荧光研究表明去辅基天青蛋白突变体M121L至少存在两种不同的构象

以往对绿脓杆菌去辅基天青蛋白变性机制的研究认为它经历了一个复杂的反应过程,相比之下,锌离子替代的天青蛋白的变性符合简单的二态模型。以脲为变性剂对去辅基天青蛋白突变体M121L的变性过程进行了研究。结果表明,虽然稳态条件下突变体的变性／复性符合二态模型,但其动力学过程复杂,并可用溶液中存在着两种可以相互转化的构象的变性／复性来解释。天然态N1去折叠的速度快,其重折叠的速度也快,N1的折叠机制可用存在着折叠途径上的快速折叠中间体模型来描述;天然态N2的去折叠速度慢,其重折叠主要是首先生成天然态N1,然后再缓慢地转化成N2。添加Zn^2 能够把两种构象整合成一种构象,相应地,Zn^2 替代的天青蛋白突变体的变性过程简化为单指数过程。对该突变体的研究加深了对天青蛋白去折叠机制的理解。     

鸡肝二氢叶酸还原酶在盐酸胍溶液中构象的序变与激活

用蛋白质内源荧光、疏水荧光探针TNS及蛋白酶K限制性酶解等方法研究了二氢叶酸还原酶在盐酸胍变性过程中的构象变化及动力学,并与活力变化进行了比较.TNS可以监测到与激活同步的构象变化;盐酸胍浓度大于0.75mol/L时,二氢叶酸还原酶被蛋白酶K水解速度增大;当盐酸胍浓度大于1.2mol/L时,才能监测到酶分子整体构象的变化.以上结果表明二氢叶酸还原酶在盐酸胍溶液中的变性并不符合标准的二态模型,而是先经历构象逐步松散的序变过程,然后发生协同的构象伸展.二氢叶酸还原酶在低浓度盐酸胍溶液中的激活是由于酶活性部位构象的微小变化引起的.酶活性部位构象的变化虽然降低了酶与废物的结合能力,但加快了酶促反应限速步骤,即底物解离速度而使酶活力升高.     

二氢叶酸还原酶天然状态可能存在两种构象

鸡肝二氢叶酸还原酶（ＤＨＦＲ）平衡态的去折叠曲线符合二态模型,但在４．０ｍｏｌ／Ｌ脲中的去折叠动力学为两相。该酶的ＡｒｒｅｈｉｕｓＰｌｏｔ有一个拐点,但在低浓度变性剂存在下,拐点消失。用二氢叶酸还原酶的天然状态存在两种构象可以很好地解释上述现象。二氢叶酸还原酶去折叠过程中没有稳定存在的中间体,动力学过程中的两相可能是两种天然构象态的去折叠速度常数不同造成的。ＡｒｒｅｈｉｕｓＰｌｏｔ的拐点是由于在不同的温度条件下,两种天然构象的含量不同造成的,低浓度变性剂对两种构象的影响不同,使拐点消失。ＫＣｌ可以改变两种构象的平衡     

锌离子对氨基酰化酶构象及其稳定性的影响

天然氨基酰化酶和脱谷氨基酰化酶无论在二级结构（用ＣＤ和ＦＴＩＲ监测）还是三级结构上（以荧光发射光谱监测）都有明显的差异,表明了脱锌后酶的有序度降低;当比较天然和脱锌氨基酸化酶对去圬剂的稳定性时,结果表明脱锌后酶的构象的稳定性明显降低．因此可以认为锌离子对维持酶分子活性部位的特定构象以及构象的稳定性具有重要的作用．     

碱提工艺对茶树菇多糖形貌特征和构象及其生物活性的影响

利用传统水提及碱提的方法得到茶树菇粗多糖S-ACP和J-ACP,经CTAB法和Sephadex G-150凝胶层析法对其分离纯化,分别得到S-ACP2-1和S-ACP2-2以及J-ACP2-1和J-ACP2-2两组主要组分,用扫描电子显微镜（SEM）和原子力显微镜（AFM）对多糖的形貌进行表征并测定其体外抗氧化活性和抗肿瘤活性;对多糖S-ACP2-2、J-ACP2-2进行刚果红实验测定及圆二色谱仪（CD）分析。SEM观测结果：S-ACP2-1为较粗的表面光滑的丝状,J-ACP2-1呈较细的有少量碎屑的丝状;S-ACP2-2为较大的片状,J-ACP2-2在大的片状周围有很多细小的碎屑。AFM观测结果：碱液可以使多糖分子部分断裂成小片段。刚果红实验：S-ACP2-2、J-ACP2-2在水溶液中为自由卷曲构型。CD分析：S-ACP2-2的空间构型中有序结构较少,J-ACP2-2在水溶液中为无序构型。对比4种多糖的活性,碱液作用的多糖J-ACP2-2活性高于S-ACP2-2。     

肺炎链球菌血红素结合脂蛋白PiuA的表达、纯化和表征

【背景】肺炎链球菌是社区获得性肺炎最常见的致病菌之一,它也会引起脑膜炎、鼻窦炎、中耳炎、菌血症等一系列疾病,对人类(特别是儿童、老人、免疫缺陷患者)健康造成重大威胁。铁是肺炎链球菌生存和感染所必需的元素之一,其中血红素转运系统PiuABCD是肺炎链球菌最重要的铁转运系统。【目的】克隆、表达和纯化肺炎链球菌血红素转运系统脂蛋白PiuA,并在体外表征PiuA蛋白的血红素结合特性。【方法】将肺炎链球菌D39菌株中的piuA(spd_1652)基因连接到载体pBAD-HisA上,在大肠杆菌Top10菌株中进行异源表达,然后运用Ni-NTA亲和层析纯化PiuA-His蛋白,并用肠激酶切掉His标签获得不含标签的PiuA蛋白,最后运用圆二色谱、紫外光谱和荧光光谱表征PiuA蛋白的血红素结合特性。【结果】构建了pBAD/HisA-PiuA重组表达载体,获得了纯度大于95%的PiuA蛋白,圆二色谱显示PiuA蛋白与Hemin结合后,其二级结构不发生改变;紫外光谱结果显示PiuA蛋白具有血红素结合能力;荧光光谱结果显示apo-PiuA蛋白与Hemin结合常数K=3.4×10~5 L/mol。【结论】肺炎链球菌血红素转运系统脂蛋白PiuA能够特异地结合血红素,为肺炎链球菌的生存和感染提供必需的铁源,PiuA蛋白的体外表征结果为针对PiuABCD血红素转运系统设计抗菌药物奠定了基础。     

圆二色谱激子手笥法进展及其在天然产物绝对构型确定中的应用

本文综述了圆二色谱激子手性法进展及其在天然产物绝对构型确定中地应用。     

圆二色谱激子手性法进展及其在天然产物绝对构型确定中的应用

本文综述了圆二色谱激子手性法进展及其在天然产物绝对构型确定中的应用.     

天花粉蛋白小结构域N端同源片段的溶液构象：T18肽的圆二色性和荧光研究

应用圆二色性,内源荧光和疏水荧光探针法进一步研究Ｔｌ８肽的溶液构象及其相互转化。发现Ｔ１８肽在水溶液中为β折叠结构,且在高浓度（＞１ｍｇ／ｍｌ）时形成疏水聚合物;Ｌｙｓ１５和Ｉｌｅ３－Ｉｌｅ４是形成β折叠疏水簇的关键因素。并讨论了蛋白质肽链和溶剂环境对肽段二级结构的调制作用。     

马槟榔甜味蛋白的圆二色谱和激光拉曼光谱

测定了自马槟榔(Capparis masaikai Levl.)种子分离的二种甜味蛋白MaⅠ和MaⅡ的远紫外区域圆二色谱,并按Yang和Chen的方法用最小二乘法计算了它们的构象单元含量,结果表明α螺旋含量最多:对MaⅠf_H=0.43、f_β=0.24、f_R=0.33;对MaⅡ,f_H=0.37、f_β=0.33、f_R=0.30;其相关系数均为0.9876;计算所得理论曲线与实验曲钱其本吻合。二种甜蛋白的激光拉曼光谱测定结果也表明其主要构象单元为α螺旋,此外,均无SH谱带:MaⅠ的Tyr残基暴露于分子表面;与MaⅡ相比,MaⅠ有明显的545cm~(-1)和1101cm~(-1)谱带,这可能从构象上说明MaⅠ与MaⅡ对热和变性剂处理表现不同的原因。     

圆二色光谱揭示光系统Ⅱ在热处理过程中叶绿素荧光动力学变化的原因

研究了光系统Ⅱ(PSⅡ)在热处理过程中的叶绿素a荧光和圆二色(CD)光谱.在30℃～40℃热处理过程中,PSⅡ的叶绿素荧光参数Fo′保持稳定不变;当温度大于40℃时,Fo'逐渐升高并在55℃达到最大值.在PSⅡ颗粒和富含捕光色素(LHCⅡ)的复合物的热处理过程中,具有超大振幅的CD异常信号出现,并且在40℃时,677 nm的异常CD峰强度达到最大.这些结果暗示在PS Ⅱ颗粒热处理过程中,PSⅡ颗粒中的LHCⅡ的聚集状态和异常CD信号相关,并且也可能是影响Fo′的一个重要因素.     

Spectroscopic and chemical studies of the interaction between nerve growth factor (NGF) and the extracellular domain of the low affinity NGF receptor.

Nerve growth factor (NGF) interacts with a cell surface receptor on responsive neurons to initiate a series of cellular events leading to neuronal survival and/or differentiation. The first step in this process is the binding of NGF to a low affinity and/or a high affinity receptor. In the present report, we have studied the conformation and stability of recombinant receptor extracellular domain (RED) from the human low affinity receptor and the structural basis of its interaction with NGF. Circular dichroism (CD) studies indicate that the RED is primarily random coil in nature with little regular secondary structure. Thermal stability studies have shown that this irregular conformation is a specific structure that can undergo a reversible two-state thermal denaturation with a concomitant fluorescent and CD change. During heating at 100 degrees C for 15 min, the structure of RED is sufficiently unfolded for a reducing agent, dithiothreitol, to inactivate the receptor toward NGF binding and cross-linking. The complex formation between the RED and NGF has been examined by differential CD measurements, and we have shown that a small, reproducible change in conformation occurs in RED or NGF upon interaction. These results are interpreted in terms of the initiation of NGF cell surface binding and possible modes of signal transduction.     

Structural studies on the freezing-point-depressing protein of the winter flounder Pseudopleuronectes americanus

The freezing-point-depressing protein from the winter flounder, $\text{Pseudopleuronectes americanus}$ has been shown from circular dichroism measurements to possess a large proportion (～85%) of the α-helical conformation in aqueous solution (pH 8.0) at ?1°C. The helical content decreases as the temperature is raised. Viscosity data at ?1°C indicate an asymmetric shape for the protein molecule compatible with its high helical content. Thus, the secondary and tertiary structure of this freezing-point-depressing protein as well as its primary structure (reported elsewhere), are found to be different from its counterpart glycoproteins isolated from the Antarctic fish.     

Pseudomonas aeruginosa EftM Is a Thermoregulated Methyltransferase

Pseudomonas aeruginosa is a Gram-negative opportunistic pathogen that trimethylates elongation factor-thermo-unstable (EF-Tu) on lysine 5. Lysine 5 methylation occurs in a temperature-dependent manner and is generally only seen when P. aeruginosa is grown at temperatures close to ambient (25 °C) but not at higher temperatures (37 °C). We have previously identified the gene, eftM (for EF-Tu-modifying enzyme), responsible for this modification and shown its activity to be associated with increased bacterial adhesion to and invasion of respiratory epithelial cells. Bioinformatic analyses predicted EftM to be a Class I S-adenosyl-l-methionine (SAM)-dependent methyltransferase. An in vitro methyltransferase assay was employed to show that, in the presence of SAM, EftM directly trimethylates EF-Tu. A natural variant of EftM, with a glycine to arginine substitution at position 50 in the predicted SAM-binding domain, lacks both SAM binding and enzyme activity. Mass spectrometry analysis of the in vitro methyltransferase reaction products revealed that EftM exclusively methylates at lysine 5 of EF-Tu in a distributive manner. Consistent with the in vivo temperature dependence of methylation of EF-Tu, preincubation of EftM at 37 °C abolished methyltransferase activity, whereas this activity was retained when EftM was preincubated at 25 °C. Irreversible protein unfolding at 37 °C was observed, and we propose that this instability is the molecular basis for the temperature dependence of EftM activity. Collectively, our results show that EftM is a thermolabile, SAM-dependent methyltransferase that directly trimethylates lysine 5 of EF-Tu in P. aeruginosa.     

Trichotoxin A-40, a new membrane-exciting peptide. Part A. Isolation,characterization and conformation

The new polypeptide antibiotic trichotoxin A-40 is isolated by chloroform/methanol extraction from the dry mycelium of Trichoderma viride NRRL 5242. The lipophilic peptide is purified by chromatography on Kieselgel H-60 and reverse-phase chromatography on Lichrosorb RP-8. The new antibiotic differs in amino acid composition and various chemical and physicochemical properties from similar peptides such as trichotoxin A, the suzukacillins or alamethicins. The amino acid composition is (Pro)₁ (Gly)₁ (Ala)₂ (Leu)₂ (Aib)₁₀ (Glx)₂. (Aib, α-aminoisobutyric acid.) The antibiotic has a carboxyl group which can be esterified by diazomethane, which results in slightly enhanced membrane-modifying activities.The peptide exhibits a right-handed α-helical conformation increasing about two-fold from aqueous to lipophilic media as shown by solvent-dependent circular dichroism measurements. Most of the ¹³C-NMR resonances can be assigned unequivocally and amino acids situated in the α-helical part show characteristic shift differences from those in the non-helical regions. No β-phenylalaninol residue could be identified by ¹³C-NMR and ultraviolet spectroscopy, as can be for alamethicins and suzukacillins. A pronounced hemolytic action is found on human erythrocytes, which develops at micromolar concentrations. Trichotoxin A-40 induces a voltage-dependent ionic conductance in bilayer lipid membranes and it can serve as a new pore-forming model system for structure/activity studies in membrane excitation by peptides.     

An ochre suppressor of bacteriophage T4 that is associated with a transfer RNA

Ultraviolet circular dichroism spectra have been obtained for native and heat-denatured Drosophila virilis satellite DNAs I, II and III. Gall &; Atherton (1974) have found that these DNAs have simple, unique sequences. We compare here the circular dichroism spectra of these satellite sequences with the circular dichroism spectra of synthetic DNAs of simple sequences which are combined in first-neighbor calculations. We also apply an analytical procedure for determining nearest-neighbor frequencies from the DNA spectra (Allen et al., 1972). The results are an indication of the potential usefulness and present limitations of circular dichroism measurements in confirming or determining the nearestneighbor frequencies of satellite DNAs of simple sequences.     

Optical studies of a conformational change in DNA before melting

The circular dichroism of double-stranded DNA is temperature dependent prior to its melting. As the temperature is increased the spectrum becomes more nonconservative. This is certainly due to a conformational change within the framework of the double helix. To ascertain the nature of the conformational change, a series of synthetic and natural DNA's from a variety of sources was investigated. The same qualitative changes were seen for all the DNA samples, independent of base composition. However, there were definite quantitative differences, with poly [d(A-T)] manifesting the largest effect. Oligomers of the form [d(A-T)]_n with n = 10 to 21 behaved in a manner similar to the polymer. There is no observed chain-length dependence. The breadth of the pre-melt transition indicates a low ΔH (less than 5 kcal./mole); the lack of dependence on chain length indicates that the co-operative unit is smaller than eight base pairs.