers, individual protein components in a solution mixture followed a cosinusoidal electrophoretic interferogram as a function of its unique electrophoretic migration rate. After Fourier transformation in the electrophoretic flow dimension, the protein signals were resolved at different resonant frequencies proportional to their electrophoretic mobilities. Currently, the mobility resolution of the proteins in the electrophoretic flow dimension is limited by severe truncations of the electrophoretic interferograms due to the finite electric field strength available before the onset of heat-induced convection. In this article, we present a successful signal processing method, the Burg's maximum entropy method (MEM), to analyze the truncated ENMR signals (MEM-ENMR). Significant enhancement in flow resolution was demonstrated using two-dimensional ENMR of two protein samples: a lysozyme solution and a solution mixture of bovine serum albumin (BSA) and ubiquitin. The electrophoretic mobilities of lysozyme, BSA and ubiquitin were measured from the MEM analysis as 7.5x10(-5), 1.9x10(-4) and 8.7x10(-5) cm2 V-1 s-1, respectively. Results from computer simulations confirmed a complete removal of truncation artifacts in the MEM-ENMR spectra with 3- to 6-fold resolution enhancement.     

Protein Binding on the Surface of Magnetic Nanoparticles

Magnetic nanoparticles (MNPs) are widely used in the areas of biology and biomedicine. The interaction between MNPs and proteins plays a crucial role in the bioapplication of MNPs, and the binding affinity of protein–MNPs is the manifestation of this interaction. The binding affinity of some proteins with MNPs modified in various ways is determined by fluorescence quenching. The results show that the binding affinity depends on the properties of both the MNPs and the proteins. The higher the surface curvature of MNPs, the larger the MNP, and the higher the binding affinity. No significant difference is found in binding affinity between MNPs with different modification methods. For proteins, the binding affinity depends on the properties of individual proteins, such as the amino acid sequence, the native protein conformation in solution, the isoelectric point, and surface potential. In general, the binding affinity is higher for proteins with cysteine residues on the surface. In addition, pH affects the binding affinity between proteins and MNPs; positively charged proteins and lower pH are more suitable for MNP binding due to electrostatic forces.     

大豆硒蛋白构象的光谱法研究

采用荧光光谱、紫外光谱并结合红外光谱对比研究了大豆硒蛋白和大豆蛋白的结构区别,并用荧光相图法分析了脲诱导下两种蛋白的去折叠过程。考察了温度、pH值对大豆硒蛋白质构象的影响,同时对其乳化稳定性能进行了测定。结果表明,与大豆分离蛋白相比,大豆硒蛋白分子中的共价二硫键受到破坏,分子内氢键作用减弱,疏水相互作用增强,蛋白质分子发生伸展。大豆硒蛋白在溶液中只呈现“折叠”和“松散”两种状态,与大豆蛋白相比更容易被水解变性。升高温度,大豆硒蛋白溶液存在明显荧光热猝灭效应,疏水性逐渐增强,蛋白质分子趋于折叠。在pH值2.8～8.0的范围内,大豆硒蛋白的Trp残基主要分布于其分子外部的极性环境中,并随pH值变化在等电点两侧呈现不同的构象变化。在酸性环境中大豆硒蛋白较易发生从松散到折叠的构象转变,碱性条件则较有利于大豆硒蛋白以松散的结构存在。此外,基于紫外光谱数据分析了大豆硒蛋白乳化特性,结果表明降低温度有利于增强大豆硒蛋白的乳化性能,但使其稳定性能下降。     

NTO及其铷盐水溶液光谱研究

用Raman光谱和FTIR光谱对NTO晶体和不同pH值的水溶液以及配合物Rb(NTO)·H₂O的水溶液进行了研究。从已测定的单晶结构表明金属离子与NTO-的CNO,CO基团和水分子中的氮、氧原子键合。Raman和FTIR光谱特征的主强峰也表明是在NTO-的CNO₂和CO基团的氧和氮原子与金属离子形成弱配位键。该配合物饱和水溶液的振动光谱峰与晶体的振动光谱峰基本上吻合。表明该配合物在水中键合未发生变化,因而NTO的碱金属配合物在水溶液中的振动光谱峰归属可以用晶体中键的形成来说明。讨论了金属离子与NTO必需在碱性水中才能形成NTO的金属配合物机理。