Identification of PDZ5, a candidate universal minicircle sequence binding protein of Trypanosoma cruzi

The dodecamer universal minicircle sequence is a conserved sequence present in minicircles of trypanosomatid kinetoplast DNA studied so far. This sequence is recognised by a protein named universal minicircle sequence binding protein, described for Crithidia fasciculata, involved in minicircle DNA replication. We have identified a Trypanosoma cruzi gene homologue of the Crithidia fasciculata universal minicircle sequence binding protein. Similar to the Crithidia fasciculata universal minicircle sequence binding protein, the Trypanosoma cruzi protein, named PDZ5, contains five zinc finger motifs. Pulsed field gel electrophoresis indicated that the pdz5 gene is located in the chromosomal band XX of the Trypanosoma cruzi genome. The predicted amino acid sequence of PDZ5 shows a high degree of similarity with several trypanosomatid zinc finger proteins. Specific antibody raised against Crithidia fasciculata universal minicircle sequence binding protein recognises both the recombinant and endogenous PDZ5. The complete pdz5 coding sequence cloned in bacteria expresses a recombinant PDZ5 protein that binds specifically to the universal minicircle sequence dodecamer. These data strongly suggest that PDZ5 represents a Trypanosoma cruzi universal minicircle sequence binding protein.     

The quaternary structure, antigenicity, and aggregational behavior of the secretory core protein of human hepatitis B virus are determined by its signal sequence.

Human hepatitis B virus encodes a secretory core protein, referred to as the HBe protein, whose secretion is mediated by the pre-C signal sequence. Here we examined whether this sequence is important only for translocation of the HBe precursor (the precore protein) or whether it also contributes to the structural and biophysical properties of the mature HBe protein. When a truncated hepatitis B virus precore protein, lacking the basic C-terminal domain which is cleaved from the wild-type protein during its conversion into HBe, was expressed in human hepatoma cells, only a small amount of HBe-like protein was produced. This protein was slightly smaller than the wild-type HBe protein, suggesting that C-terminal cleavage of the precore protein does not occur at the suggested site. When the authentic signal sequence of the precore protein (the pre-C sequence) was replaced by the unrelated signal sequence of an influenza virus hemagglutinin, not only the full-length but also the C-terminally truncated protein was expressed and secreted with high efficiency. Western blot (immunoblot) analyses with nonreducing gels and conformation-specific monoclonal antibodies revealed that the HBe protein secreted under control of the pre-C signal sequence was a monomer with HBe antigenicity, whereas the HBe-like protein secreted under control of the hemagglutinin signal sequence was a disulfide-bridge-linked dimer with both HBe and HBc antigenicity. Electron microscopic examination of gradient-purified particulate core gene products showed that HBe protein secreted under control of the hemagglutinin signal sequence forms core particles, whereas HBe protein secreted under control of the pre-C sequence does not. Thus, the pre-C sequence not only mediates the secretion but also determines the structural and aggregational properties of the HBe protein.     

A database of unique protein sequence identifiers for proteome studies

In proteome studies, identification of proteins requires searching protein sequence databases. The public protein sequence databases (e.g., NCBInr, UniProt) each contain millions of entries, and private databases add thousands more. Although much of the sequence information in these databases is redundant, each database uses distinct identifiers for the identical protein sequence and often contains unique annotation information. Users of one database obtain a database-specific sequence identifier that is often difficult to reconcile with the identifiers from a different database. When multiple databases are used for searches or the databases being searched are updated frequently, interpreting the protein identifications and associated annotations can be problematic. We have developed a database of unique protein sequence identifiers called Sequence Globally Unique Identifiers (SEGUID) derived from primary protein sequences. These identifiers serve as a common link between multiple sequence databases and are resilient to annotation changes in either public or private databases throughout the lifetime of a given protein sequence. The SEGUID Database can be downloaded (http://bioinformatics.anl.gov/SEGUID/) or easily generated at any site with access to primary protein sequence databases. Since SEGUIDs are stable, predictions based on the primary sequence information (e.g., pI, Mr) can be calculated just once; we have generated approximately 500 different calculations for more than 2.5 million sequences. SEGUIDs are used to integrate MS and 2-DE data with bioinformatics information and provide the opportunity to search multiple protein sequence databases, thereby providing a higher probability of finding the most valid protein identifications.     

一种苦荞主要过敏原基因cDNA的克隆及序列分析

为了获得苦荞中主要过敏原的cDNA和由此推导的蛋白质序列 ,分析其结构特点 ,以苦荞幼根根尖为材料 ,提取总RNA并反转录mRNA为cDNA第一链 .通过RT PCR、3′RACE、基因克隆及序列测定 ,获得一种苦荞主要过敏蛋白基因的cDNA片段 (GenBank登录号为AY0 4 4918) .该cDNA片段由 76 8bp组成 ,包括 3′端非编码区 180个bp ,开放阅读框 5 88bp .可编码一个由 195个氨基酸残基组成的功能蛋白及一个终止密码 .苦荞主要过敏原基因与甜荞 2 2kD过敏蛋白、豆球类蛋白的核苷酸序列分别有 95 %和 93%的同源性 .其推导的氨基酸序列与甜荞球蛋白、刀豆蛋白、甜橙柠檬素分别有 93%、83%和 5 7%的同源性 .该过敏蛋白 183～ 188位氨基酸残基KEEEKE在多数不同过敏原中均存在 ,推测可能为其中的抗原决定簇序列     

Improvement in protein functional site prediction by distinguishing structural and functional constraints on protein family evolution using computational design

The prediction of functional sites in newly solved protein structures is a challenge for computational structural biology. Most methods for approaching this problem use evolutionary conservation as the primary indicator of the location of functional sites. However, sequence conservation reflects not only evolutionary selection at functional sites to maintain protein function, but also selection throughout the protein to maintain the stability of the folded state. To disentangle sequence conservation due to protein functional constraints from sequence conservation due to protein structural constraints, we use all atom computational protein design methodology to predict sequence profiles expected under solely structural constraints, and to compute the free energy difference between the naturally occurring amino acid and the lowest free energy amino acid at each position. We show that functional sites are more likely than non-functional sites to have computed sequence profiles which differ significantly from the naturally occurring sequence profiles and to have residues with sub-optimal free energies, and that incorporation of these two measures improves sequence based prediction of protein functional sites. The combined sequence and structure based functional site prediction method has been implemented in a publicly available web server.     

Correction of the cDNA-derived protein sequence of prostatic spermine binding protein: pivotal role of tandem mass spectrometry in sequence analysis

Spermine binding protein (SBP) is a rat ventral prostate protein that binds various polyamines, and the level of this protein and its mRNA is regulated by androgens. Previously, the cDNA for SBP was cloned and sequenced and an amino acid sequence deduced from the cDNA. Data from cloned and sequenced and an amino acid sequence deduced from the cDNA. Data from partial amino acid sequencing of the purified protein were consistent with the amino acid sequence deduced from the cDNA. However, the amino terminus of the protein was blocked, and therefore, direct protein sequence information confirming the cDNA reading frame of this region could not be obtained by Edman degradation. We have now employed an integrated approach using fast atom bombardment mass spectrometry, tandem mass spectrometry, and conventional sequencing methodologies to establish the amino-terminal sequence of the protein and to identify an amino acid sequence (35 residues) present in the purified protein but missing from the amino acid sequence deduced from cDNA clones for this protein. The missing piece of cDNA corresponds to an exon found in mouse genomic clones for a protein similar to rat SBP. Therefore, the cDNA clones for rat SBP may represent splicing variants that lack the sequence information of one exon. The blocked amino terminus of the protein was identified as 5-oxopyrrolidine-2-carboxylic acid. Mass spectrometry also provided evidence regarding glycosylation of the protein. The first of two potential glycosylation sites clearly carries carbohydrate; the second site is, at most, only partially glycosylated.     

Fusion of pro region of subtilisin to staphylococcal protein A and its secretion by Bacillus subtilis

Subtilisin is synthesized as a preproenzyme in Bacillus subtilis. We fused that region of the subtilisin gene, (apr[BamP]), which encodes the signal sequence and pro region, to the mature gene sequence (spa) for a heterologous protein (staphylococcal protein A). B. subtilis cells harboring this gene fusion synthesized a fusion protein consisting of the signal and pro sequence of subtilisin fused to the protein A; the signal sequence was processed and a fusion protein (pro + protein A) was secreted into the growth medium.     

Amino-terminal amino acid sequence of the nonspecific phospholipid exchange protein from bovine liver

The amino-terminal amino acid sequence of the nonspecific phospholipid exchange protein from bovine liver has been determined. The first 52 amino-terminal residues in the sequence were identified. The sequence determined failed to show statistically significant homology to any previously published protein sequence. However, a stretch of 12 amino acids at the end of the sequence displays homology to the phosphatidylcholine-specific phospholipid exchange protein.     

The ultimate localization of an outer membrane protein of Escherichia coli K-12 is not determined by the signal sequence

To study the role of the signal sequences in the biogenesis of outer membrane proteins, we have constructed two hybrid genes: a phoE-ompF hybrid gene, which encodes the signal sequence of outer membrane PhoE protein and the structural sequence of outer membrane OmpF protein, and a bla-phoE hybrid gene which encodes the signal sequence as well as 158 amino acids of the structural sequence of the periplasmic enzyme beta-lactamase and the complete structural sequence of PhoE protein. The products of these genes are normally transported to and assembled into the outer membrane These results show: (i) that signal sequences of exported proteins are export signals which function independently of the structural sequence, and (ii) that the information which determines the ultimate location of an outer membrane protein is located in the structural sequence of this protein, and not in the signal sequence.     

Sequences of Escherichia coli uvrA gene and protein reveal two potential ATP binding sites

We have determined the nucleotide sequence of the uvrA gene of Escherichia coli. The coding region of the gene is 2820 base pairs which specifies a protein of 940 amino acids and Mr = 103,874. The polypeptide sequence predicted from the DNA sequence was confirmed by analyzing the UvrA protein: the sequence of the first 7 NH2-terminal amino acids as well as the amino acid composition of the pure protein agreed with those predicted from the nucleotide sequence. By comparing the sequence of UvrA protein to the amino acid sequences of other ATPases, we found that two regions in the UvrA protein, separated from one another by about 600 amino acids, have the highly conserved G-X4-GKT(S)-X6-I(V) sequence found at the active sites of many, but not all, ATPases. Our findings suggest that UvrA protein may have two ATP binding sites.     

超级杂交稻乙烯反应元件结合蛋白cDNA的生物信息学分析与克隆

目的：从超级杂交稻中克隆乙烯反应元件结合蛋白（EREBP）的cDNA。方法：利用模式植物拟南芥中编码乙烯反应元件结合蛋白的cDNA,对现有的水稻基因组数据库进行搜索,获得一条高同源的未知序列。对这条未知序列的核酸序列蛋白质序列及其结构、性质、功能等进行生物信息学分析后,以超级杂交稻为材料,用未知序列设计一对简并引物,用RTPCR技术扩增后进行T-A克隆。结果：生物信息学分析结果表明,这个未知序列应为水稻中编码EREBP的cDNA;克隆后经测序获得一条915bp的cDNA,BLAST表明这条cDNA序列与未知序列的部分核酸序列的同源性达到了99％;提NCBI的GenBank后被接受,登录号为EF507537。结论：以生物信息学分析为基础,结合RT-PCR和T-A克隆技术,成功地从超级杂交稻中克隆了EREBP cDNA。     

The imprint of codons on protein structure

The "central dogma" of biology outlines the unidirectional flow of interpretable data from genetic sequence to protein sequence. This has led to the idea that a protein's structure is dependent only on its amino acid sequence and not its genetic sequence. Recently, however, a more than transient link between the coding genetic sequence and the protein structure has become apparent. The two interact at the ribosome via the process of co-translational protein folding. Evidence for co-translational folding is growing rapidly, but the influence of codons on the protein structure attained is still highly contentious. It is theorised that the speed of codon translation modulates the time available for protein folding and hence the protein structure. Here, past and present research regarding synonymous codons and codon translation speed are reviewed within the context of protein structure attainment.     

Applications of sequence coevolution in membrane protein biochemistry

Recently, protein sequence coevolution analysis has matured into a predictive powerhouse for protein structure and function. Direct methods, which use global statistical models of sequence coevolution, have enabled the prediction of membrane and disordered protein structures, protein complex architectures, and the functional effects of mutations in proteins. The field of membrane protein biochemistry and structural biology has embraced these computational techniques, which provide functional and structural information in an otherwise experimentally-challenging field. Here we review recent applications of protein sequence coevolution analysis to membrane protein structure and function and highlight the promising directions and future obstacles in these fields. We provide insights and guidelines for membrane protein biochemists who wish to apply sequence coevolution analysis to a given experimental system.     

The amino acid sequence of human beta-microseminoprotein

The complete amino acid sequence of beta-microseminoprotein of human seminal plasma was determined by automated Edman degradation of the protein and peptides which were obtained by enzymatic cleavage with trypsin, chymotrypsin and Staphylococcus aureus V8 proteinase. The carboxyl-terminal sequence of the protein was established with the aid of carboxypeptidase A. The amino acid sequence of this protein proved to be as follows: (sequence; see text) Thus, beta-microseminoprotein consisting of 93 amino acid residues has a molecular mass of 10 652 Da. The linear structure of this protein represents the first complete amino acid sequence of a sperm-coating protein specific to human seminal plasma.     

Latent Periodicity of Protein Sequences

This article is in the area of protein sequence investigation. It studies protein sequence periodicity. The notion of latent periodicity is introduced. A mathematical method for searching for latent periodicity in protein sequences is developed. Implementation of the method developed for known cases of perfect and imperfect periodicity is demonstrated. Latent periodicity of many protein sequences from the SWISS-PROT data bank is revealed by the method and examples of latent periodicity of amino acid sequences are demonstrated for: the translation initiation factor EIF-2B (epsilon subunit) of Saccharomyces cerevisiae from the E2BE_YEAST sequence; the E.coli ferrienterochelin receptor from the FEPA_ECOLI sequence; the lysozyme of Bacteriophage SF6 from the LY_BPSF6 sequence; lipoamide dehydrogenase of Azotobacter vinelandii from the DLDH_AZOVI sequence. These protein sequences have latent periods equal to six, two, seven and 19 amino acids, respectively. We propose that a possible purpose of the amino acid sequence latent periodicity is to determine certain protein structures.     

Complete cDNA-derived amino acid sequence of rat brown fat uncoupling protein

Cloned cDNAs corresponding to the mitochondrial uncoupling protein of rat brown adipose tissue have been sequenced and the complete amino acid sequence of this unique membranous component is given. The N-terminal sequence of this protein is almost identical to the 14-residue N-terminal sequence previously determined by others for the hamster uncoupling protein. The uncoupling protein has no N-terminal signal extension. We found a significant sequence homology between the uncoupling protein and the ADP/ATP carrier and propose that the nucleotide binding site of the uncoupling protein is localized at the C-terminal end.     

Interaction between the replication origin and the initiator protein of the filamentous phage f1. Binding occurs in two steps

The replication initiator protein of bacteriophage f1 (gene II protein) binds to the phage origin and forms two complexes that are separable by polyacrylamide gel electrophoresis. Complex I is formed at low gene II protein concentrations, and shows protection from DNase I of about 25 base-pairs (from position +2 to +28 relative to the nicking site) at the center of the minimal origin sequence. Complex II is produced at higher concentrations of the protein, and has about 40 base-pairs (from -7 to +33) protected. On the basis of gel mobility, complex II appears to contain twice the amount of gene II protein as does complex I. The 40 base-pair sequence protected in complex II corresponds to the minimal origin sequence as determined by in-vivo analyses. The central 15 base-pair sequence (from +6 to +20) of the minimal origin consists of two repeats in inverted orientation. This sequence, when cloned into a plasmid, can form complex I, but not complex II. We call this 15 base-pair element the core binding sequence for gene II protein. Methylation interference with the formation of complex I by the wild-type origin indicates that gene II protein contacts six guanine residues located in a symmetric configuration within the core binding sequence. Formation of complex II requires, in addition to the core binding sequence, the adjacent ten base-pair sequence on the right containing a third homologous repeat. A methylation interference experiment performed on complex II indicates that gene II protein interacts homologously with the three repeats. In complex II, gene II protein protects from DNase I digestion not only ten base-pairs on the right but also ten base-pairs on the left of the sequence that is protected in complex I. Footprint analyses of various deletion mutants indicate that the left-most ten base-pairs are protected regardless of their sequence. The site of nicking by gene II protein is located within this region. A model is presented for the binding reaction involving both protein-DNA and protein-protein interactions.     

Recapitulation of protein family divergence using flexible backbone protein design

We use flexible backbone protein design to explore the sequence and structure neighborhoods of naturally occurring proteins. The method samples sequence and structure space in the vicinity of a known sequence and structure by alternately optimizing the sequence for a fixed protein backbone using rotamer based sequence search, and optimizing the backbone for a fixed amino acid sequence using atomic-resolution structure prediction. We find that such a flexible backbone design method better recapitulates protein family sequence variation than sequence optimization on fixed backbones or randomly perturbed backbone ensembles for ten diverse protein structures. For the SH3 domain, the backbone structure variation in the family is also better recapitulated than in randomly perturbed backbones. The potential application of this method as a model of protein family evolution is highlighted by a concerted transition to the amino acid sequence in the structural core of one SH3 domain starting from the backbone coordinates of an homologous structure.     

Overproduction, purification, and ATPase activity of the Escherichia coli RuvB protein involved in DNA repair.

The ruvA and ruvB genes of Escherichia coli constitute an operon which belongs to the SOS regulon. Genetic evidence suggests that the products of the ruv operon are involved in DNA repair and recombination. To begin biochemical characterization of these proteins, we developed a plasmid system that overproduced RuvB protein to 20% of total cell protein. Starting from the overproducing system, we purified RuvB protein. The purified RuvB protein behaved like a monomer in gel filtration chromatography and had an apparent relative molecular mass of 38 kilodaltons in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which agrees with the value predicted from the DNA sequence. The amino acid sequence of the amino-terminal region of the purified protein was analyzed, and the sequence agreed with the one deduced from the DNA sequence. Since the deduced sequence of RuvB protein contained the consensus sequence for ATP-binding proteins, we examined the ATP-binding and ATPase activities of the purified RuvB protein. RuvB protein had a stronger affinity to ADP than to ATP and weak ATPase activity. The results suggest that the weak ATPase activity of RuvB protein is at least partly due to end product inhibition by ADP.     

The molecular cloning of the complementary deoxyribonucleic acid for bovine vitamin D-dependent calcium-binding protein: structure of the full-length protein and evidence for homologies with other calcium-binding proteins of the troponin-C superfamily of proteins

We have cloned the cDNA for bovine intestinal vitamin D-dependent calcium-binding protein and, based on the sequence of the DNA, have deduced the structure of the full-length protein. The sequence of the cDNA clone predicts a protein comprised of 78 amino acids with a mol wt of 8788. The mRNA for the protein in bovine duodenum is about 500-600 bases in length. The protein sequence of bovine intestinal calcium-binding protein is 87% homologous with the sequence of porcine intestinal vitamin D-dependent calcium-binding protein and 81% homologous with the sequence of rat intestinal vitamin D-dependent calcium-binding protein. Hydrophilicity plots of the proteins noted above show that despite differences in amino acid sequence the proteins have similar patterns. In addition, the predicted secondary structure of the proteins is similar. Bovine intestinal calcium-binding protein shows 48.6% homology with the alpha-chain and 38.2% homology with the beta-chain of bovine S-100 protein and a similar high degree of homology with the beta-chain of human S-100 protein. The protein also demonstrates 36-43% homology with parvalbumin alpha and beta from various species and with troponin-C. There is some homology with the 28K vitamin D-dependent calcium-binding proteins. Vitamin D-dependent bovine intestinal calcium-binding protein is closely related to other mammalian intestinal calcium-binding proteins and to the S-100 proteins, parvalbumins, and troponin-C.