Assessment of the CASP4 fold recognition category.

We present the assessment of the CASP4 fold recognition category. The tasks we had to execute include the splitting of multidomain targets into single domains, the classification of target domains in terms of prediction categories, the numerical evaluation of predictions, the mapping of numerical scores to quality indices, the ranking of predictors, the selection of top-performing groups, and the analysis and critical discussion of the state of the art in this field. The 125 fold recognition groups were assessed by a total score that summarizes their performance over all targets and a quality score reflecting the average quality of the submitted models. Most of the top-performing groups achieved respectable results on both scores simultaneously. Several groups submitted models that were much closer to the respective target structures than any of the known folds in the Protein Data Bank. The CASP4 assessment included the automated servers of the parallel CAFASP experiment. For the total score, the highest rank achieved by a fully automated server is 12. Two thirds of the predictors have rather low scores.

Structure-derived substitution matrices for alignment of distantly related sequences.

Sequence alignment is a standard method to infer evolutionary, structural, and functional relationships among sequences. The quality of alignments depends on the substitution matrix used. Here we derive matrices based on superimpositions from protein pairs of similar structure, but of low or no sequence similarity. In a performance test the matrices are compared with 12 other previously published matrices. It is found that the structure-derived matrices are applicable for comparisons of distantly related sequences. We investigate the influence of evolutionary relationships of protein pairs on the alignment accuracy.

The role of protein structure in genomics.

The genome projects produce an enormous amount of sequence data that needs to be annotated in terms of molecular structure and biological function. These tasks have triggered additional initiatives like structural genomics. The intention is to determine as many protein structures as possible, in the most efficient way, and to exploit the solved structures for the assignment of biological function to hypothetical proteins. We discuss the impact of these developments on protein classification, gene function prediction, and protein structure prediction.

Structure-based evaluation of sequence comparison and fold recognition alignment accuracy.

The biological role, biochemical function, and structure of uncharacterized protein sequences is often inferred from their similarity to known proteins. A constant goal is to increase the reliability, sensitivity, and accuracy of alignment techniques to enable the detection of increasingly distant relationships. Development, tuning, and testing of these methods benefit from appropriate benchmarks for the assessment of alignment accuracy.Here, we describe a benchmark protocol to estimate sequence-to-sequence and sequence-to-structure alignment accuracy. The protocol consists of structurally related pairs of proteins and procedures to evaluate alignment accuracy over the whole set. The set of protein pairs covers all the currently known fold types. The benchmark is challenging in the sense that it consists of proteins lacking clear sequence similarity. Correct target alignments are derived from the three-dimensional structures of these pairs by rigid body superposition. An evaluation engine computes the accuracy of alignments obtained from a particular algorithm in terms of alignment shifts with respect to the structure derived alignments. Using this benchmark we estimate that the best results can be obtained from a combination of amino acid residue substitution matrices and knowledge-based potentials.

Characterization of novel proteins based on known protein structures.

The genome sciences face the challenge to characterize structure and function of a vast number of novel genes. Sequence search techniques are used to infer functional and structural information from similarities to experimentally characterized genes or proteins. The persistent goal is to refine these techniques and to develop alternative and complementary methods to increase the range of reliable inference.Here, we focus on the structural and functional assignments that can be inferred from the known three-dimensional structures of proteins. The study uses all structures in the Protein Data Bank that were known by the end of 1997. The protein structures released in 1998 were then characterized in terms of functional and structural similarity to the previously known structures, yielding an estimate of the maximum amount of information on novel protein sequences that can be obtained from inference techniques. The 147 globular proteins corresponding to 196 domains released in 1998 have no clear sequence similarity to previously known structures. However, 75 % of the domains have extensive structure similarity to previously known folds, and most importantly, in two out of three cases similarity in structure coincides with related function. In view of this analysis, full utilization of existing structure data bases would provide information for many new targets even if the relationship is not accessible from sequence information alone. Currently, the most sophisticated techniques detect of the order of one-third of these relationships.

ProSup: a refined tool for protein structure alignment.

We investigated and optimized a method for structure comparison which is based on rigid body superimposition. The method maximizes the number of structurally equivalent residues while keeping the root mean square deviation constant. The resulting number of equivalent residues then provides an adequate similarity measure, which is easy to interpret. We demonstrate that the approach is able to detect remote structural similarity. We show that the number of equivalent residues is a suitable measure for ranking database searches and that the results are in good agreement with expert knowledge protein structure classification. Structure comparison frequently has multiple solutions. The approach that we use provides a range of alternative alignments rather a single solution. We discuss the nature of alternative solutions on several examples.

Automated large scale evaluation of protein structure predictions.

Evaluation and assessment are critical issues in CASP experiments. Automated procedures are necessary to compare a large number of predictions with the target folds. The evaluation has to reveal the maximum extent of similarity between predictions and targets, it should be applicable across prediction categories, and it should be transparent and accessible to a wide community. Here we present an automated evaluation scheme which is an attempt to meet these requirements. In the implementation and execution of this scheme we had to solve or circumvent problems of convergence, where algorithms fail to find optimum solutions, problems of ambiguity where no unique optimum solution exists, and problems in ranking and interpretation. Key features of this implementation are (1) the root mean square deviation of structure superimposition is kept close to a constant value throughout the evaluation and (2) all structural matches found between two folds are taken into account. We discuss these points in detail and describe the numerical criteria used in the CASP3 evaluation.

Sustained performance of knowledge-based potentials in fold recognition.

We describe the results obtained using fold recognition techniques in our third participation in the CASP experiment. The approach relies on knowledge-based potentials for alignment production and fold identification. As indicated by the increase in alignment quality and fold identification reliability, the predictions improved from CASP1 to CASP3. In particular, we identified structural relationships in which no known evolutionary link exists. Our predictions are based on single sequences rather than multiple sequence alignments. Additionally, we voluntarily submitted only a single model for each target because, in our view, submission of a single model is the most stringent test. We describe the methods used, the strategy adopted in the predictions, and the prediction results and discuss future work.

An attempt to analyse progress in fold recognition from CASP1 to CASP3.

The Critical Assessment of Techniques for Protein Structure Prediction (CASP) experiment has been conducted for the third time. An obvious question is whether there has been progress from CASP1 to CASP3. An analysis depends on many variables, including prediction category, number and difficulty of targets, methods used to evaluate prediction success, and the rules for submission. It also depends on whether progress is measured in terms of all predictions submitted or in terms of the best predictions for each target. The progress made by individual groups is another interesting issue. In view of this complexity and the limited amount of data, an objective estimate of progress is difficult to obtain. Despite such difficulties, some estimate of progress is desirable. Here, we present an attempt to quantify progress in the fold-recognition category from CASP1 to CASP3. The numbers indicate clear progress from CASP1 to CASP2 but no improvement from CASP2 to CASP3. However, we argue that the targets in CASP3 are more difficult compared with CASP2, which translates into better performance of CASP3 over CASP2.

Isoforms of the major allergen of birch pollen induce different immune responses after genetic immunization.

BACKGROUND: Recent publications indicate that immunization with plasmid DNA encoding allergens might represent a potential approach in allergen-specific immunotherapy. OBJECTIVE: In the present study we have compared the immune responses induced by plasmid DNA encoding for two isoforms of Bet v 1, the major allergen of birch pollen. METHODS: BALB/c mice were injected intradermally with plasmid DNA encoding for the genes of Bet v 1a (pCMV-Beta) and Bet v 1d (pCMV-Betd). In addition, the effect of immunostimulatory DNA sequences was investigated by appending and/or coinjecting CpG motifs. Antibody responses and IFN-gamma and IL-4 levels were measured by ELISA. Allergen-specific proliferation was determined by incorporation of [(3)H]-thymidine. RESULTS: The two isoforms induced a similar humoral response. The lack of any IgE production and the ratio of IgG1 to IgG2a clearly indicated a Th-1-type response. The antisera against both isoforms were highly cross-reactive, which was supported by the energy plot indicating similar folding of the two protein isoforms. However, determination of IFN-gamma and IL-4 in the serum elicited a strikingly different cytokine profile during the course of the immune response. In contrast to pCMV-Beta, pCMV-Betd caused no significant allergen-specific proliferation and induced only marginal levels of the key cytokines. CONCLUSIONS: Based on the assumption that the induction of a strong Th-1 type response is a prerequisite for successful treatment of allergy, our results favor the use of isoform Bet v 1a in combination with CpG motifs for a novel type of allergen immunotherapy based on plasmid DNA immunization. Additionally, the data also confirm the assumption that the antigen itself can have a marked influence on the immune response after genetic immunization.

Fold recognition study of alpha3-galactosyltransferase and molecular modeling of the nucleotide sugar-binding domain.

The structure and fold of the enzyme responsible for the biosynthesis of the xenotransplantation antigen, namely pig alpha3 galactosyltransferase, has been studied by means of computational methods. Secondary structure predictions indicated that alpha3-galactosyltransferase and related protein family members, including blood group A and B transferases and Forssman synthase, are likely to consist of alternating alpha-helices and beta-strands. Fold recognition studies predicted that alpha3-galactosyltransferase shares the same fold as the T4 phage DNA-modifying enzyme beta-glucosyltransferase. This latter enzyme displays a strong structural resemblance with the core of glycogen phosphorylase b. By using the three-dimensional structure of beta-glucosyltransferase and of several glycogen phosphorylases, the nucleotide binding domain of pig alpha3-galactosyltransferase was built by knowledge-based methods. Both the UDP-galactose ligand and a divalent cation were included in the model during the refinement procedure. The final three-dimensional model is in agreement with our present knowledge of the biochemistry and mechanism of alpha3-galactosyltransferases.

Who solved the protein folding problem?

For the third time, techniques for the prediction of three-dimensional structures of proteins were critically assessed in a worldwide blind test. Steady progress is undeniable. How did this happen and what are the implications?

Knowledge-based potentials--back to the roots.

Applications of knowledge-based quantities in protein structure theory are well established but their theoretical foundation, physical interpretation, and range of applicability seems unclear or even controversial. Moreover, the current literature contains terms like "pseudo-energy", "energy-like quantity", or "true energy" which are vague and unclear and terms like "mean-force potential" corresponding to well defined concepts. Seemingly contradictory results are often caused by inconsistent terminology. Often such problems are resolved when the physical nature of the involved quantities is properly defined. We summarize the fundamental principles of mean-force potentials and radial distribution functions as defined in statistical mechanics and put these into perspective with the term "knowledge-based potential".

Neutral networks in protein space: a computational study based on knowledge-based potentials of mean force.

BACKGROUND: Many protein sequences, often unrelated, adopt similar folds. Sequences folding into the same shape thus form subsets of sequence space. The shape and the connectivity of these sets have implications for protein evolution and de novo design. RESULTS: We investigate the topology of these sets for some proteins with known three-dimensional structure using inverse folding techniques. First, we find that sequences adopting a given fold do not cluster in sequence space and that there is no detectable sequence homology among them. Nevertheless, these sequences are connected in the sense that there exists a path such that every sequence can be reached from every other sequence while the fold remains unchanged. We find similar results for restricted amino acid alphabets in some cases (e. g. ADLG). In other cases, it seems impossible to find sequences with native-like behavior (e.g. QLR). These findings seem to be independent of the particular structure considered. CONCLUSIONS: Amino acid sequences folding into a common shape are distributed homogeneously in sequence space. Hence, the connectivity of the set of these sequences implies the existence of very long neutral paths on all examined protein structures. Regarding protein design, these results imply that sequences with more or less arbitrary chemical properties can be attached to a given structural framework. But we also observe that designability varies significantly among native structures. These features of protein sequence space are similar to what has been found for nucleic acids.

Empirical potentials and functions for protein folding and binding.

Simplified models and empirical potentials are being increasingly used for the analysis of proteins, frequently augmenting or replacing molecular mechanics approaches. Recent folding simulations have employed potentials that, in addition to terms assuring proper polypeptide geometry, include only two noncovalent effects-hydrogen bonding and hydrophobicity, with extremely simple approximations to the latter. The potentials that have been used in the free-energy ranking of protein-ligand complexes have generally been more involved. These potentials have more detailed solvation models and account for both local (hydrophobic and polar) solute-solvent phenomena and long range electrostatic solvation effects. The models of solvation that have been used most frequently are surface area related atomic parameters, knowledge-based models extracted from protein-structure data, and continum electrostatics with an additional area-related parameter. The knowledge-based approaches to solvation, although convenient and accurate enough, are suspect of double counting certain free-energy terms.

Protein folds from pair interactions: a blind test in fold recognition.

We submitted nine predictions to CASP2 using our fold recognition program ProFIT. Two of these structures were still unsolved by the end of the experiment, six had a recognizable fold, and one fold was new. Four predictions of the six recognizable folds were correct. Two models were excellent in terms of alignment quality (T0031, T0004): in one the alignment was partially correct (T0014), and one fold was correctly identified (T0038). We discuss improvements of the program and analyze the prediction results.

Molecular and structural analysis of a continuous birch profilin epitope defined by a monoclonal antibody.

The interaction of a mouse monoclonal antibody (4A6) and birch profilin, a structurally well conserved actin- and phosphoinositide-binding protein and cross-reactive allergen, was characterized. In contrast to serum IgE from allergic patients, which shows cross-reactivity with most plants, monoclonal antibody 4A6 selectively reacted with tree pollen profilins. Using synthetic overlapping peptides, a continuous hexapeptide epitope was identified. The exchange of a single amino acid (Gln-47 --> Glu) within the epitope was found to abolish the binding of monoclonal antibody 4A6 to other plant profilins. The NMR analyses of the birch and the nonreactive timothy grass profilin peptides showed that the loss of binding was not due to major structural differences. Both peptides adopted extended conformations similar to that observed for the epitope in the x-ray crystal structure of the native birch profilin. Binding studies with peptides and birch profilin mutants generated by in vitro mutagenesis demonstrated that the change of Gln-47 to acidic amino acids (e.g. Glu or Asp) led to electrostatic repulsion of monoclonal antibody 4A6. In conclusion the molecular and structural analyses of the interaction of a monoclonal antibody with a continuous peptide epitope, recognized in a conformation similar to that displayed on the native protein, are presented.

Helmholtz free energy of peptide hydrogen bonds in proteins.

We estimate the Helmholtz free energy of peptide hydrogen bonds in native protein structures as a function of spatial separation between donor and acceptor atoms. The resulting potential function has a deep narrow well at H-bond contact but bond formation is hindered by a barrier and the net change in free energy is close to zero. The barrier provides a molecular lock mechanism acting as a kinetic trap. Once formed, H-bonds keep protein chains in a precise orientation. However, bond formation requires energy input and opposes protein folding. In contrast, the free energy functions of most side-chain interactions have no energy barriers. They lack spatial precision but free energy differences of contact formation are substantial. These interactions drive folding and stabilize structures but precision is mediated and maintained by H-bonds.