Insertion mutagenesis of Escherichiacoli GroEL

To gain insights into the in vivo folding and assembly of bacterial chaperonins, groEL was subjected to insertion mutagenesis using transposon ISlacZ/in. Four GroEL-LacZ fusions and the corresponding insertion mutants were obtained after residues 34, 90, 291, and 367. Apical domain insertion mutants GroEL291 and GroEL367 were degraded into monomeric 30- and 40-kDa fragments, respectively. Only the latter was fully soluble, suggesting that proper isomerization of an essentially complete apical domain is required for efficient protomer folding. Truncated variants were inactive as minichaperones as they failed to restore the growth of groEL140 cells at 43 degrees C whether or not GroES was co-expressed. A 31-residue insertion in equatorial helix D led to complete degradation of GroEL90. By contrast, extraneous amino acids were tolerated at equatorial position 34, indicating that this region is highly flexible. Nevertheless, GroEL34 did not fold as efficiently as authentic GroEL and reached only a heptameric conformation.     

易位子辅助膜蛋白插入热力学

易位子辅助膜蛋白插入内质网膜是膜蛋白质生物生成的关键过程。了解不同类分子插入生物膜的机制是预测溶质分子透膜速度的先决条件,这也是药物设计和药理学领域的关键因素。根据插入机制,可以设计插膜肽直接用于疾病治疗,或者作为载体有选择性地将药物靶向特定细胞。自从2004年第1个易位子通道蛋白（Sec）的晶体结构被解析后,近十几年来大量的实验和理论研究,都在致力于揭示Sec辅助膜蛋白插入过程的分子机制。本文总结了过去该领域的实验和分子动力学模拟研究进展,从热力学方面重点分析了造成膜蛋白插入自由能分子动力学模拟计算值,以及实验值间偏差的原因。其中,根据研究条件精确设置模拟参数、插入造成的膜变形对自由能计算有很大的影响;核糖体为新生肽插入到Sec通道过程提供了能量,核糖体与Sec的结合影响Sec侧门的开放程度和Sec通道的结构,从而降低膜插入自由能。Sec辅助膜蛋白插入是一个极其复杂的过程,但整个过程仍然符合热力学和动力学的基本原理,尽管疏水性是Sec辅助膜蛋白质插入的关键性因素,但也不能忽略动力学因素的影响。     

Collection of Arabidopsis thalianaMorphological Insertion Mutants

A collection of transgenic Arabidopsis thalianaplants has been obtained by Agrobacterium-mediated transformation. The genomes of the transgenic plants contain insertions of T-DNA of the vector plasmids pLD3 or pPCVRN4. Genes bearing T-DNA insertions were shown to constitute 12–18% of the total number of A. thalianagenes. Seventy-five lines have been chosen from the collection and subjected to genetic and molecular-genetic analysis. Of these, 5 were dominant mutants, and 70, recessive insertion mutants with various morphological defects. Identification of mutant phenotypes and genetic characterization of the transgenic lines have been performed with the use of nutrient media supplemented with exogenous hormones, which revealed five recessive lethal mutants and one dominant sterile mutant.     

Specificity of Transposon Tn5 Insertion

Genetic mapping studies had shown that the bacterial transposon Tn5 can insert into many sites in a gene, but that some sites are preferred. To begin understanding Tn5's insertion specificity at the molecular level, we selected transpositions of Tn5 from the Escherichia coli chromosome to the plasmid pBR322 and analyzed the resultant pBR322::Tn5 plasmids by restriction endonuclease digestion and DNA sequencing. Seventy-five insertions in the tet gene were found at 28 sites including one major hotspot (with 21 insertions) and four lesser hotspots (with four to ten insertions each). All five hotspots are within the first 300 of the 1250-base pair (bp) tet gene. In contrast, 31 independent insertions in the amp gene were found in at least 27 distinct sites.—Tn5 generates 9 bp target sequence duplications when it transposes. Such transposon-induced duplications are generally taken to indicate that cleavages of complementary target DNA strands are made 9 bp apart during transposition. DNA sequence analysis indicated that GC base pairs occupy positions 1 and 9 in the duplications at each of the five hotspots examined, suggesting a GC-cutting preference during Tn5 transposition.     

Insertion of isocyanides into Re---C bonds

Isocyanide-substituted Re alkyl complexes cis-p-XC₆H₄CH₂Re(CO)₄(CN-p-tolyl) (X = Cl, OMe) were prepared from the PdO-catalyzed reaction of p-XC₆H₄CH₂Re(CO)₅ (X = Cl, OMe) with p-tolyl isocyanide. On heating in toluene these complexes undergo isocyanide insertion into the Re---C bond to afford iminoacyl complexes which further react to orthometallate the p-tolyl ring. An X-ray crystal structure determination on (CO)₄

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(3a) revealed that C₁₉H₁₃ClO₄NRe crystallizes in the monoclinic space group P2₁/c with 4 formulas per unit cell. Unit cell parameters are a = 9.799 (1), B = 15.252 (2), C = 13.569 (2) Å and β = 110.788 (8)°. The structure shows Re---C bond distances indicative of substantial carbenoid character.     

Insertion elements in Staphylococcus intermedius

Staphylococcus intermedius cultures from dogs, pigeons, horses and mink were investigated for the prevalence of the insertion elements IS 256 and IS 257 in relation to their antibiotic resistance. Copies of IS 256 could not be detected in any of the Staph. intermedius isolates tested whereas single copies of IS 257 occurred in the isolates from dogs and horses. The mink strains did not harbour IS 257 elements, whereas Staph. intermedius isolates from pigeons carried multiple copies of IS 257 as predicted from the hybridization patterns obtained with a gene probe derived from the internal part of the IS 257 -encoded transposase gene. Independently of the origin of the Staph. intermedius isolates, all IS 257 copies were found to be located in the chromosomal DNA. The large number of chromosomal IS 257 copies in the pigeon strains might help to explain chromosomal multiresistance in many of those strains.     

A Monte Carlo Study of Peptide Insertion into Lipid Bilayers: Equilibrium Conformations and Insertion Mechanisms

The membrane insertion behavior of two peptides, Magainin2 and M2δ, was investigated by applying the Monte Carlo simulation technique to a theoretical model. The model included many novel aspects, such as a new semi-empirical lipid bilayer model and a new set of semi-empirical transfer energies, which reproduced the experimental insertion behavior of Magainin2 and M2δ without parameter fitting. Additionally, we have taken into account diminished internal (intramolecular) hydrogen bonding at the N- and C-termini of helical peptides. All simulations were carried out at 305 K, above the membrane thermal phase transition temperature, and at pH 7.0. The peptide equilibrium conformations are discussed for a range of bilayers with tail polarities varying from octanol-like to alkane-like. Probability distributions of the individual amino-acid-residue positions show the dynamic nature of these equilibrium conformations. Two different insertion mechanisms for M2δ, and a translocation mechanism for Magainin2, are described. A study of the effect of bilayer thickness on M2δ insertion suggests a critical thickness above which insertion is unfavorable. Additionally, we did not need to use an orientational potential or array of hard cylinders to persuade M2δ to insert perpendicular to the membrane surface. Instead, we found that diminished internal hydrogen bonding in the helical conformation anchored the termini in the headgroups and resulted in a nearly perpendicular orientation.     

Insertion sequences in prokaryotic genomes

Insertion sequences (ISs) are small DNA segments that are often capable of moving neighbouring genes. Over 1500 different ISs have been identified to date. They can have large and spectacular effects in shaping and reshuffling the bacterial genome. Recent studies have provided dramatic examples of such IS activity, including massive IS expansion during the emergence of some pathogenic bacterial species and the intimate involvement of ISs in assembling genes into complex plasmid structures. However, a global understanding of their impact on bacterial genomes requires detailed knowledge of their distribution across the eubacterial and archaeal kingdoms, understanding their partition between chromosomes and extra-chromosomal elements (e.g. plasmids and viruses) and the factors which influence this, and appreciation of the different transposition mechanisms in action, the target preferences and the host factors that influence transposition. In addition, defective (non- autonomous) elements, which can be complemented by related active elements in the same cell, are often overlooked in genome annotations but also contribute to the evolution of genome organisation.     

Insertion of apocytochrome c into lipid vesicles

Apocytochrome c (cytochrome c without the heme) is synthesized in the cell cytoplasm without a cleaved signal sequence, then transported across the outer mitochondrial membrane. We have studied the interaction of apocytochrome c with lipid vesicles as a model for understanding protein translocation across membranes. Apocytochrome c (but not holocytochrome c) that has been incubated with vesicles at 37 degrees C in 0.2 M NaCl binds to the vesicles. Under these conditions, as well as upon incubation with detergent or at high protein concentrations, all the added protein remains partly accessible to externally added protease, but a COOH-terminal fragment of some of the protein molecules becomes protected against digestion. When apocytochrome c is added to azolectin vesicles with internally trapped proteases, most of the added protein can be digested, even in the presence of a large excess of protease inhibitor external to the vesicles. Thus, in spite of a lack of nonpolar stretches in its amino acid sequence, apocytochrome c is capable of binding to and inserting into lipid membranes. In this model system, transport may be driven by trapping of protease-digested apocytochrome c on one side of the membrane.     

插入突变在功能基因组学研究中的应用

插入突变库的构建是功能基因组学研究的一个重要内容,可为确定基因的功能提供最直接的证据。构建插入突变库的方法有T-DNA插入突变、转座子插入突变和质粒介导的插入突变。本文分别介绍三种方法的原理及其在功能基因组学研究中的应用和研究进展。     

Insertion of heteroallenes into the rhenium-hydride bond

Dithioformato [Re{η²-SC(H)S}(NO)P₃]BPh₄ (1), thioformamido [Re{η²-RNC(H)S}(NO)P₃]BPh₄ (2) (R = Et, p-tolyl), formamido [Re{η²-PhNC(H)O}(NO)P₃]BPh₄ (3) and formamidinato [Re{η²-p-tolylNC(H)Np-tolyl}(NO)P₃]BPh₄ (4) (P = PPh₂OEt) complexes were prepared by allowing the hydride ReH₂(NO)P₃ to react first with triflic acid and then with the appropriate heteroallene CS₂, RNCS, PhNCO and p-tolylNCNp-tolyl. Treatment of the ReH₂(NO)L(PPh₃)₂ [L = P(OEt)₃, PPh(OEt)₂] and ReH₂(NO)(PPh₃)₃ hydrides first with triflic acid and then with isothiocyanate RNCS (R = Et, p-tolyl) gave the [Re{η²-RNC(H)S}(NO){P(OEt)₃}(PPh₃)₂]BPh₄ (5, 6) and [Re(η²-RNC(H)S)(NO)(PPh₃)₃]BPh₄ (7) derivatives. Depending on the nature of the phosphite, instead, the reaction of ReH₂(NO)L(PPh₃)₂ and ReH₂(NO)(PPh₃)₃ hydrides first with CF₃SO₃H and then with isocyanate R1NCO (R1 = Ph, p-tolyl) gave the chelate [Re{η²-R1NC(H)O}(NO){P(OEt)₃}(PPh₃)₂]BPh₄ (8) and [Re{η²-R1NC(H)O}(NO)(PPh₃)₃]BPh₄(10) complexes with P(OEt)₃ or PPh₃, while the η¹-coordinate [Re{η¹-RNC(H)S}(NO){PPh(OEt)₂}₂(PPh₃)₂]BPh₄ (9) derivative was obtained with the PPh(OEt)₂ phosphite ligand. η¹-Coordinate dithioformato [Re{η¹-SC(H)S}(NO){PPh(OEt)₂}₂(PPh₃)₂]BPh₄ (11) and formato [Re{η¹-OC(H)O}(NO){PPh(OEt)₂}₂(PPh₃)₂]BPh₄ (12) complexes, as well as the formamidinato [Re{η²-p-tolylNC(H)Np-tolyl}(NO){P(OEt)₃}(PPh₃)₂]BPh₄ (13) derivative were also prepared.     

Insertion of a Glycosylphosphatidylinositol-Anchored Enzyme into Liposomes

Incorporation of alkaline phosphatase (AP), a glycosylphosphatidylinositol (GPI)-anchored protein, into liposomes containing detergent, followed by detergent removal with hydrophobic resin was performed. Incorporation media were collected during different steps of detergent removal and were analyzed by flotation in sucrose gradient. The presence of protein was checked by measuring enzymatic activity, while the presence of ³H-radio-labelled liposomes was followed by determination of the radioactivity. The incorporation yield of the protein into liposomes increased with incubation time in presence of hydrophobic resin. Protein was also incorporated at different protein/lipid ratios. At the highest protein lipid ratio, our data showed that 260 molecules of GPI-linked AP (AP-GPI) could be associated with one liposome, corresponding to 65% vesicle coverage. Finally, observations by electron cryomicroscopy indicated (i) that the protein seemed exclusively associated with the lipid bilayer via the GPI-anchor, as shown by the distance—about 2.5 nm—between the protein core and the liposome membrane; (ii) that the AP-GPI distribution was heterogeneous on the liposome surface, forming clusters of protein. Abbreviations: AP, alkaline phosphatase; AP-GPI, glycosylphosphatidylinositol-linked alkaline phosphatase; EM, electron microscopy; EPA, egg phosphatidic acid; GPI, glycosylphosphatidylinositol; OctGlc, n-octyl -D-glucoside; PtdCho, egg yolk phosphatidylcholine; PtdIns-PLC, glycosylphosphatidylinositol-specific phospholipase C. Enzymes: Alkaline phosphatase, orthophosphoric-monoester phosphohydrolase (EC 3.1.3.1); glycosylphosphatidylinositol-specific phospholipase C (EC 3.1.4.10).