Gene synthesis, expression, structures, and functional activities of site-specific mutants of ubiquitin

To study the structure and function of ubiquitin we have chemically synthesized a ubiquitin gene that encodes the amino acid sequence of animal ubiquitin, inserting a series of restriction enzyme sites that divide the gene into eight "mutagenesis modules." A series of site-specific mutations were constructed to selectively perturb various regions of the molecule. The mutant genes were expressed in a large quantity of Escherichia coli, and the modified proteins were purified. To determine the structural effects of the amino acid substitutions, the solution structure of ubiquitin was investigated by two-dimensional NMR and each of the mutant proteins were screened for structural perturbations. With one exception, virtually no changes were seen other than at the point of mutation. Functional studies of the mutant proteins with the ubiquitin-activating enzyme E1 and in the reticulocyte protein degradation assay were used to identify regions of the molecule important to ubiquitin's activity in intracellular proteolysis.     

眼镜王蛇泛素融合蛋白基因和核糖体蛋白L30基因的克隆及分析

从广西产眼镜王蛇（Ophiophagus hannah）毒腺中抽提总RNA,经mRNA纯化后构建眼镜王蛇毒腺cDNA文库。从所构建的cDNA文库中,随机筛选200个克隆测序,得到两个在进化上高度保守的基因：泛素融合蛋白基因（GenBank登录号为AF297036）和核糖体蛋白L30基因（GenBank登录号是AF297033）。前者cDNA的开放阅读框为387bp,后者为348bp。前者编码128个氨基酸残基组成的泛素融合蛋白前体;后者编码115个氨基酸残基组成的核糖体蛋白L30前体。由cDNA序列推导出的氨基酸序列分析表明,泛素融合蛋白前体包括N-末端的泛素结构域（76个氨基酸残基）和C-末端的核糖体蛋白L40结构域（52个氨基酸残基）。该蛋白为一高碱性蛋白,C末端含有一个“锌指”模式结构。与16个物种比较的结果表明,眼镜王蛇与脊椎动物的泛素融合蛋白氨基酸序列相似度较高,具有高度的保守性。     

棉卷叶野螟泛素基因的克隆、序列分析及原核表达

本研究用RT-PCR方法,克隆了棉卷叶野螟Haritalodes derogata (Fabricius)泛素基因编码区,GenBank登录号为EU580145。序列分析表明,该编码区长228 bp,编码76个氨基酸,推测的编码蛋白的相对分子质量和等电点分别为8.53 kD和5.83。同源性比较发现,棉卷叶野螟泛素基因与其他10种昆虫泛素基因在氨基酸水平上具有93%以上的相似性。系统发育树显示棉卷叶野螟与斜纹夜蛾Spodoptera litura (Fabricius)遗传距离较近,通过同源建模获得了该棉卷叶野螟基因编码蛋白的理论三维结构。将棉卷叶野螟泛素基因与pET-32a(+)连接,构建原核表达载体pET-32a-ub,经IPTG诱导,棉卷叶野螟泛素基因在大肠杆菌BL21(DE3) 中高效表达。本研究成功克隆了棉卷叶野螟泛素基因的编码区,并经Western blotting分析证明实现了该基因的原核表达,为进一步研究其在该昆虫体内的作用机理奠定了基础。     

Complete amino acid sequence of tenebrosin-C, a cardiac stimulatory and haemolytic protein from the sea anemone Actinia tenebrosa

The complete amino acid sequence of the cardiac stimulatory and haemolytic protein tenebrosin-C, from the Australian sea anemone Actinia tenebrosa, has been determined by Edman degradation of the intact molecule and fragments produced by treatment of the polypeptide chain with cyanogen bromide and enzymatic cleavage with endoproteinase Asp-N, thermolysin and trypsin. The molecule is a single-chain polypeptide consisting of 179 amino acid residues with a calculated molecular mass of 19,797 Da. Tenebrosin-C shows a high degree of amino acid sequence similarity (63%) with Stoichactis helianthus cytolysin III [Blumenthal, K. M. and Kem, W. R. (1983) J. Biol. Chem. 258, 5574-5581] and is identical to a partial sequence (90 residues) reported for equinatoxin, a cardiostimulatory and haemolytic protein isolated from the European sea anemone Actinia equina [Ferlan, I. and Jackson, K. (1983) Toxicon Suppl. 3, 141-144]. No amino acid sequence similarity was detected between tenebrosin-C and other protein sequences stored in available databases. The predicted secondary structure of tenebrosin-C suggests that it is a compact, highly structured molecule.     

A unique primary structure, cDNA cloning and function of a galactose-specific lectin from ascidian plasma.

The complete amino acid sequence of a galactose-specific lectin from the plasma of the ascidian Halocynthia roretzi has been determined by sequential Edman degradation analysis of peptide fragments derived by proteolytic fragmentation and chemical cleavage of the reductive S-pyridylethylated lectin. Peptide fragments were separated by reverse-phase HPLC. The N-terminal and C-terminal amino acid sequences were determined by Edman degradation and enzymatic digestion. The H. roretzi plasma lectin is a single-chain protein consisting of 327 amino acids and four disulfide bonds, one of which was found to be cross-linked intramolecularly. A comparison of the amino acid sequence of the H. roretzi plasma lectin with the sequences of other proteins reveals that the H. roretzi lectin has a structure consisting of a twice-repeated sequence, a fibrinogen-related sequence and a C-type lectin-homologous sequence. The above amino acid sequence was verified by cDNA cloning of this lectin. Three cDNA clones that have single ORFs encoding the lectin precursor were isolated from an H. roretzi hepatopancreas cDNA library. The deduced amino acid sequences in the three cDNA clones contain the same sequence of the mature lectin molecule and the same putative signal sequence. In addition, it was demonstrated that this lectin can enhance phagocytosis by H. roretzi hemocytes. Thus, the plasma lectin is constructed into an oligomer structure via intermolecular disulfide bonds and plays a role in the biological defense of H. roretzi as a defense molecule.     

Cloning of a polyubiquitin gene fromNicotiana tabacum and comparison to other polyubiquitin genes

Using a tobacco cDNA clone as a probe, a genomic clone named TUQG-4, coding for a tobacco polyubiquitin protein with the five head-to-tail repeats of ubiquitin monomer was isolated. The five ubiquitin units were completely conserved except for the extra phenylalanine at the carboxy terminus of the last ubiquitin monomer. The putative open reading frame identified from the nucleotide sequence showed two possible intron sequences in the coding region for the first ubiquitin monomer. When the amino acid sequence deduced from the nucleotide sequence of TUQG-4 was compared to the amino acid sequences coded by other polyubiquitin genes of tobacco, there were three or four amino acid differences in the sequence. When the nucleotide sequences coding for the ubiquitin monomers were compared for various species origins, the degree of identity was at the highest between the ubiquitin monomers in one polyubiquitin and did not reflect the distance of the phylogenetic relationship.     

Determination and analysis of the primary structure of the nerve terminal specific phosphoprotein, synapsin I.

A rat brain cDNA clone containing an open reading frame encoding the neuron-specific protein synapsin I has been sequenced. The sequence predicts a protein of 691 amino acids with a mol. wt of 73 kd. This is in excellent agreement with the size of rat brain synapsin Ib measured by SDS--polyacrylamide gel electrophoresis. Inspection of the predicted primary structure has revealed the probable sites for synapsin I phosphorylation by the cAMP-dependent and Ca2+/calmodulin-dependent protein kinases. All of the biochemically observed intermediates of synapsin I digestion by collagenase can be verified by inspection of the sequence, and the collagenase-resistant fragment has been defined as the amino-terminal 439 amino acids of the molecule. Predictions of sequence secondary structure and hydrophobicity suggest that a central domain of approximately 270 amino acids may exist as a folded, globular core. The carboxyl-terminal domain of the protein (the region sensitive to collagenase digestion) contains sites for Ca2+/calmodulin-dependent protein kinase phosphorylation. These sites are flanked by three regions of repeating amino acid sequence that are proposed to be the synaptic vesicle-binding domain of synapsin I. This region also shares homology with the actin-binding proteins profilin and villin. The characteristics of the synapsin I sequence do not support extensive homology with the erythrocyte cytoskeletal protein 4.1.     

Engineering a thermostable protein via optimization of charge-charge interactions on the protein surface

A simple theoretical model for increasing the protein stability by adequately redesigning the distribution of charged residues on the surface of the native protein was tested experimentally. Using the molecule of ubiquitin as a model system, we predicted possible amino acid substitutions on the surface of this protein which would lead to an increase in its stability. Experimental validation for this prediction was achieved by measuring the stabilities of single-site-substituted ubiquitin variants using urea-induced unfolding monitored by far-UV CD spectroscopy. We show that the generated variants of ubiquitin are indeed more stable than the wild-type protein, in qualitative agreement with the theoretical prediction. As a positive control, theoretical predictions for destabilizing amino acid substitutions on the surface of the ubiquitin molecule were considered as well. These predictions were also tested experimentally using correspondingly designed variants of ubiquitin. We found that these variants are less stable than the wild-type protein, again in agreement with the theoretical prediction. These observations provide guidelines for rational design of more stable proteins and suggest a possible mechanism of structural stability of proteins from thermophilic organisms.     

Comparison of the three-dimensional structures of human, yeast, and oat ubiquitin

The crystal structure of human ubiquitin has been solved by x-ray diffraction methods and refined by standard procedures to a conventional crystallographic R factor of 0.176 at 1.8-A resolution (Vijay-Kumar, S., Bugg, C.E., and Cook, W.J. (1987) J. Mol. Biol. 194, 525-538). Crystals of yeast and oat ubiquitin have been grown using human ubiquitin crystals as seeds. Diffraction data for yeast and oat ubiquitin have been collected to a resolution of 1.9 and 1.8 A, respectively. Difference Fourier electron-density maps reveal that the structures of yeast and oat ubiquitin are quite similar to human ubiquitin. All the amino acid changes are clustered in two small patches on one surface of the molecule. This surface is probably not involved in conjugation with proteins destined for ATP-dependent proteolysis.     

Comparison of amino acid sequences of sturgeon triprotamines using protamines from Acipenser stellatus gonads as an example]

The amino acid sequence of the triprotamine stelline C from mature sperm nuclei of Acipenser stellatus has been established by automated sequence analysis of the protein and from data provided by thermolysine peptides. The complete amino acid sequence of stelline C is: R-R-R-R-R-H-A-S-T-K-L-K-R-R-R-R-R-R-R-H-G-K-K-S-H-K. The comparison of the primary structure of stelline C with that of other triprotamines from Acipenser stellatus shows that they are similar except for the absence of N-terminal alanine in the stelline A molecule. The main structure difference between stelline C and other fish protamines is that the stelline C molecule begins with five arginine residues.     

Shark Myelin Basic Protein: Amino Acid Sequence, Secondary Structure, and Self-Association

Myelin basic protein (MBP) from the Whaler shark (Carcharhinus obscurus) has been purified from acid extracts of a chloroform/methanol pellet from whole brains. The amino acid sequence of the majority of the protein has been determined and compared with the sequences of other MBPs. The shark protein has only 44% homology with the bovine protein, but, in common with other MBPs, it has basic residues distributed throughout the sequence and no extensive segments that are predicted to have an ordered secondary structure in solution. Shark MBP lacks the triproline sequence previously postulated to form a hairpin bend in the molecule. The region containing the putative consensus sequence for encephalitogenicity in the guinea pig contains several substitutions, thus accounting for the lack of activity of the shark protein. Studies of the secondary structure and self-association have shown that shark MBP possesses solution properties similar to those of the bovine protein, despite the extensive differences in primary structure.     

Complete cDNA sequence of a Dictyostelium ubiquitin with a carboxy-terminal tail and identification of the protein using an anti-peptide antibody

The complete sequence of a Dictyostelium discoideum cDNA is presented that codes for monoubiquitin extended at its C-terminus by a 52 amino acid tail. The sequence of both the ubiquitin portion and the tail is highly homologous to the one of Saccharomyces cerevisiae and to a partial mouse sequence. The highly basic tail sequence contains a putative metal and nucleic acid-binding motif. The gene encoding the 0.6 kb mRNA of the C-terminally extended ubiquitin is represented only once in the haploid genome. The 0.6 kb mRNA as well as its translation product, a 15 kDa protein, is expressed in exponentially growing cells and remains present for at least 5 h of development. Using antibodies against a synthetic peptide that corresponds to the C-terminal amino acid sequence, a 15 kDa protein containing the extension a synthetic peptide that corresponds to the C-terminal amino acid sequence, a 15 kDa protein containing the extension was also detected in yeast.     

Molecular cloning and expression of a Tetrahymena pyriformis ubiquitin fusion gene coding for a 53-amino-acid extension protein.

Summary The genome of Tetrahymena pyriformis has been shown to contain a ubiquitin multigene family consisting of several polyubiquitin genes and at least one ubiquitin fusion gene. We report here the isolation and characterization of one genomic clone (pTUl1), that encodes a ubiquitin extension protein. A comparison of the predicted amino acid sequence of the ubiquitin extension protein gene of T. pyriformis with those from other organisms indicated a high degree of homology. However, the Tetrahymena ubiquitin extension protein contains 53 and not 52 amino acids. This feature is different from all ubiquitin 52-amino-acid extension protein genes thus far sequenced. Furthermore, we found an array of four cysteine residues similar to those found in nucleic acid binding proteins. Also, the C-terminal sequence possesses a conserved motif which may represent a nuclear translocation signal. The ubiquitin 53-amino-acid extension protein gene encodes the smallest class of ubiquitin mRNAs in T. pyriformis.     

The primary structure of the cytotoxin restrictocin

The complete amino acid sequence of the single polypeptide chain of cytotoxin restrictocin has been determined. Its structure was established by automated Edman degradation of the intact molecule reduced and [14C]carboxymethylated and of fragments obtained by chemical cleavage of the protein with cyanogen bromide and BNPS-skatole and by enzymatic cleavage of the polypeptide chain with trypsin. The molecule consists of 149 amino acid residues with a calculated relative molecular mass of 16836. The protein presents two disulfide bridges, one between cysteine residues at positions 5 and 147 and the other one formed by cysteine residues at positions 75 and 131. The amino acid sequence of restrictocin shows a high degree of homology (86%) with that of the cytotoxin named alpha-sarcin.     

Protein synthesis rhythm.

The three-dimensional structure of a protein molecule appears to depend on the amino acid sequence of the protein in an as yet incompletely described manner. If the amino acid sequence is replaced by a numerical sequence of values representing a physical or chemical property of amino acids, the resulting numerical sequence is amenable to autocorrelation analysis. Further, if certain geometrical parameters are calculated from the three-dimensional structure of a protein to form a configurational series, pairs of property series and configurational series can be analyzed by cross-correlation techniques. The data base for the analysis was the three-dimensional structures of ten proteins as determined by X-ray crystallography. Such analysis yields the result that the hydrophobicity of an amino acid residue in a protein influences the orientation angle of the amino acid side chain. This result is consistent with the widely current “oil-drop” model of protein structure. Hydrophobicity also appears to influence the backbone dihedral angle φ, but not ψ Such a directional effect cannot be explained by a current model of information transfer in protein helices. The magnitude of the cross correlations does not appear to be satisfactory for construction of a transfer function model for the prediction of general features of protein structure from amino acid sequences.     

Identification, cloning, and initial characterization of a novel mouse testicular germ cell-specific antigen

A monoclonal antibody, designated TES101, was raised by immunizing BALB/c mice with an allogenic mouse testicular homogenate followed by immunohistochemical selection as the initial screening method. By searching the expressed sequence tag (EST) database with the N-terminal amino acid sequence of TES101 reactive protein, we found that the predicted amino acid sequence encoded by a mouse testicular EST clone matched the TES101 protein sequence. Sequence analysis of the clone revealed no homologous molecule in the DNA/protein database. Based on data obtained from N-terminal amino acid analysis of the TES101 protein, the derived amino acid sequence contained a signal peptide region of 25 amino acids and a mature protein region of 225 amino acids, which translated into a protein with a molecular weight of 24 093. Northern blot analysis showed that mRNA of the TES101 protein was found in testis but not in any other mouse tissues examined. Western blot analysis revealed that TES101 reacted with a 38-kDa band on SDS-PAGE under nonreducing conditions, and this reactivity was abrogated under reducing conditions. Immunoelectron microscopic studies demonstrated that the molecule was predominantly located on the plasma membrane of spermatocytes and spermatids but not in Sertoli cells or interstitial cells, including Leydig cells. Thus, the TES101 protein is a novel molecule present primarily on the surface of developing male germ cells. TES101 protein may play a role in the processes underlying male germ cell formation.