A scheme for multiple sequences alignment optimization-an improvement based on family representative mechanics features

As a basic tool of modern biology, sequence alignment can provide us useful information in fold, function, and active site of protein. For many cases, the increased quality of sequence alignment means a better performance. The motivation of present work is to increase ability of the existing scoring scheme/algorithm by considering residue–residue correlations better. Based on a coarse-grained approach, the hydrophobic force between each pair of residues is written out from protein sequence. It results in the construction of an intramolecular hydrophobic force network that describes the whole residue–residue interactions of each protein molecule, and characterizes protein's biological properties in the hydrophobic aspect. A former work has suggested that such network can characterize the top weighted feature regarding hydrophobicity. Moreover, for each homologous protein of a family, the corresponding network shares some common and representative family characters that eventually govern the conservation of biological properties during protein evolution. In present work, we score such family representative characters of a protein by the deviation of its intramolecular hydrophobic force network from that of background. Such score can assist the existing scoring schemes/algorithms, and boost up the ability of multiple sequences alignment, e.g. achieving a prominent increase (50%) in searching the structurally alike residue segments at a low identity level. As the theoretical basis is different, the present scheme can assist most existing algorithms, and improve their efficiency remarkably.

Unnatural amino acids in the synthesis and semisynthesis of metalloprotein motifs

The design, synthesis, and characterization of two novel metalloprotein motifs is presented. The first project involved the design and construction of a protein motif which was programmed to form a tetradentate metal complex upon the addition of metal cations. The overall structure of the motif was based on a ββ super-secondary structure consisting of a flexible peptide sequence flanked by metal binding regions located at the carboxy and amino termini. The metal binding region near the amino terminus was constructed from a reverse turn motif with two metal ligating residues, (2R, 3R)-β-methyl-cysteine and histidine. Selection of the peptide sequence for this region was based on the conformational analysis of a series of tetrapeptides designed to form reverse turns in solution.

The stereospecific syntheses of a series of novel bipyridyl- and phenanthrolylsubstituted amino acids was carried out to provide ligands for the carboxy terminus metal binding region. These residues were incorporated into peptide sequences using solid phase peptide synthesis protocols, and metal binding studies indicated that the metal binding properties of these ligands was dictated by the specific regioisomer of the heteroaromatic ring and the peptide primary sequence.

Finally, a peptide containing optimized components for the metal binding regions was prepared to test the ability of the compound to form the desired intramolecular peptide:metal cation complexes. Metal binding studies demonstrated that the peptide formed monomeric complexes with very high metal cation binding affinities and that the two metal binding regions act cooperatively in the metal binding process. The use of these systems in the design of proteins capable of regulating naturally occurring proteins is discussed.

The second project involved the semisynthesis of two horse heart cytochrome c mutants incorporating the bipyridyl-amino acids at position 72 of the protein sequence. Structural studies on the proteins indicated that the bipyridyl amino acids had a neglible effect on the protein structure. One of the mutants was modified with Ru(bpy)_2^(+2) to form a redox-active protein, and the modified protein was found to have enhanced electron transfer properties between the heme and the introduced metal site.

Structural and mechanistic motifs in membrane proteins: the three-dimensional modelling of rhodopsin, band 3, and the nicotinic acetylcholine receptor

Because so little is known about the structure of membrane proteins, an attempt has been made in this work to develop techniques by which to model them in three dimensions. The procedures devised rely heavily upon the availability of several sequences of a given protein. The modelling procedure is composed of two parts. The first identifies transmembrane regions within the protein sequence on the basis of hydrophobicity, β-turn potential, and the presence of certain amino acid types, specifically, proline and basic residues. The second part of the procedure arranges these transmembrane helices within the bilayer based upon the evolutionary conservation of their residues. Conserved residues are oriented toward other helices and variable residues are positioned to face the surrounding lipids. Available structural information concerning the protein's helical arrangement, including the lengths of interhelical loops, is also taken into account. Rhodopsin, band 3, and the nicotinic acetylcholine receptor have all been modelled using this methodology, and mechanisms of action could be proposed based upon the resulting structures.

Specific residues in the rhodopsin and iodopsin sequences were identified, which may regulate the proteins' wavelength selectivities. A hinge-like motion of helices M3, M4, and M5 with respect to the rest of the protein was proposed to result in the activation of transducin, the G-protein associated with rhodopsin. A similar mechanism is also proposed for signal transduction by the muscarinic acetylcholine and β-adrenergic receptors.

The nicotinic acetylcholine receptor was modelled with four trans-membrane helices per subunit and with the five homologous M2 helices forming the cation channel. Putative channel-lining residues were identified and a mechanism of channel-opening based upon the concerted, tangential rotation of the M2 helices was proposed.

Band 3, the anion exchange protein found in the erythrocyte membrane, was modelled with 14 transmembrane helices. In general the pathway of anion transport can be viewed as a channel composed of six helices that contains a single hydrophobic restriction. This hydrophobic region will not allow the passage of charged species, unless they are part of an ion-pair. An arginine residue located near this restriction is proposed to be responsible for anion transport. When ion-paired with a transportable anion it rotates across the barrier and releases the anion on the other side of the membrane. A similar process returns it to its original position. This proposed mechanism, based on the three-dimensional model, can account for the passive, electroneutral, anion exchange observed for band 3. Dianions can be transported through a similar mechanism with the additional participation of a histidine residue. Both residues are located on M10.

Structural and Biophysical Characterization of Variants of the Mechanosensitive Channel of Large Conductance (MSCL)

The ability to sense mechanical force is vital to all organisms to interact with and respond to stimuli in their environment. Mechanosensation is critical to many physiological functions such as the senses of hearing and touch in animals, gravitropism in plants and osmoregulation in bacteria. Of these processes, the best understood at the molecular level involve bacterial mechanosensitive channels. Under hypo-osmotic stress, bacteria are able to alleviate turgor pressure through mechanosensitive channels that gate directly in response to tension in the membrane lipid bilayer. A key participant in this response is the mechanosensitive channel of large conductance (MscL), a non-selective channel with a high conductance of ~3 nS that gates at tensions close to the membrane lytic tension.

It has been appreciated since the original discovery by C. Kung that the small subunit size (~130 to 160 residues) and the high conductance necessitate that MscL forms a homo-oligomeric channel. Over the past 20 years of study, the proposed oligomeric state of MscL has ranged from monomer to hexamer. Oligomeric state has been shown to vary between MscL homologues and is influenced by lipid/detergent environment. In this thesis, we report the creation of a chimera library to systematically survey the correlation between MscL sequence and oligomeric state to identify the sequence determinants of oligomeric state. Our results demonstrate that although there is no combination of sequences uniquely associated with a given oligomeric state (or mixture of oligomeric states), there are significant correlations. In the quest to characterize the oligomeric state of MscL, an exciting discovery was made about the dynamic nature of the MscL complex. We found that in detergent solution, under mild heating conditions (37 °C – 60 °C), subunits of MscL can exchange between complexes, and the dynamics of this process are sensitive to the protein sequence.

Extensive efforts were made to produce high diffraction quality crystals of MscL for the determination of a high resolution X-ray crystal structure of a full length channel. The surface entropy reduction strategy was applied to the design of S. aureus MscL variants and while the strategy appears to have improved the crystallizability of S. aureus MscL, unfortunately the diffraction qualities of these crystals were not significantly improved. MscL chimeras were also screened for crystallization in various solubilization detergents, but also failed to yield high quality crystals.

MscL is a fascinating protein and continues to serve as a model system for the study of the structural and functional properties of mechanosensitive channels. Further characterization of the MscL chimera library will offer more insight into the characteristics of the channel. Of particular interest are the functional characterization of the chimeras and the exploration of the physiological relevance of intercomplex subunit exchange.

Engineering and Characterization of Cytochrome P450 Enzymes for Nitrogen-Atom Transfer Reactions

The creation of novel enzyme activity is a great challenge to protein engineers, but nature has done so repeatedly throughout the process of natural selection. I begin by outlining the multitude of distinct reactions catalyzed by a single enzyme class, cytochrome P450 monooxygenases. I discuss the ability of cytochrome P450 to generate reactive intermediates capable of diverse reactivity, suggesting this enzyme can also be used to generate novel reactive intermediates in the form of metal-carbenoid and nitrenoid species. I then show that cytochrome P450 from Bacillus megaterium (P450_BM3) and its isolated cofactor can catalyze metal-nitrenoid transfer in the form of intramolecular C–H bond amination. Mutations to the protein sequence can enhance the reactivity and selectivity of this transformation significantly beyond that of the free cofactor. Next, I demonstrate an intermolecular nitrene transfer reaction catalyzed by P450_BM3 in the form of sulfide imidation. Understanding that sulfur heteroatoms are strong nucleophiles, I show that increasing the sulfide nucleophilicity through substituents on the aryl sulfide ring can dramatically increase reaction productivity. To explore engineering nitrenoid transfer in P450BM3, active site mutagenesis is employed to tune the regioselectivity intramolecular C–H amination catalysts. The solution of the crystal structure of a highly selective variant demonstrates that hydrophobic residues in the active site strongly modulate reactivity and regioselectivity. Finally, I use a similar strategy to develop P450-based catalysts for intermolecular olefin aziridination, demonstrating that active site mutagenesis can greatly enhance this nitrene transfer reaction. The resulting variant can catalyze intermolecular aziridination with more than 1000 total turnovers and enantioselectivity of up to 99% ee.

Adaptive evolution of primate TRIM5 alpha, a gene restricting HIV-1 infection

Recent studies showed that nonhuman primate TRIM5 alpha can efficiently block HIV-1 infection in human cell lines. It can also restrict other retroviruses, therefore, suggested as a general defender against retrovirus infection. Here, we present an evolutionary analysis of TRIM5 alpha in primates. Our results demonstrated that TRIM5a has been evolving rapidly in primates, which is likely caused by Darwinian positive selection. The SPRY domain of TRM5 alpha, which may be responsible for recognition of incoming viral capsids showed higher nonsynonymous/synonymous substitution ratios than the non-SPRY domain, indicating that the adaptive evolution of TRIM5a ill primates might be an innate strategy developed in defending retrovirus infection during primate evolution. In addition, the comparative protein sequence analysis suggested that the amino acid substitution pattern at a single site (344R/Q/P) located in the SPRY domain may explain the differences in Susceptibilities of HIV-1 infection in diverse primate species. (c) 2005 Elsevier B.V. All rights reserved.

Molecular characterization of a weak fibrinogen-clotting enzyme from Trimeresurus jerdonii venom

A fibrinogen-clotting enzyme designed as jerdonobin-II was isolated from the venom of Trimeresurus jerdonii. It differed in molecular weight and N-terminal sequence with the previously isolated jerdonobin, a thrombin-like enzyme from the same venom. The enzyme consists of a single polypeptide chain with molecular weights of 30,000 and 32,000 under non-reducing and reducing conditions, respectively. Jerdonobin-II showed weak fibrinogen clotting activity and its activity unit on fibrinogen was calculated to be less than one unit using human thrombin as standard. The precursor protein sequence of jerodonobin-II was deduced from cloned cDNA sequence. The sequence shows high similarity (identity = 89%) to TSV-PA, a specific plasminogen activator from venom of T stejnegeri. Despite of the sequence similarity, jerdonobin-II was found devoid of plasminogen activating effect. Sequence alignment analysis suggested that the replacement of Lys(239) in TSV-PA to Gln(239) in jerdonobin-II might play an important role on their plasminogen activating activity difference. (C) 2005 Elsevier Ltd. All rights reserved.

SARS 及其它冠状病毒基因组组织结构和序列的初步比较分析

:严重急性呼吸综合症(Severe acute respiratory sydrome , SARS) 是一种全新的传染性疾病,它可能主 要是由冠状病毒的一个变种引起的. 本文比较SARS 及其它冠状病毒基因组组织结构和序列变异的情况,初步 的结果表明: ①尽管SARS 病毒与冠状病毒属的另外6 种病毒基因组序列上有较大的差异,但是它们具有较相 似的基因组组织结构; ②SARS 病毒与牛冠状病毒、鼠肝炎病毒具有较近的系统发育关系; ③虽然SARS 病毒扩 散的时间极短,但来自不同地区SARS 病毒的DNA 序列却存在一些突变,且这些突变又大多是改变氨基酸序 列的非同义突变.

Evolution of structures and specific recognitions in aminoacyl-tRNA synthetases

The aminoacyl-tRNA synthetases (AARS) are very important during the protein biosynthesis, which can make the gene sequence be accurately translated into the protein sequence by the specific recognition between AARS and tRNA/amino acids. However, the recog

Identification of a Giardia krr1 homolog gene and the secondarily anucleolate condition of Giaridia lamblia

Giaridia lamblia was long considered to be one of the most primitive eukaryotes and to lie close to the transition between prokaryotes and eukaryotes, but several supporting features, such as lack of mitochondrion and Golgi, have been challenged recently. It was also reported previously that G. lamblia lacked nucleolus, which is the site of pre-rRNA processing and ribosomal assembling in the other eukaryotic cells. Here, we report the identification of the yeast homolog gene, krr1, in the anucleolate eukaryote, G. lamblia. The krr1 gene, encoding one of the pre-rRNA processing proteins in yeast, is actively transcribed in G. lamblia. The deduced protein sequence of G. lamblia krr1 is highly similar to yeast KRR1p that contains a single-KH domain. Our database searches indicated that krr1 genes actually present in diverse eukaryotes and also seem to present in Archaea. However, only the eukaryotic homologs, including that of G. lamblia, have the single-KH domain, which contains the conserved motif KR(K)R. Fibrillarin, another important pre-rRNA processing protein has also been identified previously in G. lamblia. Moreover, our database search shows that nearly half of the other nucleolus-localized protein genes of eukaryotic cells also have their homologs in Giardia. Therefore, we suggest that a common mechanism of pre-RNA processing may operate in the anucleolate eukaryote G. lamblia and in the other eukaryotes and that like the case of "lack of mitochondrion," "lack of nucleolus" may not be a primitive feature, but a secondarily evolutionary condition of the parasite.

SynGAP isoforms exert opposing effects on synaptic strength.

Alternative promoter usage and alternative splicing enable diversification of the transcriptome. Here we demonstrate that the function of Synaptic GTPase-Activating Protein (SynGAP), a key synaptic protein, is determined by the combination of its amino-terminal sequence with its carboxy-terminal sequence. 5' rapid amplification of cDNA ends and primer extension show that different N-terminal protein sequences arise through alternative promoter usage that are regulated by synaptic activity and postnatal age. Heterogeneity in C-terminal protein sequence arises through alternative splicing. Overexpression of SynGAP α1 versus α2 C-termini-containing proteins in hippocampal neurons has opposing effects on synaptic strength, decreasing and increasing miniature excitatory synaptic currents amplitude/frequency, respectively. The magnitude of this C-terminal-dependent effect is modulated by the N-terminal peptide sequence. This is the first demonstration that activity-dependent alternative promoter usage can change the function of a synaptic protein at excitatory synapses. Furthermore, the direction and degree of synaptic modulation exerted by different protein isoforms from a single gene locus is dependent on the combination of differential promoter usage and alternative splicing.

Cloning and characterization of a new multi-stress inducible metallothionein gene in Tetrahymena pyriformis

A new multi-stress-inducible metallothionein (MT) gene isoform has been cloned and characterized from the ciliate Tetrahymena pyriformis. Both the 5'- and 3'-UT regions of the Tp-MT2 gene are very different from the previously reported Tp-MT1 isoform in this organism and from other described MT genes in Tetrahymena pigmentosa and Tetrahymena thermophila. The putative protein sequence of Tp-MT2 contains cysteine clusters with characteristics of the typical Tetrahymena Cd-inducible MT genes. However, the sequence has a special feature of four intragenic tandem repeats within its first half, with a conserved structural pattern x(5/8)CCCx(6)CCx(6)CxCxNCxCCK. To investigate the transcriptional activities of both Tp-MT2 and Tp-MT1 genes toward heavy metals (Cd, Hg, Cu, Zn) and H2O2, the mRNA levels of these two isoforms were evaluated by means of real-time quantitative PCR. Results showed that Tp-MT2 had a higher basal expression level than Tp-MT1 and both genes were induced by Cd, Hg, Cu, and Zn ions after short exposure (I h), although to different extents. Cd was the most effective metal inducer of both two isoforms, but the relative expression level of Tp-MT2 was much lower than that of Tp-MT1. Different expression patterns were also shown between the two genes when treated with Cd over a period of 24 h. We suggest that TpMT-1 plays the role of a multi-inducible stress gene, while TpMT-2 may have a more specific function in basal metal homeostasis although it may have undergone a functional differentiation process. The putative functional significance and evolutionary mode of the TpMT-2 isoform are discussed. (c) 2006 Elsevier GmbH. All rights reserved.

小麦高分子量谷蛋白新亚基基因1Dx5’的克隆与分子标记

小麦是世界第一大粮食作物, 而HMW- GS 是直接影响小麦品质的重要因子。我国小麦面粉的烘烤品质普遍较差, 这与我国品种缺少优质的HMW- GS 有关，因此创造与发掘新的优质谷蛋白亚基编码基因，并开展相关生化、农学、分子生物学等方面研究、探讨优质的分子机理，对于培育优质小麦新品种具有重要意义。W958是我们培育的种间远缘杂交（T.durum Desf. ×T.aestivum L）优良品系，该品系在1D染色体上具有父母本没有的新型亚基，由于此亚基在SDS- PAGE电泳中具有和1Dx5亚基一样的电泳迁移率, 因此我们将该亚基命名为1Dx5’亚基。为了进一步从分子水平确证该亚基为新亚基和在育种中利用该亚基，本研究对该亚基的遗传规律、基因分子结构、品质特性和农艺性状等进行了分析。结果表明1Dx5’亚基在品质上与1Dx5亚基一样优质，对于品质的贡献大于1Dx2亚基。1Dx5’亚基具有特异的遗传规律，在分离群体中，此亚基占有极大的比例，该特性十分有利于将其导入高产小麦遗传背景中。此外，本研究扩增出了1Dx5’亚基基因的启动子区域、N－端区域和部分中间重复区域，并比较了1Dx5’和传统的1Dx5、1Dx2亚基在此区域氨基酸序列。结果进一步证明了1Dx5’是一个新的基因。通过蛋白质结构模拟分析，认为1Dx5’亚基的优良特性可能是由于1Dx5’亚基的的中部重复区域能形成分子间较强的氢键，加大了分子间的相互作用，使1Dx5’亚基的面团具有优良的品质，这为1Dx5’亚基的应用提供了理论基础。同时，本研究还设计用于区分1Dx5’和1Dx5等位基因的分子标记，解决了利用SDS-PAGE生化标记难以将二者区分的问题。Wheat is one of the major crops utilized worldwide. Nevertheless, due to the lackof the superior HMW- GS, the wheat quality in China is not satisfying. Therefore,identification and characterization of the superior HMW- GS will lay good foundation to the wheat breeding.W958 is a new bread wheat line developed by interspecific cross (T.durum Desf.×T.aestivum L). It contains a novel HMW- GS. We have designated such subunit as1Dx5’ here for its unique character. To confirm that such subunit is innovative andbeneficial for wheat breeding program on the molecular level, we have investigated itin terms of inheritance, DNA and protein sequence, dough property and agronomictrait associated with it. The result shows that 1Dx5’is as superior as 1Dx5 in terms of dough property.In addition, we have cloned the promoter, N- terminal as well as partial centralrepetitive domain of the allele coding for this subunit. Comparison of the amino acidsequence of 1Dx5’ with that of 1Dx5 and 1Dx2 showed that the superior quality of1Dx5’ subunit may result from the degree of conservation of the repetitive sequencesby which the glutenin polymers interact via inter-chain hydrogen bonds formedbetween the subunit repetitive domains with longer subunits forming more stableinteractions. In addition, we have developed two dominant molecular markers tofacilitate the discrimination of 1Dx5’ and 1Dx5 which could no be achieved by SDS-PAGE.

Analysis of the elements of catabolite repression in Clostridium acetobutylicum.

The ptsH gene, encoding the phosphotransferase protein HPr, from Clostridium acetobutylicum ATCC 824 was identified from the genome sequence, cloned and shown to complement a ptsH mutant of Escherichia coli. The deduced protein sequence shares significant homology with HPr proteins from other low-GC gram-positive bacteria, although the highly conserved sequence surrounding the Ser-46 phosphorylation site is not well preserved in the clostridial protein. Nevertheless, the HPr was phosphorylated in an ATP-dependent manner in cell-free extracts of C. acetobutylicum. Furthermore, purified His-tagged HPr from Bacillus subtilis was also a substrate for the clostridial HPr kinase/phosphorylase. This phosphorylation reaction is a key step in the mechanism of carbon catabolite repression proposed to operate in B. subtilis and other low-GC gram-positive bacteria. Putative genes encoding the HPr kinase/phosphorylase and the other element of this model, namely the catabolite control protein CcpA, were identified from the C. acetobutylicum genome sequence, suggesting that a similar mechanism of carbon catabolite repression may operate in this industrially important organism.

Cloning and characterization of the mouse homologue of the human dendritic cell maturation marker CD208/DC-LAMP

DC-LAMP, a member of the lysosomal-associated membrane protein (LAMP) family, is specifically expressed by human dendritic cells (DC) upon activation and therefore serves as marker of human DC maturation. DC-LAMP is detected first in activated human DC within MHC class II molecules-containing compartments just before the translocation of MHC class II-peptide complexes to the cell surface, suggesting a possible involvement in this process. The present study describes the cloning and characterization of mouse DC-LAMP, whose predicted protein sequence is over 50% identical to the human counterpart. The mouse DC-LAMP gene spans over 25 kb and shares syntenic chromosomal localization (16B2-B4 and 3q26) and conserved organization with the human DC-LAMP gene. Analysis of mouse DC-LAMP mRNA and protein revealed the expression in lung peripheral cells, but also its unexpected absence from mouse lymphoid organs and from mouse DC activated either in vitro or in vivo. In conclusion, mouse DC-LAMP is not a marker of mature mouse DC and this observation raises new questions regarding the role of human DC-LAMP in human DC.