A 'molten-globule' membrane-insertion intermediate of the pore-forming domain of colicin A

The 'molten' globular conformation of a protein is compact with a native secondary structure but a poorly defined tertiary structure. Molten globular states are intermediates in protein folding and unfolding and they may be involved in the translocation or insertion of proteins into membranes. Here we investigate the membrane insertion of the pore-forming domain of colicin A, a bacteriocin that depolarizes the cytoplasmic membrane of sensitive cells. We find that this pore-forming domain, the insertion of which depends on pH, undergoes a native to molten globule transition at acidic pH. The variation of the kinetic constant of membrane insertion of the protein into negatively charged lipid vesicles as a function of the interfacial pH correlates with the appearance of the acidic molten globular state, indicating that this state could be an intermediate formed during the insertion of colicin A into membranes.     

Demonstration by NMR of folding domains in lysozyme

Although there has been much speculation on the pathways of protein folding, only recently have experimental data on the topic been available. The study of proteins under conditions where species intermediate between the fully folded and unfolded states are stable has provided important information, for example about the disulphide intermediates in BPTI, cis/trans proline isomers of RNase A3 and the molten globule state of alpha-lactalbumin. An alternative approach to investigating folding pathways has involved detection and characterization of transient conformers in refolding studies using stopped-flow methods coupled with NMR measurements of hydrogen exchange. The formation of intermediate structures has been detected in the early stages of folding of cytochrome c, RNaseA and barnase. For alpha-lactalbumin, hydrogen exchange kinetics monitored by NMR proved to be crucial for identifying native-like structural features in the stable molten globule state. An analogous partially folded protein stable under equilibrium conditions has not been observed for the structurally homologous protein hen egg-white lysozyme, although there is evidence that a similar but transient state is formed during refolding. Here we describe NMR experiments based on competition between hydrogen exchange and the refolding process which not only support the existence of such a transient species for lysozyme, but enable its structural characteristics to be defined. The results indicate that the two structural domains of lysozyme are distinct folding domains, in that they differ significantly in the extent to which compact, probably native-like, structure is present in the early stages of folding.     

Characteristics of Two Intermediates Trapped in the Unfolding Pathway of Arginine Kinase Induced by Guanidinium Chloride

Equilibrium guanidinium chloride （GdmCl）-induced unfolding of arginine kinase （AK） was investigated by enzymatic activity, intrinsic fluorescence, 8-anilinonaphthalene-1-sulfonic acid （ANS） fluorescence, circular dichroism （CD） spectrum, and size-exclusion chromatography. The measurements showed that AK unfolded through two equilibrium intermediates： the molten globule state and the partly folded state. Both intermediates have no enzyme activity. The molten globule state exists at 0.4-0.8 mol/L GdmCi, perhaps after the N-terminal domain has unfolded but the C-terminal domain is still intact. The partly folded state occurs at 1.1-1.5 mol/L GdmCI with a hydrodynamic volume no more than 1.6-fold larger than the native state and a pronounced far UV-CD signal. Its ANS fluorescence intensity is about 50% of the molten globule state. This partly folded state shares similarities with the “burst” kinetic intermediate of protein folding.     

Sequence of protein disulphide isomerase and implications of its relationship to thioredoxin

The formation of disulphide bonds is essential to the structure and function of proteins. These bonds rapidly form either cotranslationally or immediately post-translationally in the lumen of the endoplasmic reticulum. Native disulphide pairing for such proteins has been achieved in vitro; however, the rates of reassembly are slow and the conditions non-physiological. To account for these observations, Anfinsen et al. proposed that a 'disulphide interchange protein' was the in vivo catalyst of disulphide bond rearrangement. Other groups discovered an activity with similar characteristics that catalysed the reductive cleavage of insulin and may be associated with insulin degradation, although this result has been disputed. The enzyme involved, protein disulphide isomerase (PDI; EC 5.3.4.1), may be the in vivo catalyst of disulphide bond formation. Here we describe the sequence of cloned rat liver PDI complementary DNA which predicts a protein with two distinct regions homologous with Escherichia coli thioredoxin, a known cofactor in oxidation-reduction reactions. Each of these regions contains the presumed active site sequence Trp-Cys-Gly-His-Cys-Lys, suggesting that PDI, similar in action to thioredoxin, catalyses disulphide bond interchange via an internal disulphide-sulphydryl interchange. The cDNA predicts a signal peptide consistent with the view that PDI is a luminal endoplasmic reticulum protein. PDI messenger RNA, although ubiquitous, is more highly concentrated in secretory cells.     

Glycoproteins form mixed disulphides with oxidoreductases during folding in living cells

The formation of intra- and interchain disulphide bonds constitutes an integral part of the maturation of most secretory and membrane-bound proteins in the endoplasmic reticulum. Evidence indicates that members of the protein disulphide isomerase (PDI) superfamily are part of the machinery needed for proper oxidation and isomerization of disulphide bonds. Models based on in vitro studies predict that the formation of mixed disulphide bonds between oxidoreductase and substrate is intermediate in the generation of the native intrachain disulphide bond in the substrate polypeptide. Whether this is how thiol oxidoreductases work inside the endoplasmic reticulum is not clear. Nor has it been established which of the many members of the PDI superfamily interacts directly with newly synthesized substrate proteins, because transient mixed disulphides have never been observed in the mammalian endoplasmic reticulum during oxidative protein folding. Here we describe the mechanisms involved in co- and post-translational protein oxidation in vivo. We show that the endoplasmic-reticulum-resident oxidoreductases PDI and ERp57 are directly involved in disulphide oxidation and isomerization, and, together with the lectins calnexin and calreticulin, are central in glycoprotein folding in the endoplasmic reticulum of mammalian cells.     

几种盐类对光系统Ⅱ33kD锰稳定蛋白溶液构象的影响

借助内源荧光和圆二色谱分析，考察了几种不同的盐溶液（CaCl2,NaCl,脲，三氯乙酸、二硫苏糖醇）对水溶液中33kD蛋白构象的影响，以295nm波长的乐激发时，33kD蛋白具有330nm的荧光发射峰，表明33kD蛋白所含唯一的^241Trp位于分子内部的疏水部位，CaCl2、脲、三氯乙酸或二硫苏糖醇的存在，均会改变33kD蛋白的内源荧光的强度和位置，而NaCl几科无影响，脲、二硫苏糖醇对33kD蛋白的二级结构的影响较大，因此维系33kD蛋白构象的主要因素包括：二硫键、疏水作用、范得华力和氢键，而离子键的贡献较小。     

Role of ATP and disulphide bonds during protein folding in the endoplasmic reticulum.

Being topologically equivalent to the extracellular space, the lumen of the endoplasmic reticulum (ER) provides a unique folding environment for newly synthesized proteins. Unlike other compartments in the cell where folding occurs, the ER is oxidizing and therefore can promote the formation of disulphide bonds. The reducing agent dithiothreitol, when added to living cells, inhibits disulphide formation with profound effects on folding. Taking advantage of this effect, we demonstrate here that folding of influenza haemagglutinin is energy dependent. Metabolic energy is required to support the correct folding and disulphide bond formation in this well characterized viral glycoprotein, to rescue misfolded proteins from disulphide-linked aggregates, and to maintain the oxidized protein in its folded and oligomerization-competent state.     

Role of a subdomain in the folding of bovine pancreatic trypsin inhibitor.

The disulphide-bonded intermediates that accumulate in the oxidative folding of bovine pancreatic trypsin inhibitor (BPTI) were characterized some time ago. Structural characterization of these intermediates would provide an explanation of the kinetically preferred pathways of folding for BPTI. When folding occurs under strongly oxidizing conditions, more than half the molecules become trapped in an intermediate, designated N*, which is similar to the native protein but lacks the 30-51 disulphide bond. We have tested the hypothesis that the precursor to N* is the one-disulphide intermediate [5-55], which contains the most stable disulphide in BPTI, and present evidence here that this is the case. A peptide model of [5-55], corresponding to a subdomain of BPTI, seems to fold into a native-like conformation, explaining why [5-55] does not lead to native protein and why it folds rapidly to N*. A native-like subdomain structure in a peptide model of [30-51], the other crucial one-disulphide intermediate, may explain the route by which [30-51] folds to native protein. Thus, much of the folding pathway of BPTI can be explained by the formation of a native-like subdomain in these two early intermediates. This suggests that a large part of the protein folding problem can be reduced to identifying and understanding subdomains of native proteins.     

Substantial increase of protein stability by multiple disulphide bonds

Disulphide bonds can significantly stabilize the native structures of proteins. The effect is presumed to be due mainly to a decrease in the configurational chain entropy of the unfolded polypeptide. In phage T4 lysozyme, a disulphide-free enzyme, engineered disulphide mutants that crosslink residues 3-97, 9-164 and 21-142 are significantly more stable than the wild-type protein. To investigate the effect of multiple-disulphide bonds on protein stability, mutants were constructed in which two or three stabilizing disulphide bridges were combined in the same protein. Reversible thermal denaturation shows that the increase in melting temperature resulting from the individual disulphide bonds is approximately additive. The triple-disulphide variant unfolds at a temperature 23.4 degrees C higher than wild-type lysozyme. The results demonstrate that a combination of disulphide bonds, each of which contributes to stability, can achieve substantial overall improvement in the stability of a protein.     

Protein-disulphide isomerase and prolyl isomerase act differently and independently as catalysts of protein folding

Two enzymes are now known that catalyse slow steps in protein folding. Peptidyl-prolyl cis-trans isomerase catalyses the cis-trans isomerization of Xaa-Pro peptide bonds in oligopeptides and during the refolding of several proteins. The other enzyme, protein-disulphide isomerase, accelerates the reactivation of reduced proteins, presumably by catalysis of thiol-disulphide exchange reactions. Recent evidence indicates that the beta-subunit of prolyl 4-hydroxylase, an enzyme involved in collagen biosynthesis, is identical with disulphide isomerase. On the basis of this important finding, it was suggested that disulphide isomerase accelerates protein folding, not by 'reshuffling' incorrect disulphide bonds, but in the same way as prolyl isomerase by catalysing proline isomerization which is known to be important for the folding of collagen and other proteins. Here we show that the catalytic activities of these two enzymes are different. Disulphide isomerase accelerates the reformation of native disulphide bonds during protein reoxidation. We find no evidence that this enzyme can catalyse the isomerization of proline peptide bonds, a reaction efficiently accelerated by prolyl isomerase. When both enzymes are present simultaneously during protein folding, they act independently of one another.     

电喷雾质谱研究乙腈诱导的溶菌酶的构象变化

首次采用电喷雾质谱结合圆二色性光谱以及荧光光谱,研究不同浓度的乙腈诱导对溶菌酶构象变化的影响.在pH2.0的酸性环境下,随着乙腈浓度的上升,电喷雾质谱的谱图中溶菌酶电荷状态分布会变宽,当乙腈浓度达到70%时,会出现15 的高电荷离子峰,电荷中心也从纯水溶剂的10 到了12 ,表明其三级结构发生了变化,即由天然构象转变为部分非折叠的构象.圆二色光谱显示在溶菌酶水溶液中加入乙腈,二级结构中无规卷曲的百分含量在增加.荧光光谱进一步证明在乙腈的作用下,溶菌酶由天然构象转变为部分非折叠的构象.     

蛋白质的挤压组织化改性─—大豆蛋白在挤压过程中的物理、化学变化

将挤压过程用于蛋白质改性，使大豆浓缩蛋白经挤压组织化处理后，成为一种具有一定组织特性的功能蛋白质。由缓冲液浸出试验、聚丙烯酰胺凝胶电泳分析，并结合傅立叶变换红外差谱测试结果可知：大豆浓缩蛋白在挤压组织化过程中，发生了二硫键交换反应，但并未形成异肽键，蛋白质的相对分子质量也无变化。维持组织蛋白结构的主要空间构象力是二硫键和疏水相互作用。     

Hyperconjugation not steric repulsion leads to the staggered structure of ethane

Many molecules can rotate internally around one or more of their bonds so that during a full 360 degrees rotation, they will change between unstable and relatively stable conformations. Ethane is the textbook example of a molecule exhibiting such behaviour: as one of its two methyl (CH3) groups rotates once around the central carbon-carbon bond, the molecule will alternate three times between an unstable eclipsed conformation and the preferred staggered conformation. This structural preference is usually attributed to steric effects; that is, while ethane rotates towards an eclipsed structure, the electrons in C-H bonds on the different C atoms are drawing closer to each other and therefore experience increased repulsion, introducing a rotation barrier that destabilizes the eclipsed structure. Stabilization of the staggered structure through rotation-induced weakening of the central C-C bond and hyperconjugation has been considered to be involved, but evaluation of the contributions of these effects to ethane's internal rotation barrier and conformational preference remains difficult. Here we report a series of ethane structure optimizations, where successive removal of different interactions indicates that ethane's staggered conformation is the result of preferential stabilization through hyperconjugation. Removal of hyperconjugation interactions yields the eclipsed structure as the preferred conformation, whereas repulsive forces, either present or absent, have no influence on the preference for a staggered conformation.     

Distal residues in the oxygen binding site of haemoglobin studied by protein engineering

The geometries of the Fe-O2 and Fe-CO bonds in myoglobin and haemoglobin differ significantly from those in free porphyrin model compounds. It has been suggested that steric hindrance by Val-E11 and His-E7 and a hydrogen bond between His-E7 and oxygen affect the geometry and electronic state of the Fe-ligand bond, and that these interactions may be important in controlling oxygen affinity. We have produced mutant haemoglobins in E. coli having Val(67 beta)E11 replaced by Ala, Met, Leu or Ile and His(58 beta)E7 by Gln, Val or Gly. We have studied the effect of these mutations on the equilibrium and kinetics of ligand binding. The conformation of the new side chains and their effect on the protein structure have been examined by X-ray crystallography, and the vibrational properties of the Fe-CO bond observed by resonance Raman spectroscopy. We found that the steric hindrance of ligand binding by the E11 residue and the polarity of the E7 residue in the beta subunit are critical for fine-tuning ligand affinity.     

Detection and characterization of a folding intermediate in barnase by NMR

Protein engineering is being developed for mapping the energetics and pathway of protein folding. From kinetic studies on wild-type and mutant proteins, the sequence and energetics of formation of tertiary interactions of side chains can be mapped and the formation of secondary structure inferred. Here we cross-check and complement results from this approach by using a recently developed procedure that traps and characterizes secondary structure in intermediate states using 1H NMR. The refolding of barnase is shown to be a multiphasic process in which the secondary structure in alpha-helices and beta-sheets and some turns is formed more rapidly than is the overall folding.     

Mimicry of ice structure by surface hydroxyls and water of a beta-helix antifreeze protein

Insect antifreeze proteins (AFP) are much more effective than fish AFPs at depressing solution freezing points by ice-growth inhibition. AFP from the beetle Tenebrio molitor is a small protein (8.4 kDa) composed of tandem 12-residue repeats (TCTxSxxCxxAx). Here we report its 1.4-A resolution crystal structure, showing that this repetitive sequence translates into an exceptionally regular beta-helix. Not only are the 12-amino-acid loops almost identical in the backbone, but also the conserved side chains are positioned in essentially identical orientations, making this AFP perhaps the most regular protein structure yet observed. The protein has almost no hydrophobic core but is stabilized by numerous disulphide and hydrogen bonds. On the conserved side of the protein, threonine-cysteine-threonine motifs are arrayed to form a flat beta-sheet, the putative ice-binding surface. The threonine side chains have exactly the same rotameric conformation and the spacing between OH groups is a near-perfect match to the ice lattice. Together with tightly bound co-planar external water, three ranks of oxygen atoms form a two-dimensional array, mimicking an ice section.     

蛋白质二级结构预测概率图模型的改进

蛋白质二级结构与蛋白质三级结构及蛋白质功能密切相关,是生物信息学研究的热点,其中概率图模型隐马尔可夫算法(HMM)是该领域研究的重要工具。但是在实际应用中,存在着HMM训练下溢、不同训练集的效果差异较大及参数优化困难等问题。对预测蛋白质二级结构时HMM遇到的训练下溢问题提出了改进方案;首次提出8-状态HMM来预测蛋白质二级结构,并且将参数B改进成为包含状态转移信息的三维参数;为了改进最优HMM模型的确定方法,用每个样本分别对初始HMM模型进行训练,得到一系列新的模型,然后对这些新模型的参数求均值,将求得的均值作为最优模型的参数。这些改进方法提高了HMM预测蛋白质二级结构的准确率,为HMM的进一步优化打下良好的基础。     

A peptide model of a protein folding intermediate

It is difficult to determine the structures of protein folding intermediates because folding is a highly cooperative process. A disulphide-bonded peptide pair, designed to mimic the first crucial intermediate in the folding of bovine pancreatic trypsin inhibitor, contains secondary and tertiary structure similar to that found in the native protein. Peptide models like this circumvent the problem of cooperativity and permit characterization of structures of folding intermediates.     

Catalysis of protein folding by prolyl isomerase

Rates of protein folding reactions vary considerably. Some denatured proteins regain the native conformation within milliseconds or seconds, whereas others refold very slowly in the time range of minutes or hours. Varying folding rates are observed not only for different proteins, but can also be detected for single polypeptide species. This originates from the co-existence of fast- and slow-folding forms of the unfolded protein, which regain the native state with different rates. The proline hypothesis provides a plausible explanation for this heterogeneity. It assumes that the slow-folding molecules possess non-native isomers of peptide bonds between proline and another residue, and that crucial steps in the refolding of the slow-folding molecules are limited in rate by the slow reisomerization of such incorrect proline peptide bonds. Recently the enzyme peptidyl-prolyl cis-trans isomerase (PPIase) was discovered and purified from pig kidney. It catalyses efficiently the cis in equilibrium trans isomerization of proline imidic peptide bonds in oligopeptides. Here we show that it also catalyses slow steps in the refolding of a number of proteins of which fast- and slow-folding species have been observed and where it was suggested that proline isomerization was involved in slow refolding. The efficiency of catalysis depends on the accessibility for the isomerase of the particular proline peptide bonds in the refolding protein chain.