Biomedical subjects
Publications and source records attributed to K Pawłowski.
A new protein domain was found in several proteins involved in apoptosis, inflammation, cancer and immune responses. Its location within these proteins and predicted fold suggests that it functions as a protein-protein interaction domain, possibly uniting different signaling pathways.
With the rapidly increasing pace of genome sequencing projects and the resulting flood of predicted amino acid sequences of uncharacterized proteins, protein sequence analysis, and in particular, protein structure prediction is quickly gaining in importance. Prediction algorithms can be used for preliminary annotation of newly sequenced proteins and, at least in some cases, provide insights into their function and specific mode of action. Such annotations for several microbial genomes were performed by several groups and placed in public domain for evaluation. An example presented in this work comes from a related project of structural and functional predictions for proteins involved in the process of controlled cell death (apoptosis). The BID protein belongs to an important class of regulators of apoptosis identified by short sequence motifs. Here, several fold prediction methods are used to build a series of three-dimensional models. Structure analysis of the models with reference to the biological data available allows selection of the most appropriate model. It is found that the most likely structural model of BID is built on the structure of Bcl-X(L). The model is discussed in terms of experimental data on specific proteolytic cleavage of BID and its effect on BID interactions with other proteins and membranes.
MOTIVATION: Two proteins can have a similar 3-dimensional structure and biological function, but have sequences sufficiently different that traditional protein sequence comparison algorithms do not identify their relationship. The desire to identify such relations has led to the development of more sensitive sequence alignment strategies. One such strategy is the Intermediate Sequence Search (ISS), which connects two proteins through one or more intermediate sequences. In its brute-force implementation, ISS is a strategy that repetitively uses the results of the previous query as new search seeds, making it time-consuming and difficult to analyze. RESULTS: Saturated BLAST is a package that performs ISS in an efficient and automated manner. It was developed using Perl and Perl/Tk and implemented on the LINUX operating system. Starting with a protein sequence, Saturated BLAST runs a BLAST search and identifies representative sequences for the next generation of searches. The procedure is run until convergence or until some predefined criteria are met. Saturated BLAST has a friendly graphic user interface, a built-in BLAST result parser, several multiple alignment tools, clustering algorithms and various filters for the elimination of false positives, thereby providing an easy way to edit, visualize, analyze, monitor and control the search. Besides detecting remote homologies, Saturated BLAST can be used to maintain protein family databases and to search for new genes in genomic databases.
Protein function assignments based on postulated homology as recognized by high sequence similarity are used routinely in genome analysis. Improvements in sensitivity of sequence comparison algorithms got to the point, that proteins with previously undetectable sequence similarity, such as for instance 10-15% of identical residues, sometimes can be classified as similar. What is the relation between such proteins? Is it possible that they are homologous? What is the practical significance of detecting such similarities? A simplified analysis of the relation between sequence similarity and function similarity is presented here for the well-characterized proteins from the E. coli genome. Using a simple measure of functional similarity based on E.C. classification of enzymes, it is shown that it correlates well with sequence similarity measured by statistical significance of the alignment score. Proteins, similar by this standard, even in cases of low sequence identity, have a much larger chance of having similar function than the randomly chosen protein pairs. Interesting exceptions to these rules are discussed.
Fold assignments for proteins from the Helicobacter pylori genome are carried out using BASIC, a profile-profile alignment algorithm recently tested on the Mycoplasma genitalium and Escherichia coli genomes. The fold assignments are followed by automated function evaluation, based on the multilevel description of functional sites in proteins. Over 40% of the proteins encoded in the H. pylori genome can be recognized as belonging to a protein family with known structure. Previous estimates suggested that only 10-15% of genome proteins could be characterized this way. This dramatic increase in the number of recognized homologies between H. pylori proteins and structurally characterized protein families is partly due to the rapid increase of the database of known protein structures, but mostly it is due to the significant improvement in prediction algorithms. Knowledge of a protein fold adds a new dimension to our understanding of its function and, similarly, structure prediction can also add to understanding, verification, and/or prediction of function for uncharacterized proteins. Several examples analyzed in more detail in this article illustrate insights that can be achieved from structure and detailed function prediction.
A database of functional sites for proteins with known structures, SITE, is constructed and used in conjunction with a simple pattern matching program SiteMatch to evaluate possible function conservation in a recently constructed database of fold predictions for Escherichia coli proteins (Rychlewski L et al., 1999, Protein Sci 8:614-624). In this and other prediction databases, fold predictions are based on algorithms that can recognize weak sequence similarities and putatively assign new proteins into already characterized protein families. It is not clear whether such sequence similarities arise from distant homologies or general similarity of physicochemical features along the sequence. Leaving aside the important question of nature of relations within fold superfamilies, it is possible to assess possible function conservation by looking at the pattern of conservation of crucial functional residues. SITE consists of a multilevel function description based on structure annotations and structure analyses. In particular, active site residues, ligand binding residues, and patterns of hydrophobic residues on the protein surface are used to describe different functional features. SiteMatch, a simple pattern matching program, is designed to check the conservation of residues involved in protein activity in alignments generated by any alignment method. Here, this procedure is used to study conservation of functional features in alignments between protein sequences from the E. coli genome and their optimal structural templates. The optimal templates were identified and alignments taken from the database of genomic structural predictions was described in a previous publication (Rychlewski L et al., 1999, Protein Sci 8:614-624). An automated assessment of function conservation is used to analyze the relation between fold and function similarity for a large number of fold predictions. For instance, it is shown that identifying low significance predictions with a high level of functional residue conservations can be used to extend the prediction sensitivity for fold prediction methods. Over 100 new fold/function predictions in this class were obtained in the E. coli genome. At the same time, about 30% of our previous fold predictions are not confirmed as function predictions, further highlighting the problem of function divergence in fold superfamilies.
Helix-loop-helix fragments of EF-hand proteins are known to dimerize in solution, re-producing the characteristic structure of native protein domains [Shaw, G.S., Hodges, R.S., & Sykes, B. D. (1990) Science 249, 280-283]. In this paper we present evidence that isolated calcium-binding loops can also dimerize, when saturated with lanthanide ions, interacting with each other in a similar way as do loops in intact proteins. A synthetic analogue of calcium binding loop III of calmodulin, AcDKDGDGYISAAE-NH2, has been studied by 1H NMR spectroscopy. For the La(3+)-saturated peptide, concentration dependent broadenings and shifts of certain signals have been observed indicating dimerization process of intermediate rate on the NMR time scale. Analysis of signal shape and position of the Tyr7 ring protons as a function of concentration makes it possible to determine the association and dissociation rate constants of the process for various temperatures within the range of 10-80 degrees C. The dimerization constant changes according to van't Hoff relationship with delta S = 233 J/mol.K and delta H = 62 kJ/mol. A distance of 11.4 +/- 0.4 A between the ions coordinated by dimer molecules has been determined by measurements of Tb(3+)-->Ho3+ luminescence energy transfer. This value suggests that the dimer structure is similar to that of two-loop structural elements in native EF-hand proteins. From a thermodynamic cycle it can be shown that La3+ ion binding to the peptide dimers must be highly cooperative. Therefore, cooperativity of ion binding to domains of EF-hand proteins is, at least partly, due to local interactions between binding loops.
Sequence alignments for distantly homologous proteins are often ambiguous, which creates a weak link in structure prediction by homology. We address this problem by using several plausible alignments in a modeling procedure, obtaining many models of the target. All are subsequently evaluated by a threading algorithm. It is shown that this approach can identify best alignments and produce reasonable models, whose quality is now limited only by the extent of the structural similarity between the known and predicted protein. Using a similar approach structure prediction for the oxidized dimer of S100A1 protein, for which the structure is not known, is presented.
Principles of contemporary theoretical description of alpha-helix formation by polypeptide chains in water solution are shortly presented and critically discussed. The theory treats the unfolded state of a peptide as "random coil"--an ideal conformation quite distant from reality. We suggest that for this reason the helix propagation parameters of amino-acid residues, determined using series of model peptides with different sequential patterns, are not the same. Interpretation of the so called "nucleation parameter" is erroneous. In fact, it is not determined by the helix nucleation process but rather by a specific situation of residues at the helix N- and C-termini, and it strongly depends on solvation of their NH and CO groups, respectively. Consequently, helical segments with terminal sequences dominated by residues with strongly hydrophobic, bulky side chains can be very unstable. We postulate that an unexpectedly high stability of very short, pre-nucleated helices studied by us arises from a "helix end separation effect": separated helix termini are better solvated than when they overlap each other. Because of this effect, helix initiation may be much more difficult than predicted by the theoretical "helix nucleation parameters".
An interesting example of a structurally diverse group of sequentially homologous proteins is analyzed at the level of molecular interactions. In this family, the EF-hand calcium-binding proteins, there are examples of at least three distinct mutual positions of the N and C-terminal domains, despite significant sequence homology between all members of this family. Why does a particular protein choose one arrangement over another? To answer this question, detailed models of all proteins in their native structures as well as all alternative sequence/structure combinations are built by comparative modeling. By studying and comparing interactions stabilizing native structures and destabilizing alternative conformations, it is possible to gain insight into how such conformational diversity is achieved. It is shown that some mechanisms used to achieve it are: correlated mutations on the surface of two units and the presence of additional domains/chain fragments stabilizing desired topologies. The implications of these findings, both for structure predictions for other members of this family as well as the general problem of quaternary structure formation, are discussed.
Antiinflammatory effect of ibuprofen was studied in the model of adjuvant arthritis in rats. Its effectiveness was evaluated on the basis of morphological and biochemical indices. The values of proteolytic and beta-glucuronidase activity in the blood serum were shown to correlate well with clinical exacerbation of adjuvant disease. Both enzymes, especially the beta-glucuronidase activity appeared to be the indices of particular value.
Anti-inflammatory effect of ibuprofen was studied on the model of adjuvant arthritis. Its effectiveness was evaluated on the basis of morphological and biochemical indices. The values of haptoglobin and copper in the blood serum were shown to well correlate to clinical exacerbation of adjuvant disease. Haptoglobin appeared to be the index of particular value.
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