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.

Iron scavenging from the host is essential for the growth of pathogenic bacteria. In this study, we further characterized two staphylococcal cell wall proteins previously shown to bind hemoproteins. HarA and IsdB harbor homologous ligand binding domains, the so called NEAT domain (for "near transporter") present in several surface proteins of gram-positive pathogens. Surface plasmon resonance measurements using glutathione S-transferase (GST)-tagged HarAD1, one of the ligand binding domains of HarA, and GST-tagged full-length IsdB proteins confirmed high-affinity binding to hemoglobin and haptoglobin-hemoglobin complexes with equilibrium dissociation constants (K D ) of 5 to 50 nM. Haptoglobin binding could be detected only with HarA and was in the low micromolar range. In order to determine the fold of this evolutionarily conserved ligand binding domain, the untagged HarAD1 protein was subjected to nuclear magnetic resonance spectroscopy, which revealed an eight-stranded, purely antiparallel ␤-barrel with the strand order (-␤1 2 -␤2 1 -␤3 2 -␤6 1 -␤5 2 -␤4 1 -␤7 2 -␤8 1 ), forming two Greek key motifs. Based on structural-homology searches, the topology of the HarAD1 domain resembles that of the immunoglobulin (Ig) fold family, whose members are involved in protein-protein interactions, but with distinct structural features. Therefore, we consider that the HarAD1/NEAT domain fold is a novel variant of the Ig fold that has not yet been observed in other proteins.

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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. Proteins Suppl 1999;3:112-120.

MicroArrays of Gel Immobilized Compounds on a Chip (MAGIChipsTM)are produced by immobilizing oligonucleotides, DNA, enzymes, antibodies, and other compounds on a photopolymerized micromatrix of polyacrylamide gel pads 1OOX1OOX20 pm and smaller in size. Alternatively, allyl-modified compounds can be embedded within polyacrylamide gel pads by copolymerization. MAGIChips have been shown to be efficient for analysis of nucleic acid hybridization, specific binding of DNA with proteins, and low-molecular-weight compounds, and proteinprotein interactions. The three-dimensional gel pads of the microarrays can be used as nanoliter-sized microtest tubes to perform ligation, singlebase extension, PCR amplification of DNA, and other reactions. The fluorescence microscope has been devised for analyzing reactions on MAGIChips, including quantitative and real-time monitoring of hybridization, measuring the thermodynamic parameters of DNA duplexes containing different modified nucleotides, and measuring the kinetics of enzymatic reaction. On-chip MALDI-TOF mass spectrometry was successfully tested for analysis of DNA and protein interactions. Application of these biochips for detection of human polymorphism and mutations, identification of microorganisms and their drug resistant and toxin-bearing strains, and in different fields of biotechnology and medicine will be described.