Location of ''continuous'' antigenic determinants in the protruding regions of proteins.

A simple method is described to locate 'antigenic' peptides from the alpha-carbon co-ordinates of a protein, based on protrusion from the protein's globular surface. A good correlation is found between those parts of a protein which protrude and the experimentally determined antigenic peptides in myoglobin, lysozyme and myohemerythrin. A comparison is made between the use of protrusion index, mobility, solvent accessibility and hydrophilicity for predicting the most likely antigenic peptides.     

Prediction of antigenic determinants and secondary structures of the major AIDS virus proteins

Criteria for the design of peptide vaccines to prevent AIDS are presented. The best vaccine candidates contain both B and T lymphocyte-defined epitopes in regions conserved in sequence between viral isolates. We propose that attention should focus on proteins specified by the gag and, possibly, pol genes in addition to the env gene envelope glycoproteins being actively studied. The predictions of B- and T-epitopes are refined by consideration of secondary structure prediction and inter-isolate sequence variability to suggest peptides from env, gag and pol that would be the best vaccine candidates.     

HCV抗原表位预测

应用网络生物信息资源查找丙型肝炎病毒基因组全序列,用软件Lasergene中的EditSeq将来自中国河北株mRNA序列翻译为氨基酸序列,尔后用程序Protean进行氨基酸序列分析,对HCV各区段的B细胞抗原指数进行预测。同时又在两个网站对中国汉族人中频率较高的HLA基因型进行CD8和CD4T细胞表位预测。B细胞和T细胞抗原表位预测结果对于HCV诊断试剂和疫苗研制有重要的指导意义。     

Trypanosoma cruzi proteins which are antigenic during human infections are located in defined regions of the parasite

Polyclonal antibodies obtained against antigenic proteins encoded by six recombinant DNA clones of Trypanosoma cruzi were used for the ultrastructural localization of the respective antigens in thin sections of parasites (epimastigote, amastigote and trypomastigote forms of T. cruzi) embedded at low temperature in Lowicryl K4M resin. Antigens of high molecular weight containing tandemly repeated amino acid sequence motifs and recognized by sera from patients with Chagas' disease, were located only in the flagellum, where it contacts the parasite cell body. Other antigens were located on the surface of the parasite while still others were found within the flagellar pocket, as is the case with an antigen released during the acute phase of Chagas' disease. Thus, we conclude that some of the T. cruzi proteins which are antigenic in human infections are located in defined regions of the parasite. Some of the antigens were not expressed to the same extent in the three different developmental stages of the parasite.     

Quantitative sequential chromatin immunoprecipitation, a method for analyzing co-occupancy of proteins at genomic regions in vivo

Sequential chromatin immunoprecipitation (SeqChIP) is a procedure in which formaldehyde-crosslinked, protein–DNA complexes from living cells are subjected to two sequential immunoprecipitations with antibodies of different specificity. SeqChIP has been used to address, in a qualitative manner, whether two proteins can simultaneously co-occupy a stretch of DNA in vivo. Here, we expand on our earlier work and describe theoretical and practical considerations for performing and interpreting SeqChIP experiments in a quantitative manner. We provide a detailed experimental procedure for designing and performing SeqChIP experiments as well as experimental examples of the three possible outcomes: full co-occupancy, no co-occupancy and partial co-occupancy. In some cases of partial co-occupancy, the order of immunoprecipitations in SeqChIP can strongly influence the outcome. We experimentally confirm a quantitative parameter that provides a measure of co-occupancy of two proteins on a given region of DNA and provide information on how to interpret the results of SeqChIP experiments. Our quantitative treatment of SeqChIP data substantially expands the usefulness of the technique for elucidating molecular mechanisms in vivo.     

Conformation of two antigenic regions in myelin basic protein

Four different regions of myelin basic protein from various species have been reported to be the antigenic sites (epitopes) for seven monoclonal antibodies evoked in rats or mice by guinea pig or monkey basic protein. The structures of the epitopes located in the amino-terminal region and in the eight-residue sequence including S-133, were examined by proton n. m. r at 400 MHz in aqueous solutions of peptides obtained by enzymatic cleavage of the rabbit protein. The data suggest conformational similarities between the two regions.     

Prediction of secondary structure, spatial organization and distribution of antigenic determinants for hepatitis A virus proteins

On the basis of the secondary structure calculations from the known amino acid sequence we came to the conclusion that hepatitis A virus capsid proteins have the typical antiparallel beta-sheet bilayer structure. The predicted secondary structure of the HAV proteins can be well aligned with those of the poliovirus (type 1 Mahoney) and human rhinovirus (type 14). It enabled us to use the X-ray structure of the PV-1M and HRV-14 proteins as a template and then, firstly, to localize the positions of alpha and beta regions in the architecture of the HAV protein molecules and, secondly, to discover the amino acid homologies of the secondary structure regions aligned. The obtained model of the three-dimensional structure for HAV proteins helped us to indicate the exposed regions of the polypeptide chains and to pinpoint the potential neutralizing antigenic sites.     

Prediction of "aggregation-prone" and "aggregation-susceptible" regions in proteins associated with neurodegenerative diseases

Increasing evidence indicates that many peptides and proteins can be converted in vitro into highly organised amyloid structures, provided that the appropriate experimental conditions can be found. In this work, we define intrinsic propensities for the aggregation of individual amino acids and develop a method for identifying the regions of the sequence of an unfolded peptide or protein that are most important for promoting amyloid formation. This method is applied to the study of three polypeptides associated with neurodegenerative diseases, Abeta42, alpha-synuclein and tau. In order to validate the approach, we compare the regions of proteins that are predicted to be most important in driving aggregation, either intrinsically or as the result of mutations, with those determined experimentally. The knowledge of the location and the type of the "sensitive regions" for aggregation is important both for rationalising the effects of sequence changes on the aggregation of polypeptide chains and for the development of targeted strategies to combat diseases associated with amyloid formation.     

Prediction of loop regions in protein sequence

We suggest an algorithm that inputs a protein sequence and outputs a decomposition of the protein chain into a regular part including secondary structures and a nonregular part corresponding to loop regions. We have analyzed loop regions in a protein dataset of 3,769 globular domains and defined the optimal parameters for this prediction: the threshold between regular and nonregular regions and the optimal window size for averaging procedures using the scale of the expected number of contacts in a globular state and entropy scale as the number of degrees of freedom for the angles phi, psi, and chi for each amino acid. Comparison with known methods demonstrates that our method gives the same results as the well-known ALB method based on physical properties of amino acids (the percentage of true predictions is 64% against 66%), and worse prediction for regular and nonregular regions than PSIPRED (Protein Structure Prediction Server) without alignment of homologous proteins (the percentage of true predictions is 73%). The potential advantage of the suggested approach is that the predicted set of loops can be used to find patterns of rigid and flexible loops as possible candidates to play a structure/function role as well as a role of antigenic determinants.     

Predicting antigenic sites on proteins.

The ability to predict antigenic sites on proteins is of major importance for the production of synthetic peptide vaccines and synthetic peptide probes of antibody structure. Many predictive methods, based on various assumptions about the nature of the antigenic response have been proposed and tested. This review will discuss the principles underlying the various approaches to predicting antigenic sites and will attempt to answer the question of how well they work.     

Tyrosine specific sequential labeling of proteins

We report (a) on the synthesis of a long-wavelength fluorescent coumarin containing an allyloxy acetate moiety, (b) the synthesis of two linkers containing an allyloxy acetate and an alkyne or azide function, respectively, and (c) the selective modification human serum albumin by a sequential method involving Pd(II) catalyzed modification of the phenolic side chain of tyrosine residues with an alkyne bearing linker and a subsequent azide–alkyne click reaction with an azide functionalized long-wavelength emitting coumarin dye. The method is likely to be applicable to various kinds of azido-modified fluorophores, and the Pd(II)-catalyzed modification of the tyrosines may also be used to introduce other kinds of tags. With these reagents, tyrosine specific modulation of proteins and peptides becomes possible either directly or in a sequential manner.     

Visualization of antigenic proteins on Western blots

A new technique for the detection of antibodies bound to proteins blotted onto nitrocellulose paper was developed. The method is rapid, sensitive, and does not require radioactive probes. Proteins transferred to nitrocellulose paper are first reacted with primary antibody followed by reaction with an alkaline phosphatase conjugated second antibody. The phosphatase activity is then visualized using an agar gel impregnated with the histochemical phosphatase stain 5-bromo-4-chloro-3-indolyl phosphate (BCIP) (J. P. Horwitz, J. Chua, M. Noel, J. T. Donatti, and J. Freisler (1966) J. Med. Chem. 9, 447; Sigma Chemical Co., Technical bulletin No. 710-EP (1978]. Antigen-antibody complexes give rise to sharp, permanent blue stained bands both on the nitrocellulose paper and in the agar overlay gel. This procedure allows detection of bands containing less than 20 ng of protein.     

Prediction of natively unfolded regions in protein chains

Analysis showed that the globular or natively unfolded state of a protein can be inferred not only from a lower hydrophobicity or a higher charge, but also from the average environment density (average number of close residues located within a certain distance of a given one) of its residues. A database of 6626 protein structures was used to construct a statistical scale of the average number of close residues in globular structures for the 20 amino acids. The portion of false predictions in distinguishing between 80 globular and 90 natively unfolded proteins was 11% with the new scale and 17% with a hydrophobicity scale. The new scale proved suitable for predicting the folded or unfolded state for native proteins or the natively unfolded regions for protein chains. In comparisons with the available algorithms, the new method yielded the highest portion of true predictions (87 and 77% with averaging over residues and over proteins, respectively).     

Prediction of natively unfolded regions in protein chain

We have shown that the ability of a protein to be in globular or in natively unfolded state (under native conditions) may be determined (besides low overall hydrophobicity and a large net charge) by such a property as the average environment density, the average number of residues enclosed at the given distance. A statistical scale of the average number of residues enclosed at the given distance for 20 types of amino acid residues in globular state has been created on the basis of 6626 protein structures. Using this scale for separation of 80 globular and 90 natively unfolded proteins we fail only in 11% of proteins (compared with 17% of errors which are observed if to use hydrophobicity scale). The present scale may be used both for prediction of form (folded or unfolded) of the native state of protein and for prediction of natively unfolded regions in protein chains. The results of comparison of our method of predicting natively unfolded regions with the other known methods show that our method has the highest fraction of correctly predicted natively unfolded regions (that is 87% and 77% if to make averaging over residues and over proteins correspondingly).     

Prediction of antigenic determinants of trichosanthin by molecular modeling

The antigenic determinants of trichosanthin were predicted by molecular modeling.First,the threedimensional structure model of the antigen-binding fragment of anti-trichosanthin immunoglobulin E was built on the basis of its amino-acid sequence and the known three-dimensional structure of an antibody with similar sequence.Secondly,the preferable antigen-antibody interactions were obtained based on the known three-dimensional structure of trichosanthin and of the hypervariable regions of anti-trichosanthin immunoglobulin E.Two regions in the molecular surface of trichosanthin were found to form extensive interactions with the hypervariable regions of the antibody and have been predicted to be the possible antigenic determinants:one is composed of two polypeptide segments,Ile201-Glu210 and Ile225-Asp229,which are close to each other in the three-dimensional structure;and the other is the segment Lys173-Thr178.The former region seems to be the more reasonable antigenic determinant than the latter one.     

Conserved pore-forming regions in polypeptide-transporting proteins

Transport of solutes and polypeptides across membranes is an essential process for every cell. In the past, much focus has been placed on helical transporters. Recently, the beta-barrel-shaped transporters have also attracted some attention. The members of this family are found in the outer bacterial membrane and the outer membrane of endosymbiotically derived organelles. Here we analyze the features and the evolutionary development of a specified translocator family, namely the beta-barrel-shaped polypeptide-transporters. We identified sequence motifs, which characterize all transporters of this family, as well as motifs specific for a certain subgroup of proteins of this class. The general motifs are related to the structural composition of the pores. Further analysis revealed a defined distance of two motifs to the C-terminal portion of the proteins. Furthermore, the evolutionary relationship of the proteins and the motifs are discussed.