Direct evidence that two cysteines in the dopamine transporter form a disulfide bond

We have generated a fully functional dopamine transporter (DAT) mutant (dmDATx7) with all cysteines removed except the two cysteines in extracellular loop 2 (EL2). Random mutagenesis at either or both EL2 cysteines did not produce any functional transporter mutants, suggesting that the two cysteines cannot be replaced by any other amino acids. The cysteine-specific reagent MTSEA-biotin labeled dmDATx7 only after a DTT treatment which reduces disulfide bond. Since there are no other cysteines in dmDATx7, the MTSEA-biotin labeling must be on the EL2 cysteines made available by the DTT treatment. This result provides the first direct evidence that the EL2 cysteines form a disulfide bond. Interestingly, the DTT treatment had little effect on transport activity suggesting that the disulfide bond is not necessary for the uptake function of DAT. Our results and previous results are consistent with the notion that the disulfide bond between EL2 cysteines is required for DAT biosynthesis and/or its delivery to the cell surface.     

Selective dimerization of cysteines in glycopeptides and phosphopeptides

Summary To study the influence of phosphorylation and oxidation on the repeat domains of human Tau protein, we faced the challenge to selectively dimerize two cysteine-containing peptides in the presence of a nearby phosphate group. To this end, we were able to demonstrate the utility of a selective dimerization approach by forming disulfide bonds in unprotected phosphopeptides and extended the methodology to unprotected glycopeptides. Activation of one cysteine of a peptide chain with 2,2-dithiodipyridine and coupling this thiopyridyl-peptide to another peptide chain, containing an unprotected cysteine residue, yielded the mixed dimers in high purities and reasonable yields. Phosphate or sugar side chains on either peptide component remained unaffected during the activation and dimerization processes. While for mixed dimers the activated peptides were isolated by chromatography, homodimers were obtained by a simple one-pot reaction after 1 h. We demonstrate that cysteines can be dimerized in unprotected phosphopeptides and glycopeptides, without any side reactions affecting these posttranslational modifications.Abbreviations DCM dichloromethane - DMF N,N-dimethylformamide - DTP 2,2-dithiodipyridine - Fmoc 9-fluorenylmethyloxycarbonyl - HPLC high-performance liquid chromatography - MALDI matrix-assisted laser desorption/ionization - MS mass spectrometry - NFT neurofibrillary tangles - PHF paired helical filaments - PKC protein kinase C - RP reversed phase - human Tau protein - TFA trifluoroacetic acid Parts of this paper were presented at the 24th European Peptide Symposium in Edinburgh, Scotland, U.K., September 8–13, 1996.     

The evolutionary consequences of plasticity in host-pathogen interactions

Interactions between individuals such as hosts and pathogens are often characterized by substantial phenotypic plasticity. Pathogens sometimes alter their exploitation strategies in response to defensive strategies adopted by their host and vice versa. Nevertheless, most game-theoretic models developed to explain the evolution of pathogen and host characteristics assume that no such plasticity occurs. Allowing for phenotypic plasticity in these models is difficult because one must focus on the evolution of pathogen and host reaction norms, and then allow for the potentially indefinite reciprocal changes in pathogen and host behaviour that occur during an infection as a result of their interacting reaction norms. Here, we begin to address these issues for a simple host-pathogen system in which the pathogen exhibits a level of virulence and the host exhibits a level of immune clearance. We find, quite generally, that plasticity promotes the evolution of higher levels of cooperation, in this case leading to reduced levels of both virulence and clearance.     

The function of conserved cysteine residues in the extracellular domain of human receptor-activity-modifying protein

The receptor-activity-modifying protein (RAMP) 1 is a single-transmembrane-domain protein associated with the calcitonin-like receptor (CLR) to reveal a calcitonin gene-related peptide (CGRP) receptor. The extracellular region of RAMP1 contains six conserved cysteines. Here, Cys(27) in myc-tagged human (h) RAMP1 was deleted (hRAMP1Delta1), and Cys(40), Cys(57), Cys(72), Cys(82) and Cys(104) were each replaced by Ala. In COS-7 cells expressing hCLR/myc-hRAMP1Delta1 or -C82A, cell surface expression, [(125)I]halphaCGRP binding and cAMP formation in response to halphaCGRP were similar to those of hCLR/myc-hRAMP1. Cell surface expression of myc-hRAMP1-C72A was reduced to 24+/-7% of myc-hRAMP1, and that of -C40A, -C57A and -C104A was below 10%. [(125)I]halphaCGRP binding of hCLR/myc-hRAMP1-C72A was 13+/-3% of hCLR/myc-hRAMP1 and it was undetectable in hCLR/myc-hRAMP1-C40A-, -C57A- and -C104A-expressing cells. Maximal cAMP stimulation by halphaCGRP in hCLR/myc-hRAMP1-C40A- and -C72A-expressing cells was 14+/-1% and 33+/-2% of that of the hCLR/myc-hRAMP1 with comparable EC(50). But cAMP stimulation was abolished in cells expressing hCLR/myc-hRAMP1-C57A and -C104A. In conclusion, CGRP receptor function was not affected by the deletion of Cys(27) or the substitution of Cys(82) by Ala in hRAMP1, but it was impaired by the substitution of Cys(40), Cys(57), Cys(72) and Cys(104) by Ala. These four cysteines are required for the transport of hRAMP1 together with the CLR to the cell surface.     

Exploring synonymous codon usage preferences of disulfide-bonded and non-disulfide bonded cysteines in the E. coli genome

High-quality data about protein structures and their gene sequences are essential to the understanding of the relationship between protein folding and protein coding sequences. Firstly we constructed the EcoPDB database, which is a high-quality database of Escherichia coli genes and their corresponding PDB structures. Based on EcoPDB, we presented a novel approach based on information theory to investigate the correlation between cysteine synonymous codon usages and local amino acids flanking cysteines, the correlation between cysteine synonymous codon usages and synonymous codon usages of local amino acids flanking cysteines, as well as the correlation between cysteine synonymous codon usages and the disulfide bonding states of cysteines in the E. coli genome. The results indicate that the nearest neighboring residues and their synonymous codons of the C-terminus have the greatest influence on the usages of the synonymous codons of cysteines and the usage of the synonymous codons has a specific correlation with the disulfide bond formation of cysteines in proteins. The correlations may result from the regulation mechanism of protein structures at gene sequence level and reflect the biological function restriction that cysteines pair to form disulfide bonds. The results may also be helpful in identifying residues that are important for synonymous codon selection of cysteines to introduce disulfide bridges in protein engineering and molecular biology. The approach presented in this paper can also be utilized as a complementary computational method and be applicable to analyse the synonymous codon usages in other model organisms.     

The evolution and evolutionary consequences of marginal thermostability in proteins

When we seek to explain the characteristics of living systems in their evolutionary context, we are often interested in understanding how and why certain properties arose through evolution, and how these properties then affected the continuing evolutionary process. This endeavor has been assisted by the use of simple computational models that have properties characteristic of natural living systems but allow simulations over evolutionary timescales with full transparency. We examine a model of the evolution of a gene under selective pressure to code for a protein that exists in a prespecified folded state at a given growth temperature. We observe the emergence of proteins with modest stabilities far below those possible with the model, with a denaturation temperature tracking the simulation temperature, despite the absence of selective pressure for such marginal stability. This demonstrates that neither observations of marginally stable proteins, nor even instances where increased stability interferes with function, provide evidence that marginal stability is an adaptation. Instead the marginal stability is the result of a balance between predominantly destabilizing mutations and selection that shifts depending on effective population size. Even if marginal stability is not an adaptation, the natural tendency of proteins toward marginal stability, and the range of stabilities that occur during evolution, may have significant effect on the evolutionary process.     

毕赤酵母优化表达外源蛋白策略

毕赤酵母(Pichia pastoris)表达系统是一种异源蛋白表达的理想系统,但目前并非所有的外源蛋白都能在毕赤酵母中成功高效表达,不同的蛋白表现为不同表达水平、生物活性及稳定性。从遗传因素和表达条件综述了外源蛋白在毕赤酵母中的优化表达策略。     

毕赤酵母高效表达策略概述

毕赤酵母表达系统是外源蛋白表达的较为理想的系统,但是并不是所有蛋白都能利用此系统获得高效表达,不同来源的蛋白,其表达水平、生物活性和稳定性均存有明显差别。概述了影响毕赤酵母高效表达的主要因素以及外源蛋白在毕赤酵母中的高效表达策略。     

Specific lipid requirements of membrane proteins—a putative bottleneck in heterologous expression

Membrane proteins are mostly protein-lipid complexes. For more than 30 examples of membrane proteins from prokaryotes, yeast, plant and mammals, the importance of phospolipids and sterols for optimal activity is documented. All crystallized membrane protein complexes show defined lipid-protein contacts. In addition, lipid requirements may also be transitory and necessary only for correct folding and intercellular transport. With respect to specific lipid requiremnts of membrane proteins, the phospholipid and glycolipid as well as the sterol content of the host cell chosen for heterologous expression should be carefully considered. The lipid composition of bacteria, archaea, yeasts, insects,Xenopus oocytes, and typical plant and mamalian cells are given in this review. A few examples of heterologous expression of membrane proteins, where problems of speific lipid requirements have been noticed or should be thought of, have been chosen.     

重组毕赤酵母甲醇利用表型与基因拷贝数对外源基因表达的影响

毕赤酵母是目前最优秀的外源蛋白表达系统之一。本文着重对重组毕赤酵母甲醇利用表型（Mut+型、MutS型和Mut-型）、基因剂量对外源蛋白高效表达的影响机理进行综述。MutS型的比生长速率和蛋白产率比Mut+型低、发酵周期长、副产物（如乙醇、乙酸等）形成速率不同。外源基因拷贝数对外源蛋白的影响主要有三种情况：（1）高基因拷贝数对外源蛋白表达水平有明显的正效应作用;（2）基因拷贝数增加反而降低了表达水平,即负效应作用;（3）重组蛋白表达与基因剂正相关,之后则表现负相关关系,这可能与外源蛋白翻译后加工有关（如二硫键形成、折叠等）,而与分子伴侣共表达可促进外源蛋白的高表达。     

Structural features of plant chitinases and chitin-binding proteins

Structural features of plant chitinases and chitin-binding proteins are discussed. Many of these proteins consist of multiple domains, of which the chitin-binding hevein domain is a predominant one. X-ray and NMR structures of representatives of the major classes of these proteins are available now, and are used to describe the structures of the other ones. Conserved positions of Cys residues can be taken as evidence for identically located disulfide bridges or cysteine residues. The current classification of chitinases is unsatisfactory and needs to be replaced by an evolutionarily more correct one. As the currently known three-dimensional structures of chitinases are those from barley and the rubber tree, Hevea brasiliensis, it is proposed to adopt the designation b-type (classes I, II and IV) and h-type (classes III and V) chitinases, respectively.     

The basics of thiols and cysteines in redox biology and chemistry

Cysteine is one of the least abundant amino acids, yet it is frequently found as a highly conserved residue within functional (regulatory, catalytic, or binding) sites in proteins. It is the unique chemistry of the thiol or thiolate group of cysteine that imparts to functional sites their specialized properties (e.g., nucleophilicity, high-affinity metal binding, and/or ability to form disulfide bonds). Highlighted in this review are some of the basic biophysical and biochemical properties of cysteine groups and the equations that apply to them, particularly with respect to pK_a and redox potential. Also summarized are the types of low-molecular-weight thiols present in high concentrations in most cells, as well as the ways in which modifications of cysteinyl residues can impart or regulate molecular functions important to cellular processes, including signal transduction.     

Genetic and evolutionary consequences of symbiosis

Permanent stable symbioses, primarily microbial, are analyzed as parasexual phenomena from the evolutionary point of view. Such associations bring together in single individuals heritable traits of high selective advantage in certain environments. By convergent evolution several types of associations have repeatedly arisen: motile photosynthetic forms, nitrogen fixing and wood digesting complexes, and so forth. Many examples are discussed from the point of view of the number of originally independent genomes that comprise the recognizable individuals.The level of partner integration in many associations is analyzed. Examples of many levels: genic, gene product, metabolite, behavioral, and the methods by which they can be distinguished are discussed.The literature concerning a large number of associations is reviewed: Methanobacillus; predatory and consortia bacteria; blue green algal sheath dwelling bacteria; anaerobic worm-bacterial; algal, and foreign chloroplast retention by heterotrophic eukaryotes (ciliates, coelenterates, mollusks); the double nucleated photosynthetic dinoflagellate (Peridinium balticum); hindgut microbes in termites and woodroaches (Pyrsonympha, Barbulanympha and their associated spirochetes and other bacteria); sand dwelling and other ciliates and their associated bacteria; and so forth. The status of observations and artificial systems claiming evidence for transfer of genes between very distantly related organisms is critically discussed.A continuum from nearly completely autonomous partners (e.g., zoochlorellae in invertebrate animals) to nearly unrecognizable merged components (e.g., gamma particles in Blastocladiella) is found to exist among examples of extant organisms. The diversity and prevalence of such associations support the concept that there are many precedents for the steps hypothesized in the serial endosymbiotic theory of the origin of eukaryotic cells.     

Human evolutionary research in eastern germany

Human evolutionary research in Eastern Germany, limited in this paper to the origins and evolution of Pleistocene man, has mainly focused during the last decade on theoretical aspects of the evolution of man and human society from multiple and interdisciplinary perspectives. An interdisciplinary working group ‘Probleme der Menschwerdung’ was founded in 1977. About 30 specialists from various fields have discussed problems of the origins and evolution of man and human society under interdisciplinary aspects. The activities, difficulties and results of this cooperation will be outlined in this paper. There will also be given a progress report on the disciplinary initiated research on local sites as well as on general aspects and problems. Paper dedicated to Professor Dr. rer. nat. Dr.sc.med. Hans Grimm, Berlin, on his 80th birthday, February 7, 1990.     

Structural and energetic consequences of mutations in a solvated hydrophobic cavity

The structural and energetic consequences of modifications to the hydrophobic cavity of interleukin 1-beta (IL-1beta) are described. Previous reports demonstrated that the entirely hydrophobic cavity of IL-1beta contains positionally disordered water. To gain a better understanding of the nature of this cavity and the water therein, a number of mutant proteins were constructed by site-directed mutagenesis, designed to result in altered hydrophobicity of the cavity. These mutations involve the replacement of specific phenylalanine residues, which circumscribe the cavity, with tyrosine, tryptophan, leucine and isoleucine. Using differential scanning calorimetry to determine the relative stabilities of the wild-type and mutant proteins, we found all of the mutants to be destabilizing. X-ray crystallography was used to identify the structural consequences of the mutations. No clear correlation between the hydrophobicities of the specific side-chains introduced and the resulting stabilities was found.     

Efficient production of a folded and functional, highly disulfide-bonded beta-helix antifreeze protein in bacteria

The Tenebrio molitor thermal hysteresis protein has a cysteine content of 19%. This 84-residue protein folds as a compact beta-helix, with eight disulfide bonds buried in its core. Exposed on one face of the protein is an array of threonine residues, which constitutes the ice-binding face. Previous protocols for expression of this protein in recombinant expression systems resulted in inclusion bodies or soluble but largely inactive material. A long and laborious refolding procedure was performed to increase the fraction of active protein and isolate it from inactive fractions. We present a new protocol for production of fully folded and active T. molitor thermal hysteresis protein in bacteria, without the need for in vitro refolding. The protein coding sequence was fused to those of various carrier proteins and expressed at low temperature in a bacterial strain specially suited for production of disulfide-bonded proteins. The product, after a simple and robust purification procedure, was analyzed spectroscopically and functionally and was found to compare favorably to previously published data on refolded protein and protein obtained from its native source.     

Conserved extracellular cysteines differentially regulate the inhibitory effect of ethanol in rat P2X4 receptors

Relatively little information is available about the molecular mechanism of ethanol inhibition of P2X receptors. Here, we investigated the possibility that 10 conserved cysteine residues in the extracellular loop of the rat P2X₄ receptor may regulate ethanol inhibition of the receptor using a series of individual cysteine to alanine point mutations. Each of the mutated receptors generated robust inward current in response to ATP and the mutations produced less than a sixfold change in the ATP EC₅₀ value. For the C116A, C126A, C149A, and C165A mutants, 100 mM ethanol did not significantly affect the current activated by an EC₄₀ concentration of ATP. By contrast, for the C261A and C270A mutants, ethanol inhibited ATP-activated current in a competitive manner similar to that for the wild-type receptor. Interestingly, for the C132A, C159A, C217A, and C227A mutants, ethanol inhibited ATP-activated current, but decreased the maximal response to ATP by 70-75% without significantly changing the EC₅₀ value of ATP, thus exhibiting a noncompetitive-type inhibition. The results suggest that cysteines and disulfide bonds between cysteines are differentially involved in the inhibition of the rat P2X₄ receptor by ethanol.     

Heterologous expression and characterization of glycoside hydrolase with its potential applications in hyperthermic environment

With the progressive focus on renewable energy via biofuels production from lignocellulosic biomass, cellulases are the key enzymes that play a fundamental role in this regard. This study aims to unravel the characteristics of Thermotoga maritima MSB8 (Tma) (a hyperthermophile from hot springs) thermostable glycoside hydrolase enzyme. Here, a glycoside hydrolase gene of Thermotoga maritima (Tma) was heterologously expressed and characterized. The gene was placed in the pQE-30 expression vector under the T5 promotor, and the construct pQE-30-Gh was then successfully integrated into Escherichia coli BL21 (DH5α) genome by transformation. Sequence of the glycoside hydrolase contained an open reading frame of 2.124 kbp, encoded a polypeptide of 721 amino acid residues. The molecular weight of the recombinant protein estimated was 79 kDa. The glycoside hydrolase was purified by Ni⁺²-NTA affinity chromatography and its enzymatic activity was investigated. The recombinant enzyme is highly stable within an extreme pH range (2.0–7.0) and highly thermostable at 80 °C for 72 h indicating its viability in hyperthermic environment and acidic nature. Moreover, the Ca²⁺ and Mn²⁺ introduction stimulated the residual activity of recombinant enzyme. Conclusively, the thermostable glycoside hydrolase possesses potential to be exploited for industrial applications at hyperthermic environment.     

刺糖多孢菌中铵载体蛋白基因amtS的克隆及功能研究

目的:铵离子是细胞内合成各种核酸、氨基酸和辅助因子等含氮化合物的重要原料之一。微生物细胞膜上的铵载体蛋白介导了铵离子的转运。通过异源表达刺糖多孢菌中铵载体蛋白基因,研究其对链霉菌产孢能力和次级代谢产物产量的影响。方法:从刺糖多孢菌S04-41菌株中克隆铵载体蛋白基因amt S,通过接合转移导入天蓝色链霉菌M145和变铅青链霉菌TK24中,分析比较amt S基因的异源表达对其产孢能力和次级代谢产物产量的影响。结果:天蓝色链霉菌重组菌株M145/p MF-amt S和变铅青链霉菌重组菌株TK24/p MF-amt S中放线紫红素的产量分别提高了2.85倍和30.02倍。结论:刺糖多孢菌中的铵载体蛋白能够提高链霉菌中次生代谢产物的产量,为进一步研究该基因的功能与对刺糖多孢菌中多杀菌素合成的作用奠定了重要基础。