Contribution of hydrophobic interactions to protein stability

A major factor in the folding of proteins is the burying of hydrophobic side chains. A specific example is the packing of alpha-helices on beta-sheets by interdigitation of nonpolar side chains. The contributions of these interactions to the energetics of protein stability may be measured by simple protein engineering experiments. We have used site-directed mutagenesis to truncate hydrophobic side chains at an alpha-helix/beta-sheet interface in the small ribonuclease from Bacillus amyloliquefaciens (barnase). The decreases in stability of the mutant proteins were measured by their susceptibility to urea denaturation. Creation of a cavity the size of a -CH2-group destabilizes the enzyme by 1.1 kcal mol-1, and a cavity the size of three such groups by 4.0 kcal mol-1.     

CFCl3与O3分子间相互作用的二级Mφiler—Plesset微扰理论研究

通过在二级Mφller-Plesset微扰理论MF2／cc—pVTZ电子相关校正水平,对CFCl3和03分子间可能存在的复合物进行全自由度能量梯度优化,得到2种稳定的几何结构A和B,用MP2／aug-cc—pVTZ方法计算出其结合能△E^CP分别为-2．39kcal·mol^-1和-2．29kcal·mol^-1,LM...     

系列小分子与碱基尿嘧啶间氢键相互作用的理论研究

核酸碱基尿嘧啶分子可以使用3个不同位点与其他分子形成多种类型的分子间氢键.笔者使用密度泛函理论B3LYP/6-31＋G（d,p）方法优化得到了系列小分子与尿嘧啶分子形成的31个氢键复合物的稳定结构,进而使用MP2/aug-cc-pVTZ方法计算了这些氢键复合物的结合能.研究结果表明,尿嘧啶分子更倾向作为氢键给体与小分子形成氢键.尿嘧啶分子最易使用1号位点与小分子形成氢键复合物,最不易使用2号位点形成氢键复合物.甲基取代小分子氢键受体上的氢原子会使氢键强度变强.为了深入理解这些氢键复合物体系中氢键作用的本质,还使用B3LYP/6-31G（d,p）方法进行了自然键轨道（NBO）分析计算.自然键轨道（NBO）分析表明,大部分氢键复合物满足二阶相互作用稳定化能之和越大,结合能绝对值越大,结合得越稳定的原则,说明共价作用在这些氢键作用中起重要作用.     

Cumulative effect of intragenic amino-acid replacements on the thermostability of a protein

The marginal net stability of a folded protein is thought to depend on a small difference between large, compensating individual forces. Therefore, the net free energy of stabilization of proteins is unexpectedly small (approximately 10 kcal mol-1). The contribution of individual forces such as hydrogen bonds and salt bridges to the stabilization is evaluated as 1-3 kcal mol-1, and several additional forces are thought to be sufficient to account for the extra thermostability of thermophilic proteins. The native conformation of a protein is determined by the total number of interatomic interactions and hence by the amino-acid sequence. If the few amino-acid residues that individually contribute to the stabilization could be implemented concurrently into the sequence, the multiple replacement would enhance the overall stability of the protein molecule. Here we report evidence to support this argument. Thermal inactivation kinetics and proteolytic resistance for mutants of a kanamycin nucleotidyltransferase reveal that a few intragenic amino-acid replacements stabilize the protein cumulatively. Our experiments not only demonstrate the feasibility of elevating the thermostability of a protein but also lead to better understanding of the forces that are responsible for protein stability.     

CFCl3与O3分子间相互作用的二级Mφiler—Plesset微扰理论研究

通过在二级Mφller-Plesset微扰理论MF2／cc—pVTZ电子相关校正水平,对CFCl3和03分子间可能存在的复合物进行全自由度能量梯度优化,得到2种稳定的几何结构A和B,用MP2／aug-cc—pVTZ方法计算出其结合能△E^CP分别为-2．39kcal·mol^-1和-2．29kcal·mol^-1,LMOEDA定域轨道能量分解分析表明其中色散能占主导。     

荧光法研究3-环丙基-3-去乙烯基红紫素18甲酯、3-环丙基-3-去乙烯基-N-甲氧基红紫素18酰亚胺甲酯和转铁蛋白的相互作用

用荧光光谱法、紫外-可见分光光度法研究了3-环丙基-3-去乙烯基红紫素18甲酯(MRNB-2)、3-环丙基-3-去乙烯基-N-甲氧基红紫素18酰亚胺甲酯(MRNB-4)与转铁蛋白(Tf)的相互结合反应.实验表明,MRNB-2,MRNB-4与Tf的相互结合作用为单一的静态猝灭过程,在溶液中二者以1∶1的摩尔比牢固结合,25℃时结合反应的平衡常数分别为KMRNB-2=7.64×105L/mol,KMRNB-4=2.09×104L/mol.根据Frster非辐射能量转移机理,求算了给体(Tf)与受体(MRNB-2,MRNB-4)间距离(r)和能量转移效率(E)分别为rMRNB-2=3.85 nm,rMRNB-4=4.14 nm;EMRNB-2=0.340,EMRNB-4=0.127.并推测二者之间的主要作用力为疏水作用力.     

氯化血红素与牛血清白蛋白的作用及表面活性剂对其的影响研究

利用荧光光谱法研究了在水溶液和表面活性剂溶液中氯化血红素(hemin)与牛血清白蛋白(BSA)的相互作用,用Stern-Volmer方程、Lineweaver-Burk方程及热力学方程对实验数据进行处理,得到了在不同的pH值、温度及表面活性剂溶液中的hemin和BSA作用的结合常数、结合点位数及热力学常数,在pH=7.00,T=303 K时的水溶液中,Ka=1.35×107,n=1.32,△G=-41.36 kJ.mol-1,△H=-51.45 kJ.mol-1,T△S=-10.09 kJ.mol-1,研究结果表明其猝灭作用为静态猝灭,作用力主要为氢键和范德华力.在实验条件下表面活性剂使实验体系在346 nm处的荧光峰兰移并且其强度有所降低.     

乙氧基血根碱与人血清白蛋白的相互作用

研究了血根碱与人血清白蛋白（HSA）之间的结合特征．采用荧光光谱法,计算在不同温度下乙氧基血根碱与HSA相互作用的结合常数与结合位点数．实验结果显示,在290K,300K,310K时乙氧基血根碱与人血清白蛋白的结合常数分别为1．081×10^5L·mol-1,7．784×10^4L·mol-1,2．397×10^5L·mol-1．结合位点数分别为1．013,0．979,1．071．二者之间的主要作用力为氢键和范德华力．这表明血根碱与人血清白蛋白之间作用生成了无荧光效应的复合物,属静态猝灭．     

荧光光谱法研究3-吡唑啉基叶绿素-a衍生物和牛血清白蛋白的相互作用

用荧光光谱法、同步荧光光谱、紫外-可见分光光度法研究3-吡唑啉卟吩f-2甲酯（Mf2-5）、3-吡唑啉卟吩f-3甲酯（Mf3-5）与牛血清白蛋白（BSA）的相互结合反应．实验表明Mf2—5、Mf3—5与牛血清白蛋白的相互结合作用为静态猝灭过程,在溶液中二者以物质的量之比1：1牢固结合,25℃时其结合反应的平衡常数分别为：KMf2-5=6．14×10^5L·mol^-1,KMf3—5=1．02×10^5L·mol^-1．根据Ft~rster非辐射能量转移机理,求算了给体（BSA）与受体（Mt2-5、Mf3-5）间距离r和能量转移效率E分别为：rMf2-5=4．48nm,rMf3-5=4．86nm,EMf2-5：0．24,EMf3—5=0．20．并推测了二者之间的主要作用力为疏水作用力．     

Binding of vanadium compounds perturbs conformation and aggregation state of insulin

The interactions between zinc-free insulin and vanadium compounds, NaVO3, VO(acac)2 and VO(ma)2, have been investigated by fluorescence spectroscopy, circular dichroism (CD) and Fourier-transformed infrared (FT-IR) spectroscopy. The results showed that binding of vanadium compounds produced a static quenching of the intrinsic fluorescence of insulin. The apparent association constants were determined to be (0.17±0.01)×104 L*mol-1 for NaVO3, (2.8±0.2)×104 L*mol-1 for VO(acac)2, and (4.0±0.1)×104 L*mol-1 for VO(ma)2, respectively. The light scattering intensity of insulin decreased upon incubation with the vanadium compounds, suggesting the disaggregation of insulin. The attenuation of the band at 273 nm of insulin CD spectra also supported the disaggregation of insulin observed above. A new band at 1650～1653 cm-1 appeared in the FT-IR spectra of insulin upon incubation with the vanadium compounds, indicating the formation of an α-helix structure at B (9-19) motif. This α-helix structure suggests a structural change of insulin from an extended conformation (T state) to a helical conformation (R state), which is essential for binding of insulin to its receptor. In conclusion, binding of vanadium compounds results in conformational changes and disaggregation of insulin. These changes might account for the enhancement of binding affinity for insulin to its receptor in the presence of vanadium compounds.     

Metarhodopsin I/metarhodopsin II transition triggers light-induced change in calcium binding at rod disk membranes

The hypothesis of Yoshikami and Hagins that calcium ions act as diffusible transmitter molecules between the photochemistry of rhodopsin and the subsequent electrical events at the outer plasma membrane of rods initiated many investigations on light-stimulated calcium release in vertebrate photoreceptor cells (see refs 2, 3). Although it not seems firmly established that light has some effect on the redistribution of calcium in various disk preparations, reconstituted systems and intact rod outer segments, the physiological significance remained unclear. We previously reported a rapid, light-triggered calcium release from binding sites at the disk membrane in the presence of calcium ionophore A23187 (refs 3, 8). However, there is no evidence for rapid calcium release into the cytosol in the absence of ionophore. On fragmentation of intact rod outer segments, calcium release due to a light-requlated change of calcium binding appeared almost completely abolished. We describe here experiments with sonicated rod outer segments in which the previously observed loss of the calcium release capacity has been prevented. Calcium release in sonicated disks in the presence of A23187 kinetically follows the metarhodopsin I/metarhodopsin II transition (tau 1/2 = 10 ms, activation energy EA = 34 kcal mol-1), suggesting that calcium release is triggered by this photochemical transition.     

Reverse hydrophobic effects relieved by amino-acid substitutions at a protein surface

It is rare for amino-acid substitutions on the surface of proteins to have large stabilizing or destabilizing effects. Nevertheless, one substitution of this type, the Tyr 26----Cys mutation in lambda Cro, increases the melting temperature of the protein by 11 degrees C and the stability by 2.2 kcal mol-1. Here we show that the stability of Cro can be increased by many different amino-acid substitutions at position 26, with increasing stability showing a good correlation with decreasing side-chain hydrophobicity. As Tyr 26 is hyper-exposed to solvent in the Cro crystal structure, we suggest that wild-type and variant proteins with other hydrophobic side chains at position 26 are destabilized as a result of a reverse hydrophobic effect caused by the side chain being more exposed to solvent in the native than in the denatured state.     

The mechanism of OTUB1-mediated inhibition of ubiquitination

Histones are ubiquitinated in response to DNA double-strand breaks (DSB), promoting recruitment of repair proteins to chromatin. UBC13 (also known as UBE2N) is a ubiquitin-conjugating enzyme (E2) that heterodimerizes with UEV1A (also known as UBE2V1) and synthesizes K63-linked polyubiquitin (K63Ub) chains at DSB sites in concert with the ubiquitin ligase (E3), RNF168 (ref. 3). K63Ub synthesis is regulated in a non-canonical manner by the deubiquitinating enzyme, OTUB1 (OTU domain-containing ubiquitin aldehyde-binding protein 1), which binds preferentially to the UBC13～Ub thiolester. Residues amino-terminal to the OTU domain, which had been implicated in ubiquitin binding, are required for binding to UBC13～Ub and inhibition of K63Ub synthesis. Here we describe structural and biochemical studies elucidating how OTUB1 inhibits UBC13 and other E2 enzymes. We unexpectedly find that OTUB1 binding to UBC13～Ub is allosterically regulated by free ubiquitin, which binds to a second site in OTUB1 and increases its affinity for UBC13～Ub, while at the same time disrupting interactions with UEV1A in a manner that depends on the OTUB1 N terminus. Crystal structures of an OTUB1-UBC13 complex and of OTUB1 bound to ubiquitin aldehyde and a chemical UBC13～Ub conjugate show that binding of free ubiquitin to OTUB1 triggers conformational changes in the OTU domain and formation of a ubiquitin-binding helix in the N terminus, thus promoting binding of the conjugated donor ubiquitin in UBC13～Ub to OTUB1. The donor ubiquitin thus cannot interact with the E2 enzyme, which has been shown to be important for ubiquitin transfer. The N-terminal helix of OTUB1 is positioned to interfere with UEV1A binding to UBC13, as well as with attack on the thiolester by an acceptor ubiquitin, thereby inhibiting K63Ub synthesis. OTUB1 binding also occludes the RING E3 binding site on UBC13, thus providing a further component of inhibition. The general features of the inhibition mechanism explain how OTUB1 inhibits other E2 enzymes in a non-catalytic manner.     

四(对甲氧基苯基)卟啉锌的合成及其与牛血清白蛋白的相互作用

合成了四(对甲氧基苯基)卟啉锌,利用荧光光谱法分别研究了pH值、溶剂和四(对甲氧基苯基)卟啉锌的浓度对四(对甲氧基苯基)卟啉锌与牛血清白蛋白相互作用的影响.由实验数据计算出了该卟啉与牛血清白蛋白的双分子猝灭过程速率常数Kq=4.23×1012L.mol-1.s-1、结合常数KA=3.48×105L.mol-1和结合数n=1.19.结果表明,四(对甲氧基苯基)卟啉锌与牛血清白蛋白之间发生了荧光猝灭作用.     

用DLTS和ODLTS技术研究ZnSe晶体中与Ga有关的深能级

本文应用深能级瞬态谱(DLTS)和光深能级瞬态谱(ODLTS)技术研究了掺Ga的ZnSe晶体中的深能级.发现ZnSe:Ga晶体中有一个与Ga有关的施主能级位于导带底下0.17eV处,两个与Ga有关的受主能级分别位于价带顶上0.65eV和0.72eV处.文中还对这些能级的起源进行了讨论.     

聚苯乙烯微球中荧光共振能量转移的研究

采用分散共聚合法制备了单分散的负载有1,8-萘酰亚胺染料的荧光微球。利用供体和受体染料间的荧光共振能量转移,荧光微球在激发供体染料时同时实现了供体和受体染料的双重荧光发射信号。结果表明:当微球中染料的浓度相对较低(质量分数小于0.2%)时,荧光共振能量就可以有效地发生转移。聚合过程中增加供体或受体染料的浓度都会提高能量转移效率,这是浓度增加使得染料分子间距离缩短造成的。通过调整微球中染料浓度可以获得不同能量转移效率、具有荧光编码信号的系列荧光微球。这类粒径均一的荧光微球在多元生物分析中有潜在的应用价值。     

Reaction of CH radical with O<Subscript>2</Subscript> by time-resolved FTIR spectroscopy

The Methylidyne radical, CH, plays a crucial role in several complex environments such as planetary atmos- pheres and interstellar clouds, and is one of the most im- portant and active intermediate in hydrocarbon flames[1]. The CH involving reactions, including chemiionization, oxidation of hydrocarbons, the soot formation[2—5], and especially the formation of prompt NO[6], are of funda- mental interest in combustion chemistry, owing to the high reactivity and the large enthalpy of formatio…     

激光场和温度对GaAs/Ga_(1-x)Al_xAs量子阱中施主杂质态的影响

利用有效质量近似和变分原理,研究了激光场和温度对GaAs/Ga1-xAlxAs量子阱中基态施主束缚能的影响.结果显示,基态施主束缚能随着激光场的增强而减小,尤其是对于量子阱中心处的杂质,激光场的影响更明显.而且激光场对窄阱中杂质的施主束缚能有明显的影响.基态施主束缚能随着温度的增加而减小,随着Al含量的增加而增加,随着量子阱宽的增大而减小.并且随着阱宽的进一步增大,束缚能减小的趋势变慢.     

一种基于综合信息的剪接位点识别方法

为提高剪接位点识别的精度,提出一种基于综合信息的剪接位点识别方法.通过分析供体位点与受体位点的剪接信号、剪接序列、位点附近序列的二级结构,以及剪接因子作用过程等特征,分别为供体位点与受体位点建立信号模型和序列模型;应用Vienna软件中的Mfold包预测每个剪接位点附近序列最稳定的二级结构,将传统的四字符核酸表转化为八字符核酸表,每个序列用八字符进行描述,用结合了结构信息的序列对信号模型和序列模型进行训练学习;最后用训练好的模型进行剪接位点的识别.实验结果证明:该方法对剪接位点的识别取得了很好的效果,其识别精度可达95%以上.     

荧光光谱法研究茜素红S与壳聚糖的结合反应

研究生物探针茜素红S和壳聚糖溶液的荧光光谱特征,用荧光光度法、分光光度法研究茜素红S与壳聚糖分子之间的结合反应,结果表明:其能量转移机制为非辐射能量转移,论证了茜素红S与壳聚糖结合作用为静态猝灭过程(Kq=2.958 1×1011L.mol-1.s-1).测定了其结合常数(KA=3.26×103)和结合位点数(n=102).