蛋白纤维作为优质蛋白的新技术和应用

蛋白纤维是构成生命的基本结构单元,它在细胞、组织和生物体的游动性、弹性、稳定性、骨架保护方面有重要作用。讨论了蛋白纤维在生物学、工业和医药上的应用,以及蛋白纤维的装配、控制和模仿蛋白纤维装配的进展。     

Immunological Characterization of Honey Proteins and Identification of MRJP 1 as an IgE-Binding Protein

We encountered a fourth case of honey allergy in Japan. We characterized and identified the IgE-binding proteins in honey using the serum of a honey-allergenic patient. Immunoblot analysis revealed that IgE in the patient serum specifically bound to four proteins in each honey sample. At least three of these IgE-binding proteins were N-linked glycoproteins. To identify the 60-kDa IgE-binding protein in dandelion honey, the N-terminal sequences of the fragmented protein were analyzed, revealing the protein to be major royal jelly protein 1 (MRJP 1). Three IgE-binding proteins removed of N-linked oligosaccharide showed a large reduction in IgE-binding activity as compared with the intact protein. This suggests that the carbohydrates in the IgE-binding proteins are a major epitope for patient IgE.     

利用一种新的网上工具Scansite进行蛋白质磷酸化预测

Scansite分析软件是近两年建立的一种新的利用因特网,基于蛋白质分子中较短的模序进行蛋白质磷酸化和蛋白质蛋白质相互作用预测的工具。这里综述了Scansite的使用方法、功能介绍及与其他磷酸化分析软件的比较,并展望了Scansite在进行磷酸化预测中面临的问题和应用前景。     

Protein Sequences of Two Keto Ester Reductases: Possible Identity as Hypothetical Proteins

We determined the amino acid sequences of two keto ester reductases (YKER-V and -VI) purified from a cell-free extract of Saccharomyces cerevisiae. The N-terminal and internal amino acid sequences of YKER-VI (AcKR) were in agreement with the sequence of hypothetical 36.4-kDa protein (S. cerevisiae chromosome X reading frame ORF YJR105w) in yeast. The N-terminal amino acid sequence of YKER-V was also identical with that of the hypothetical protein coded by yeast chromosome XIV or II. These results suggested that two hypothetical proteins were expressed as keto ester reductases in yeast cells.     

Structural Capacitance in Protein Evolution and Human Diseases

Canonical mechanisms of protein evolution include the duplication and diversification of pre-existing folds through genetic alterations that include point mutations, insertions, deletions, and copy number amplifications, as well as post-translational modifications that modify processes such as folding efficiency and cellular localization. Following a survey of the human mutation database, we have identified an additional mechanism that we term “structural capacitance,” which results in the de novo generation of microstructure in previously disordered regions. We suggest that the potential for structural capacitance confers select proteins with the capacity to evolve over rapid timescales, facilitating saltatory evolution as opposed to gradualistic canonical Darwinian mechanisms. Our results implicate the elements of protein microstructure generated by this distinct mechanism in the pathogenesis of a wide variety of human diseases. The benefits of rapidly furnishing the potential for evolutionary change conferred by structural capacitance are consequently counterbalanced by this accompanying risk. The phenomenon of structural capacitance has implications ranging from the ancestral diversification of protein folds to the engineering of synthetic proteins with enhanced evolvability.     

Modular arrangement of proteins as inferred from analysis of homology.

The structure of many proteins consists of a combination of discrete modules that have been shuffled during evolution. Such modules can frequently be recognized from the analysis of homology. Here we present a systematic analysis of the modular organization of all sequenced proteins. To achieve this we have developed an automatic method to identify protein domains from sequence comparisons. Homologous domains can then be clustered into consistent families. The method was applied to all 21,098 nonfragment protein sequences in SWISS-PROT 21.0, which was automatically reorganized into a comprehensive protein domain database, ProDom. We have constructed multiple sequence alignments for each domain family in ProDom, from which consensus sequences were generated. These nonreduntant domain consensuses are useful for fast homology searches. Domain organization in ProDom is exemplified for proteins of the phosphoenolpyruvate:sugar phosphotransferase system (PEP:PTS) and for bacterial 2-component regulators. We provide 2 examples of previously unrecognized domain arrangements discovered with the help of ProDom.     

DeepFunc: A Deep Learning Framework for Accurate Prediction of Protein Functions from Protein Sequences and Interactions

Annotation of protein functions plays an important role in understanding life at the molecular level. High‐throughput sequencing produces massive numbers of raw proteins sequences and only about 1% of them have been manually annotated with functions. Experimental annotations of functions are expensive, time‐consuming and do not keep up with the rapid growth of the sequence numbers. This motivates the development of computational approaches that predict protein functions. A novel deep learning framework, DeepFunc, is proposed which accurately predicts protein functions from protein sequence‐ and network‐derived information. More precisely, DeepFunc uses a long and sparse binary vector to encode information concerning domains, families, and motifs collected from the InterPro tool that is associated with the input protein sequence. This vector is processed with two neural layers to obtain a low‐dimensional vector which is combined with topological information extracted from protein–protein interactions (PPIs) and functional linkages. The combined information is processed by a deep neural network that predicts protein functions. DeepFunc is empirically and comparatively tested on a benchmark testing dataset and the Critical Assessment of protein Function Annotation algorithms (CAFA) 3 dataset. The experimental results demonstrate that DeepFunc outperforms current methods on the testing dataset and that it secures the highest F_max = 0.54 and AUC = 0.94 on the CAFA3 dataset.     

Structural comparison of the prokaryotic ribosomal proteins L7/L12 and L30

The structures of two prokaryotic ribosomal proteins, the carboxyterminal half of L7/L12 from Escherichia coli (L12CTF) and L30 from Bacilus stearothermophilus display a remarkably similar fold in which alpha-helices pack onto one side of an antiparallel, three-stranded, beta-pleated sheet. A detailed comparison of the structures by least-squares methods reveals that more than two-thirds of the alpha carbons can be superimposed with a root mean square distance of 2.33 A. The principal difference is an extra alpha-helix in L12CTF. The sequences of the proteins display a distinct conservation in regions which are crucial to the common fold, in particular the hydrophobic core. It is proposed that the similarity is a result of divergent evolution.     

Quantitative Structural Interpretation of Protein Crosslinks

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Structural Genomics of Minimal Organisms and Protein Fold Space

The initial aim of the Berkeley Structural Genomics Center is to obtain a near-complete structural complement of two minimal organisms, closely related pathogens Mycoplasma genitalium and M. pneumoniae. The former has fewer than 500 genes and the latter fewer than 700 genes. To achieve this goal, the current protein targets have been selected starting with those predicted to be most tractable and likely to yield new structural and functional information. During the past 3 years, the semi-automated structural genomics pipeline has been set up from cloning, expression, purification, and ultimately to structural determination. The results from the pipeline substantially increased the coverage of the protein fold space of M. pneumoniae and M. genitalium. Furthermore, about 1/2 of the structures of ‘unique’ protein sequences revealed new and novel folds, and over 2/3 of the structures of previously annotated ‘hypothetical proteins’ inferred their molecular functions.     

A HT/PEXEL Motif in Toxoplasma Dense Granule Proteins is a Signal for Protein Cleavage but not Export into the Host Cell

Apicomplexan parasites, such as Toxoplasma gondii and Plasmodium, secrete proteins for attachment, invasion and modulation of their host cells. The host targeting (HT), also known as the Plasmodium export element (PEXEL), directs Plasmodium proteins into erythrocytes to remodel the host cell and establish infection. Bioinformatic analysis of Toxoplasma revealed a HT/PEXEL‐like motif at the N‐terminus of several hypothetical unknown and dense granule proteins. Hemagglutinin‐tagged versions of these uncharacterized proteins show co‐localization with dense granule proteins found on the parasitophorous vacuole membrane (PVM). In contrast to Plasmodium, these Toxoplasma HT/PEXEL containing proteins are not exported into the host cell. Site directed mutagenesis of the Toxoplasma HT/PEXEL motif, RxLxD/E, shows that the arginine and leucine residues are permissible for protein cleavage. Mutations within the HT/PEXEL motif that prevent protein cleavage still allow for targeting to the PV but the proteins have a reduced association with the PVM. Addition of a Myc tag before and after the cleavage site shows that processed HT/PEXEL protein has increased PVM association. These findings suggest that while Toxoplasma and Plasmodium share similar HT/PEXEL motifs, Toxoplasma HT/PEXEL containing proteins interact with but do not cross the PVM .     

Structural motifs for pyridoxal-5'-phosphate binding in decarboxylases: an analysis based on the crystal structure of the Lactobacillus 30a ornithine decarboxylase.

Two of the five domains in the structure of the ornithine decarboxylase (OrnDC) from Lactobacillus 30a share similar structural folds around the pyridoxal-5''-phosphate (PLP)-binding pocket with the aspartate aminotransferases (AspATs). Sequence comparisons focusing on conserved residues of the aligned structures reveal that this structural motif is also present in a number of other PLP-dependent enzymes including the histidine, dopa, tryptophan, glutamate, and glycine decarboxylases as well as tryptophanase and serine-hydroxymethyl transferase. However, this motif is not present in eukaryotic OrnDCs, the diaminopimelate decarboxylases, nor the Escherichia coli or oat arginine decarboxylases. The identification and comparison of residues involved in defining the different classes are discussed.     

Different Structural Behaviors Evidenced in Thaumatin-Like Proteins: A Spectroscopic Study

Three proteins belonging to the thaumatin-like proteins family were compared in this study from a structural point of view: zeamatin, a new recently isolated PR-5 from Cassia didymobotrya and the commercial sweet-thaumatin. The former two proteins possess antifungal activities while commercial thaumatin is well known to be a natural sweetener. Intrinsic fluorescence studies have evidenced that the three proteins behave differently in unfolding experiments showing different structural rigidity. All the three proteins are more stable at slight acidic buffers, but sweet-thaumatin has a major tendency to destructurate itself. Similar observations were made from circular dichroism studies where a structural dependence relationship from the pH and the solvent used confirmed a hierarchic scale of stability for the three proteins. These structural differences should be considered to be significant for a functional role.     

Global phylogeny determined by the combination of protein domains in proteomes

The majority of proteins consist of multiple domains that are either repeated or combined in defined order. In this study, we survey the combination of protein domains defined at fold and fold superfamily levels in 185 genomes belonging to organisms that have been fully sequenced and introduce a method that reconstructs rooted phylogenomic trees from the content and arrangement of domains in proteins at a genomic level. We find that the majority of domain combinations were unique to Archaea, Bacteria, or Eukarya, suggesting most combinations originated after life had diversified. Domain repeat and domain repeat within multidomain proteins increased notably in eukaryotes, mainly at the expense of single-domain and domain-pair proteins. This increase was mostly confined to Metazoa. We also find an unbalanced sharing of domain combinations which suggests that Eukarya is more closely related to Bacteria than to Archaea, an observation that challenges the widely assumed eukaryote-archaebacterial sisterhood relationship. The occurrence and abundance of the molecular repertoire (interactome) of domain combinations was used to generate phylogenomic trees. These global interactome-based phylogenies described organismal histories satisfactorily, revealing the tripartite nature of life, and supporting controversial evolutionary patterns, such as the Coelomata hypothesis, the grouping of plants and animals, and the Gram-positive origin of bacteria. Results suggest strongly that the process of domain combination is not random but curved by evolution, rejecting the null hypothesis of domain modules combining in the absence of natural selection or an optimality criterion.     

Structural Aspects of Congo Red as an Inhibitor of Protease-Resistant Prion Protein Formation

Abstract: Congo red (CR) has been shown to inhibit the accumulation in scrapie-infected cells of prion protein (PrP) in the abnormal protease-resistant form (PrP-res). However, it was not clear if this effect was due to a direct interaction of CR with either PrP-res or its protease-sensitive precursor (PrP-sen) or to a less direct effect on living cells. Here we show that CR inhibits PrP-res formation in a simple cell-free reaction composed predominantly of purified PrP-res and PrP-sen. Structurally modified CR analogues were also compared in both the cell-free conversion reaction and scrapie-infected neuroblastoma cells. Methylation of the central phenyl groups at the 2,2' positions diminished the inhibitory potency by ≥10-fold. In contrast, there was little effect of 3,3' methylation of the phenyls, deletion of one phenyl, or addition of an amido group between the phenyls. The relative activities of these compounds were well correlated in both cellular and acellular systems. Molecular modeling indicated that CR and 3,3'-methyl-CR have little rotational restriction about the biphenyl bond and can readily adopt a planar conformation, as can phenyl-CR and amido-CR. In contrast, 2,2'-methyl-CR is restricted to a nonplanar conformation of the biphenyl group. Thus, planarity and/or torsional mobility of the central phenyl rings of CR and its analogues is probably important for inhibition of PrP-res formation. On the other hand, variations in the intersulfonate distance in these molecules had little effect on PrP-res inhibition. These results indicated a high degree of structural specificity in the inhibition of PrP-res formation by CR and related compounds.     

GYF结构域蛋白特性及其生物学功能研究进展

甘氨酸-络氨酸-苯丙氨酸(glycine-tyrosine-phenylalanine,GYF)结构域是一种小型的、能识别富脯氨酸序列(proline rich sequences,PRS)的保守、多功能接合结构域,GYF结构域蛋白在蛋白蛋白相互作用、翻译起始、mRNA剪接、监管与翻译抑制、泛素连接、信号转导、免疫蛋白酶调控等方面起重要作用。本文从结构域特性、与靶蛋白的相互作用及其功能等方面综述GYF结构域蛋白的研究进展。     

Structural Analysis of Metal Sites in Proteins: Non-heme Iron Sites as a Case Study

In metalloproteins, the protein environment modulates metal properties to achieve the required goal, which can be protein stabilization or function. The analysis of metal sites at the atomic level of detail provided by protein structures can thus be of benefit in functional and evolutionary studies of proteins. In this work, we propose a structural bioinformatics approach to the study of metalloproteins based on structural templates of metal sites that include the PDB coordinates of protein residues forming the first and the second coordination sphere of the metal. We have applied this approach to non-heme iron sites, which have been analyzed at various levels. Templates of sites located in different protein domains have been compared, showing that similar sites can be found in unrelated proteins as the result of convergent evolution. Templates of sites located in proteins of a large superfamily have been compared, showing possible mechanisms of divergent evolution of proteins to achieve different functions. Furthermore, template comparisons have been used to predict the function of uncharacterized proteins, showing that similarity searches focused on metal sites can be advantageously combined with typical whole-domain comparisons. Structural templates of metal sites, finally, may constitute the basis for a systematic classification of metalloproteins in databases.     

Continuous and discontinuous domains: an algorithm for the automatic generation of reliable protein domain definitions.

An algorithm is presented for the fast and accurate definition of protein structural domains from coordinate data without prior knowledge of the number or type of domains. The algorithm explicitly locates domains that comprise one or two continuous segments of protein chain. Domains that include more than two segments are also located. The algorithm was applied to a nonredundant database of 230 protein structures and the results compared to domain definitions obtained from the literature, or by inspection of the coordinates on molecular graphics. For 70% of the proteins, the derived domains agree with the reference definitions, 18% show minor differences and only 12% (28 proteins) show very different definitions. Three screens were applied to identify the derived domains least likely to agree with the subjective definition set. These screens revealed a set of 173 proteins, 97% of which agree well with the subjective definitions. The algorithm represents a practical domain identification tool that can be run routinely on the entire structural database. Adjustment of parameters also allows smaller compact units to be identified in proteins.