γ辐射对红细胞膜流动性的影响

我们实验以红细胞膜作为材料,使用ANS和DPH荧光探针测量红细胞膜的荧光强度和偏振度。发现γ辐照红细胞膜剂量从4.6×10~3拉德增至148×10~3拉德,而ANS和DPH的荧光强度随剂量的增加而降低,荧光偏振度随剂量的增加而增加,这些结果表明γ辐照引起红细胞膜流动性降低。但荧光强度的减少并不伴随荧光光谱的变化。ANS与γ辐照的膜结合量也没有改变,因此荧光强度的减少可能是由于在辐照的膜中ANS荧光量子产率的降低。在我们的实验中γ辐射对ANS探针所引起的荧光变化较DPH灵敏,看来γ辐射首先影响膜蛋白。     

高通量脂肪酶稳定性测定法-ANS法的建立

根据蛋白质变性后结构变化的特点和荧光探针8-苯胺基-1-萘磺酸 (8-Anilino-1-naphthalenesulfonic acid,ANS) 的特性,建立了一种快捷而准确的脂肪酶热稳定性 (Tm) 测定的新方法——ANS脂肪酶稳定性测定法,即将脂肪酶在25 ℃~65 ℃的不同温度下保温30 min后,加入到ANS反应体系 (0.2 mg/mL酶蛋白,0.05 mmol/L ANS,20 mmol/L Tris-HCl (pH 7.2),100~500 mmol/L NaCl) 中。在激发波长378     

辣根过氧化物酶同功酶B、C构象的毫微秒萤光研究

本文利用1,8-ANS作萤光探针,通过毫微秒萤光技术研究了辣根过氧化物酶的同功酶B、C(文中以HRP(B)、HRP(C)表示)的构象,测得ANS和脱辅基HRP(B)、HRP(C)复合物的萤光寿命分别为19.6毫微秒和17.7毫微秒(即10~(-9)秒,用ns表示),从而证实同功酶B、C分子中确有较强的疏水区域,且这个疏水区域就在分子中血红素(heme)的结合区附近。通过萤光寿命的测定和研究,我们发现,HRP(B)、HRP(C)分子中的疏水区域的疏水性比牛血红蛋白分子中的要弱,比肌红蛋白分子中的要强;HRP(B)的疏水区的疏水性要略强于HRP(C)的。我们还研究了溶液的pH条件及不同浓度的脲对该疏水区构象的影响,观察到一个颇有意思的现象,即和牛血红蛋白一样,1Mal.的脲使血红素结合区的疏水性增强,1Mal.以上浓度的脲却使该区域疏水性减弱。在不同的pH条件下,血红素结合区域的疏水性亦有变化,其中,pH7条件下该区的疏水性最强。     

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.     

膜融合剂对脂质体及血影膜膜流动性的影响

本文用荧光探针ANS,DPH与A研究了几种膜融合剂对脂质体与血影膜流动性的影响.蔗糖使PS脂质体的脂双层流动性降低,探针越是在极性区流动性越小,说明蔗糖主要作用于脂双层的极性区;蔗糖也使血影膜流动性降低,此作用是可逆的.油酸甘油脂(GMO)使PS脂质体的流动性增加,且越是在疏水区内部,流动性增加得越大,说明GMO主要是作用于脂双层的非极性区:GMO也使血影膜流动性增加,此作用是不可逆的.二甲亚砜(DMSO)对血影膜的作用,两种不同荧光探针不一样,对DPH的作用出现双相让,低浓度与高浓度的作用结果分别与蔗糖和GMO的作用一致.     

培养介质pH和EGTA对水霉(Saprolegnia ferax)菌丝细胞肌动蛋白的分布与结构的影响

菌丝在pH 5.0—8.0介质中维持顶端生长,Rhodamin-phalloidin荧光探针显示在菌丝顶端都存在F-actin的“帽子”结构;加入EGTA到培养介质中不影响菌丝的顶端生长和actin的“帽子”结构。值得注意的是:菌丝的Rhodamin-phalloidin荧光强度大小与菌丝顶端生长速率成正比;在含有或不含有EGTA的pH5.0培养条件下,菌丝的生长速率均很低,且后部颗粒状的荧光斑点消失;在pH 3.0-4.0培养介质中菌丝生长停止,不但F-actin“帽子”结构消失,整个菌丝荧光也变得非常微弱无法观察,提示酸性pH可引起F-actin的解聚,从而导致生长速率下降甚至生长停止。     

渗透胁迫对小麦根质膜膜脂物理状态的影响

以两相法纯化的小麦（TriticumsativumL．）根质膜微囊为材料,研究了渗透胁迫下质膜物理状态的变化。结果表明,随着介质蔗糖浓度增加,质膜光散射值降低,二苯己三烯（DPH）荧光偏振值升高,MC540荧光强度增强,并且DPH长寿命组分的荧光寿命和平均寿命都缩短,暗示渗透胁迫使质膜微囊收缩变小,降低了质膜流动性和表面电荷密度,并且表明质膜的疏水性减弱。进一步实验发现,质膜内源色氨酸长寿命组分的寿命缩短,质膜H －AT－Pase活力降低,暗示膜蛋白的构象和功能发生了改变。结果表明,质膜膜脂物理状态改变可能是植物感受渗透胁迫的原初响应。     

去辅基天青蛋白两种天然构象共存的热力学证据 ———差热温度扫描和圆二色的研究

天然态蛋白质能否在溶液中存在多种构象是一个有争议的问题 . 在前报道中已经鉴定出绿脓杆菌去辅基天青蛋白突变体 M121L 可以多种构象共存 . 用差热扫描量热和圆二色性的方法研究了野生型去辅基天青蛋白的热变性 . 结果表明在 pH 从 4.0 到 9.0 的范围内存在着两个摩尔热容最大值 . 较低温度下的去折叠反应在所研究 pH 范围内均部分可逆,而较高温度下的去折叠反应均不可逆 . 蛋白质去折叠的热容变化双峰用可相互转化的两种构象共存模型进行拟合 . 较低温度下能够去折叠的构象在 pH 4.0 时占 64% ,在 pH 9.0 时占 55%. 监测热变性过程中圆二色谱在 219 nm 处的信号变化也可以观测到两个独立的去折叠变化 . 信号变化的比值与在相同条件下差热扫描法测得的两种构象摩尔比一致 . 上述结果进一步支持了前文提出的去辅基天青蛋白在溶液中至少存在着两种构象的设想 .     

IgG-Eu-IDPA对抗原IOV的时间分辨荧光免疫检测

镧系螯合物已经被广泛应用于高灵敏度的时间分辨荧光免疫分析.但是,使用紫外光激发镧系螯合物会对生物分子和细胞产生很大的损害.合成了能够被可见光(最大波长565nm)激发的IgG-Eu-IDPA,并测量了IgG-Eu-IDPA的光谱属性,如荧光寿命、在不同pH值和不同浓度环境下的荧光强度.在自制的时间分辨荧光仪上使用IgG-Eu-IDPA作为荧光探针,检测IOV抗原.数据显示,它的灵敏度远远高于传统的荧光仪.结果表明,IgG-Eu-IDPA能够在高灵敏度的原位和活体分析中作为一种新的、有潜力的荧光探针.     

基于双荧光和单荧光探针的溶酶体pH值测定方法比较

目的:通过比较基于双荧光探针和单荧光探针的2种溶酶体pH值测定方法,探究一种不依赖四甲基若丹明(TMR)的、基于异硫氰酸荧光素(FITC)单荧光的检测溶酶体pH值方法的可行性。方法:利用人肺癌肺泡基底上皮来源的A549细胞,分别采用FITC/TMR双荧光探针法和FITC单荧光双激发法测定溶酶体pH值;通过计算相对荧光强度的比值FI_(FITC488)/FI_(TMR561)或FI_(FITC488)/FI_(FITC405),绘制pH值标准曲线,用于pH值测定。结果:相对于FITC在Ex488 nm/Em520 nm波长下的荧光强度对pH值极为敏感,其在Ex405 nm/Em422 nm波长下对pH值不敏感,可用于荧光矫正;不同pH值条件下,FI_(FITC488)/FI_(FITC405)比值基本呈线性关系。结论:FITC单荧光双激发法只须使用一种荧光探针即可实现pH值检测,较FITC/TMR双荧光探针法更不易产生系统误差,更适用于溶酶体pH值检测。     

A conformational change in bovine beta-lactoglobulin at low pH.

A conformational change at low pH in bovine beta-lactoglobulin A has been studied by intrinsic fluorescence and fluorescence of the bound dye 8-anilinonaphthalene-1-sulphonate. Both studies show that when the pH of beta-lactoglobulin solutions is altered between 6.5 and 2.0, a rapid change in protein conformation occurs, followed by a slower conformational change. It seems likely that the rapid changes are linked with the predominance of protein dimer at pH 6.5 and monomer at pH 2.0. The slow changes involve shifts in protein conformation of the region that includes one of the protein tryptophan residues.     

Phosphorylation alters the pH-dependent active state equilibrium of rhodopsin by modulating the membrane surface potential.

Phosphorylation reduces the lifetime and activity of activated G protein-coupled receptors, yet paradoxically shifts the metarhodopsin I-II (MI-MII) equilibrium (K(eq)) of light-activated rhodopsin toward MII, the conformation that activates G protein. In this report, we show that phosphorylation increases the apparent pK for MII formation in proportion to phosphorylation stoichiometry. Decreasing ionic strength enhances this effect. Gouy-Chapman theory shows that the change in pK is quantitatively explained by the membrane surface potential, which becomes more negative with increasing phosphorylation stoichiometry and decreasing ionic strength. This lowers the membrane surface pH compared to the bulk pH, increasing K(eq) and the rate of MII formation (k(1)) while decreasing the back rate constant (k(-)(1)) of the MI-MII relaxation. MII formation has been observed to depend on bulk pH with a fractional stoichiometry of 0.6-0.7 H(+)/MII. We find that the apparent fractional H(+) dependence is an artifact of altering the membrane surface charge during a titration, resulting in a fractional change in membrane surface pH compared to bulk pH. Gouy-Chapman calculations of membrane pH at various phosphorylation levels and ionic strengths suggest MII formation behavior consistent with titration of a single H(+) binding site with 1:1 stoichiometry and an intrinsic pK of 6.3 at 0.5 degrees C. We show evidence that suggests this same site has an intrinsic pK of 5.0 prior to light activation and its protonation before activation greatly enhances the rate of MII formation.     

Effect of deprotonation on the conformational state of the tetracycline molecule]

Conformation rearrangements in the tetracycline molecule under the effect of the aqueous medium pH were investigated by circular dichroism and absorption spectrophotometry. Possible causes of the changes in the spectrum after the molecule deprotonation are discussed. An increase in the pH value was accompanied by folding-unfolding of the A-ring of the tetracycline molecule and its transfer within the pH neutral ranges to the conformation close to planar one and after increasing of the aqueous solution polarity to the folded conformation. The BCD chromophore did not change its initial flat conformation during deprotonation. The loss of the proton (4) by the nitrogen atom was accompanied by changes in the A chromophore chirality.     

不同pH条件下R-藻红蛋白和C-藻蓝蛋白荧光寿命的研究

采用自己研制的时间分辨毫微秒荧光谱仪对R-藻红蛋白(R-PE )和C-藻蓝蛋白(C-PC)进行了荧光寿命的测定和研究,并对能量传递过程进行了分析和讨论.测得R-PE的荧光寿命为3.1±0.1ns,C-PC的荧光寿命为1.3±0.1ns,且在pH5—pH9的范围内不变;当pH<5时,两者的荧光寿命都有变短的趋势.我们还测定了R-PE和C-PC混合溶液中R-PE的荧光寿命不变,而C-PC的荧光寿命变长,从而表明存在着R-PE向C-PC的辐射能量传递.     

Spectrally silent light induced conformation change in photosynthetic reaction centers

Spectrally silent conformation change after photoexcitation of photosynthetic reaction centers isolated from Rhodobacter sphaeroides R-26 was observed by the optical heterodyne transient grating technique. The signal showed spectrally silent structural change in photosynthetic reaction centers followed by the primary P+BPh- charge separation and this change remains even after the charge recombination. Without bound quinone to the RC, the conformation change relaxes with about 28micros lifetime. The presence of quinone at the primary quinone (QA) site may suppress this conformation change. However, a weak relaxation with 30-40micros lifetime is still observed under the presence of QA, which increases up to 40micros as a function of the occupancy of the secondary quinone (QB) site.     

An asymmetric dimer of beta-lactoglobulin in a low humidity crystal form--structural changes that accompany partial dehydration and protein action

Dimeric lactoglobulin molecules exist in the open conformation at basic pH, whereas they exist in the closed conformation at acidic pH, after undergoing the Tanford transition around neutral pH. Orthorhombic crystals consisting of molecules in the open conformation, grown close to neutral pH, undergo a water-mediated transformation when the relative humidity around the crystals is reduced. The two subunits in the dimer are related by a crystallographic twofold axis in the native crystals while the dimer is asymmetric in the low humidity form. Interestingly, one of the subunits in the dimer in the low humidity form is in an open conformation while the other is in a closed conformation. This is the first observation of such an asymmetric dimer. A hydrogen bond between the side chains of Gln35 and Tyr42 exists and the side chain of Glu89 is substantially buried in the closed subunit of the asymmetric unit, as in other structures with molecules in the closed conformation. However, the closure of the EF loop is not complete; its conformation can be described as half-closed. A comparison of different crystal structures of beta-lactoglobulin indicates that the conformation of the loops in the molecule is substantially influenced by other factors such as crystal packing, the pH, and the composition of the medium, while the change in the conformation of the EF loop follows the Tanford transition. The mutual disposition of the two subunits in the low humidity form is halfway between those in the open and closed structures. The present work further demonstrates that structural changes that occur during partial dehydration could mimic those that occur during the action of proteins.     

Simulation of pH-dependent edge strand rearrangement in human beta-2 microglobulin

Amyloid fibrils formed from unrelated proteins often share morphological similarities, suggesting common biophysical mechanisms for amyloidogenesis. Biochemical studies of human beta-2 microglobulin (beta2M) have shown that its transition from a water-soluble protein to insoluble aggregates can be triggered by low pH. Additionally, biophysical measurements of beta2M using NMR have identified residues of the protein that participate in the formation of amyloid fibrils. The crystal structure of monomeric human beta2M determined at pH 5.7 shows that one of its edge beta-strands (strand D) adopts a conformation that differs from other structures of the same protein obtained at higher pH. This alternate beta-strand arrangement lacks a beta-bulge, which may facilitate protein aggregation through intermolecular beta-sheet association. To explore whether the pH change may yield the observed conformational difference, molecular dynamics simulations of beta2M were performed. The effects of pH were modeled by specifying the protonation states of Asp, Glu, and His, as well as the C terminus of the main chain. The bulged conformation of strand D is preferred at medium pH (pH 5-7), whereas at low pH (pH < 4) the straight conformation is observed. Therefore, low pH may stabilize the straight conformation of edge strand D and thus increase the amyloidogenicity of beta2M.     

Effect of pH on the conformation of diphtheria toxin and its implications for membrane penetration

The pH-triggered change in diphtheria toxin conformation and the physical properties of the toxin above and below the transition pH have been examined. Exposure to low pH (less than or equal to 5 at 23 degrees C, less than or equal to 5.3 at 37 degrees C) triggers a rapid (t1/2 less than 30 s) change in toxin conformation; the transition occurs over a narrow pH range (0.2 unit). Below the transition pH, buried tryptophans become exposed, and the toxin becomes hydrophobic, binding very tightly to detergent. Aggregation is observed at low pH, probably due to this extreme hydrophobicity. Circular dichroism and fluorescence properties show that the low-pH conformation is not extensively unfolded. Therefore, the toxin "opens" at low pH without becoming a random coil. The conformation change is partly irreversible, and the degree of irreversibility parallels the degree of aggregation. Reduction of the disulfide bonds does not increase hydrophobicity at neutral pH. Furthermore, none of the structural variants of toxin (monomer or dimer, bound to ApUp or free, and nicked between subunits or intact) are hydrophobic at neutral pH or differ in transition pH markedly. Therefore, these factors do not mimic the effect of low pH. These observations are consistent with a functional role for the pH-triggered changes during penetration of the membranes of acidic organelles. The toxin may have adapted a conformational change similar to partial denaturation for a critical role in function. The possible nature of the pH-sensitive interactions and the effects of aggregation are discussed briefly.     

Light-scattering and potentiometric-titration studies of poly-L-tyrosine in aqueous solution

Aqueous solutions of poly-L -tyrosine were studied by potentiometric titrations, light-scattering measurements, and infrared spectroscopy in order to characterize the conformational changes the macromolecules exhibit when the hydrogen-ion concentration of the solutions is varied. It was found that when the pH of the system at 25°C is lowered from a value of about 13 the poly-L -tyrosine molecules undergo a random-coil to β-structure conformation change at pH 11.5. It appears, that under the experimental conditions used, the formation of the β structure is an intramolecular process which involves the folding of the polymer backbone into several hairpins of antiparallel β structure. On further lowering of the pH of the solutions aggregation (pH 11.35) and subsequently precipitation (pH 11.2) takes place. Since the apparent pK of the polymer does not show a drastic change during the random-coil to β-structure conformation change, it was concluded, that the therinodynamic driving force for this conformation change is mainly due to the electrostatic effect of the partially charged side chains of the poly-L -tyrosine molecules.     

Dependence of the conformation of a colicin E1 channel-forming peptide on acidic pH and solvent polarity

The secondary structure content of the COOH-terminal tryptic peptide of colicin E1 has been measured by analysis of UV circular dichroism spectra as a function of pH in aqueous medium and in the presence of the nonionic detergents octyl glucoside and Triton X-100. The alpha-helical content of the peptide increased by approximately 10%, from 45-47% to 56-57%, in the presence of the nonionic detergents, but not in aqueous medium, as the pH was decreased from 4.5 to 3.5. This pH dependence of conformation is similar to that reported elsewhere for the in vitro activity and binding of this peptide. A smaller increase in helical content was observed for the peptide in aqueous medium or in Triton X-100 as the pH was decreased from 6.5 to 4.5. The letter change in helical content was not seen in octyl glucoside which was present at a detergent:peptide stoichiometry 100 times that of Triton. The mean residue ellipticity measured at 222 nm for peptide added to asolectin vesicles by a freeze-thaw treatment was slightly larger at pH 3.5, and substantially larger at pH 4.5, than found at these pH values in the detergent solutions. Changes in helical content at the former, but not the latter pH, could be attributed to peptide insertion. It appears that protonation of one or more acidic amino acid residues in the COOH-terminal region of the molecule causes a conformational change that can be attributed to an extra helical domain that is stabilized in a nonpolar environment. From the similar pH dependence of the conformational change and in vitro binding and activity, it is inferred that interaction of this domain with the membrane is essential for binding and insertion.