Functional characterization of Ape1 variants identified in the human population.

Apurinic/apyrimidinic (AP) sites are common mutagenic and cytotoxic DNA lesions. Ape1 is the major human repair enzyme for abasic sites and incises the phosphodiester backbone 5' to the lesion to initiate a cascade of events aimed at removing the AP moiety and maintaining genetic integrity. Through resequencing of genomic DNA from 128 unrelated individuals, and searching published reports and sequence databases, seven amino acid substitution variants were identified in the repair domain of human Ape1. Functional characterization revealed that three of the variants, L104R, E126D and R237A, exhibited approximately 40-60% reductions in specific incision activity. A fourth variant, D283G, is similar to the previously characterized mutant D283A found to exhibit approximately 10% repair capacity. The most common substitution (D148E; observed at an allele frequency of 0.38) had no impact on endonuclease and DNA binding activities, nor did a G306A substitution. A G241R variant showed slightly enhanced endonuclease activity relative to wild-type. In total, four of seven substitutions in the repair domain of Ape1 imparted reduced function. These reduced function variants may represent low penetrance human polymorphisms that associate with increased disease susceptibility.

Structure-based sequence alignment for the beta-trefoil subdomain of the clostridial neurotoxin family provides residue level information about the putative ganglioside binding site.

Clostridial neurotoxins embrace a family of extremely potent toxins comprised of tetanus toxin (TeNT) and seven different serotypes of botulinum toxin (BoNT/A-G). The beta-trefoil subdomain of the C-terminal part of the heavy chain (H(C)), responsible for ganglioside binding, is the most divergent region in clostridial neurotoxins with sequence identity as low as 15%. We re-examined the alignment between family sequences within this subdomain, since in this region all alignments published to date show obvious inconsistencies with the beta-trefoil fold. The final alignment was obtained by considering the general constraints imposed by this fold, and homology modeling studies based on the TeNT structure. Recently solved structures of BoNT/A confirm the validity of this structure-based approach. Taking into account biochemical data and crystal structures of TeNT and BoNT/A, we also re-examined the location of the putative ganglioside binding site and, using the new alignment, characterized this site in other BoNT serotypes.

Synthesis and antiarrhythmic properties of novel 3-selena-7-azabicyclo[3.3.1]nonanes and derivatives. Single-crystal X-ray diffraction analysis of 7-benzyl-3-selena-7-azabicyclo[3.3.1]nonan-9-one and 7-benzyl-3-selena-7-azabicyclo[3.3.1]nonane hydroperchlorate.

Several members of the heterocyclic family 3-selena-7-azabicyclo[3.3.1]nonane have been synthesized and characterized via IR, 1H, 13C, 15N, and 77Se NMR spectroscopy and, in some cases, by X-ray diffraction analysis. Select members, namely the hydroperchlorates of the amines, were examined for antiarrhythmic properties in anesthetized dogs in which myocardial infarctions were induced by techniques previously described. In the predrug, or control state, sustained ventricular tachycardia were induced by ventricular paced beats at rates above 300/min. When 7-benzyl-3-selena-7-azabicyclo[3.3.1]nonane hydroperchlorate was administered at 3 and 6 mg/kg, the sustained ventricular tachycardia could no longer be induced. Similar doses of lidocaine, a commonly used antiarrhythmic, caused slowing of the sustained ventricular tachycardia below 300/min but did not abolish their inducibility. In addition, select members of the hydroperchlorates caused a moderate 10-20% increase in mean blood pressure whereas lidocaine caused either no change in or slightly reduced mean blood pressure. Some general conclusions are delineated concerning the structural requirements that appear to be necessary for activity in this family of heterocycles and that have not been reported previously.

DataFoundry: information management for scientific data.

Data warehouses and data marts have been successfully applied to a multitude of commercial business applications. They have proven to be invaluable tools by integrating information from distributed, heterogeneous sources and summarizing this data for use throughout the enterprise. Although the need for information dissemination is as vital in science as in business, working warehouses in this community are scarce because traditional warehousing techniques do not transfer to scientific environments. There are two primary reasons for this difficulty. First, schema integration is more difficult for scientific databases than for business sources, because of the complexity of the concepts and the associated relationships. While this difference has not yet been fully explored, it is an important consideration when determining how to integrate autonomous sources. Second, scientific data sources have highly dynamic data representations (schemata). When a data source participating in a warehouse changes its schema, both the mediator transferring data to the warehouse and the warehouse itself need to be updated to reflect these modifications. The cost of repeatedly performing these updates in a traditional warehouse, as is required in a dynamic environment, is prohibitive. This paper discusses these issues within the context of the DataFoundry project, an ongoing research effort at Lawrence Livermore National Laboratory. DataFoundry utilizes a unique integration strategy to identify corresponding instances while maintaining differences between data from different sources, and a novel architecture and an extensive meta-data infrastructure, which reduce the cost of maintaining a warehouse.

Addressing the issue of sequence-to-structure alignments in comparative modeling of CASP3 target proteins.

During a blind protein structure prediction experiment (the third round of the Critical Assessment of Techniques for Protein Structure Prediction; URL http://PredictionCenter.llnl.gov/casp3/) , four target proteins, T0047, T0048, T0055, and T0070, were modeled by comparison. These proteins display 62%, 29%, 24%, and 19% sequence identity, respectively, to the structurally homologous proteins most similar in sequence. The issue of sequence-to-structure alignment in cases of low sequence homology was the main emphasis. Selection of alignments was made by constructing and evaluating three-dimensional models based on series of samples produced mainly by automatic multiple sequence alignments. Sequence-to-structure alignments were correct in all but two regions, in which significant changes in target structures compared with related proteins were the source of errors. Template choice is an important determinant of model quality, and a correct selection was made of a lower homology template for modeling of T0070; however, in the case of T0055, a template with 8% greater sequence homology proved deceptive. Loops and some ungapped template regions were assigned conformations taken from other proteins. Using fragments from homologous structures led to improvement over template backbone more often than cases in which nonhomologous structures were the source. The results also indicate that side-chain prediction accuracy depends not only on sequence similarity but also on accuracy of the backbone.

Processing and evaluation of predictions in CASP4.

The Livermore Prediction Center conducted the target collection and prediction submission processes for Critical Assessment of Protein Structure Prediction (CASP4) and Critical Assessment of Fully Automated Structure Prediction Methods (CAFASP2). We have also evaluated all the submitted predictions using criteria and methods developed during the course of three previous CASP experiments and preparation for CASP4. We present an overview of the implemented system. Particular attention is paid to newly developed evaluation techniques and data presentation schemes. With the rapid increase in CASP participation and in the number of submitted predictions, special emphasis is placed on methods allowing reliable pre-classification of submissions and on techniques useful in automated evaluation of predictions. We also present an overview of our website, including target structures, predictions, and their evaluations ( http://predictioncenter.llnl.gov).

Comparison of systematic search and database methods for constructing segments of protein structure.

Two principal methods of determining the conformation of short pieces of polypeptide backbone in proteins have been developed: using a database of known structures and systematically generating all conformations. In this paper, we compare the effectiveness of these two techniques. The completeness of the database for segments of different lengths is examined and it is found to contain most conformations for segments seven residues long, but to deteriorate rapidly for longer regions. When the database segment is to be incorporated into the rest of a structure, at least seven residues are required to build four new residues, because of the need to position the segment relative to the rest of the structure. It is found that such positioning using flanking residues results in large errors in the inserted region. We conclude that the database method is currently not effective for comparative modeling, even for short segments. The systematic search procedure is found to generate almost all structures of short segments found in proteins. In contrast to the database method, low root mean square error structures are obtained for a set of trial segments embedded in the rest of a protein structure. Thus, it should be considered the method of choice.

Criteria for evaluating protein structures derived from comparative modeling.

Following the first experiment for the Critical Assessment of methods for protein Structure Prediction (CASP1), numerical criteria were devised to analyze the performance of prediction methods. We report here the criteria for comparative modeling, and how effective they were in CASP2. These criteria are intended to evolve into a set of numerical measures that provide a comprehensive assessment of the quality of a structure produced by comparative modeling, and provide a means of investigating which modeling methods are most effective, so as to establish where future effort may be most productively applied.

Processing and analysis of CASP3 protein structure predictions.

Livermore Prediction Center provides basic infrastructure for the CASP (Critical Assessment of Structure Prediction) experiments, including prediction processing and verification servers, a system of prediction evaluation tools, and interactive numerical and graphical displays. Here we outline the essentials of our approach, with discussion of the superposition procedures, definitions of basic measures, and descriptions of new methods developed to analyze predictions. Our primary focus is on the evaluation of three-dimensional models and secondary structure predictions. To put the results of the three prediction experiments held to date on the same footing, the latest CASP3 evaluation criteria were retrospectively applied to both CASP1 and CASP2 predictions. Finally, we give an overview of our website (http:/(/)PredictionCenter.llnl.gov), which makes the target structures, predictions, and the evaluation system accessible to the community.

Some measures of comparative performance in the three CASPs.

Performance in the three Critical Assessment of protein Structure Prediction (CASP) experiments has been compared in the areas of alignment accuracy for models based on homology and three-dimensional accuracy for models produced by using ab initio prediction methods. The homologous models span the comparative modeling and fold-recognition regimes. Each CASP target is assigned a relative difficulty based on the extent of sequence identity and the degree of structural overlap with the best available template. There is a clear improvement in alignment accuracy between CASP1 and CASPs 2 and 3 over much of the difficulty scale but no apparent improvement between CASP2 and CASP3. Encouragingly, the best ab initio models of small targets are clearly more accurate in CASP3 than in CASPs 1 and 2.

A modified definition of Sov, a segment-based measure for protein secondary structure prediction assessment.

We present a measure for the evaluation of secondary structure prediction methods that is based on secondary structure segments rather than individual residues. The algorithm is an extension of the segment overlap measure Sov, originally defined by Rost et al. (J Mol Biol 1994;235:13-26). The new definition of Sov corrects the normalization procedure and improves Sov's ability to discriminate between similar and dissimilar segment distributions. The method has been comprehensively tested during the second Critical Assessment of Techniques for Protein Structure Prediction (CASP2). Here, we describe the underlying concepts, modifications to the original definition, and their significance.

Comparison of performance in successive CASP experiments.

As the number of completed CASP (Critical Assessment of Protein Structure Prediction) experiments grows, so does the need for stable, standard methods for comparing performance in successive experiments. It is critical to develop methods for determining the areas in which there is progress and in which areas are static. We have added an analysis of the CASP4 results to that previously published for CASPs 1, 2, and 3. We again use a unified difficulty scale to permit comparison of performance as a function of target difficulty in the different CASPs. The scale is used to compare performance in aligning target sequences to a structural template. There was a clear improvement in alignment quality between CASP1 (1994) and CASP2 (1996). No change is apparent between CASP2 and CASP3 (1998). There is a small barely detectable improvement between CASP3 and the latest experiment (CASP4, 2000). Alignment remains the major source of error in all models based on less than about 30% sequence identity. Comparison of performance in the new fold modeling regime is complicated by issues in devising an objective target difficulty scale. We have found limited numerical support for significant progress between CASP3 and CASP4 in this area. More subjectively, most observers are convinced that there has been substantial progress. Progress is dominated by a single group.

Confronting the problem of interconnected structural changes in the comparative modeling of proteins.

Comparative models of three proteins have been built using a variety of computational methods, heavily supplemented by visual inspection. We consider the accuracy obtained to be worse than expected. A careful analysis of the models shows that a major reason for the poor results is the interconnectedness of the structural differences between the target proteins and the template structures they were modeled from. Side chain conformations are often determined by details of the structure remote in the sequence, and can be influenced by relatively small main chain changes. Almost all of the regions of substantial main chain conformational change interact with at least one other such region, so that they often cannot be modeled independently. Visual inspection is sometimes effective in correcting errors in sequence alignment and in spotting when an alternative template structure is more appropriate. We expect some improvements in the near future through the development of structure-based sequence alignment tools, side chain interconnectedness rotamer choice algorithms, and a better understanding of the context sensitivity of conformational features.

Numerical criteria for the evaluation of ab initio predictions of protein structure.

As part of the CASP2 protein structure prediction experiment, a set of numerical criteria were defined for the evaluation of "ab initio" predictions. The evaluation package comprises a series of electronic submission formats, a submission validator, evaluation software, and a series of scripts to summarize the results for the CASP2 meeting and for presentation via the World Wide Web (WWW). The evaluation package is accessible for use on new predictions via WWW so that results can be compared to those submitted to CASP2. With further input from the community, the evaluation criteria are expected to evolve into a comprehensive set of measures capturing the overall quality of a prediction as well as critical detail essential for further development of prediction methods. We discuss present measures, limitations of the current criteria, and possible improvements.

Structure and conformations of two cycloisomeric hexapeptides: cyclo(L-Leu-L-Phe-Gly-D-Phe-L-Leu-Gly-) trihydrate and cyclo(L-Phe-L-Leu-Gly-D-Leu-L-Phe-Gly-) trihydrate.

cyclo(L-Leucyl-L-phenylalanyl-glycyl-D-phenylalanyl-L-leucyl-glycyl-) trihydrate (IV), C34H46N6O6.-3H2O, Mr = 688.8, monoclinic, P2(1), a = 11.720 (2), b = 36.354 (4), c = 8.888 (1) A, beta = 103.88 (1) degree, V = 3676.3 A3, Z = 4, Dx = 1.244 g cm-3, lambda (Cu K alpha) = 1.54178 A, mu = 7.6 cm-1, F(000) = 1480, T = 138 K, final R = 0.052 for 7661 unique reflections. cyclo(L-Phenylalanyl-L-leucyl-glycyl-D-leucyl-L-phenylalanyl-glycyl-) trihydrate (V), C34H46N6O6.-3H2O, Mr = 688.8, triclinic, P1, a = 101.54 (2), b = 19.111 (5), c = 8.527 (1) A, alpha = 101.54 (2), beta = 93.42 (2), gamma = 94.27 (2) degree, V = 1852.4 A3, Z = 2, Dx = 1.235 g cm-3, lambda (Cu K alpha) = 1.54178 A, mu = 7.5 cm-1, F(000) = 740, T = 138 K, final R = 0.063 for 7574 unique reflections. Peptides IV and V both have two independent conformers (molecules A and B). The peptide ring in each case contains one beta (I) turn and one beta (II') turn. A molecules in both structures have two transannular N--H...O hydrogen bonds, while B molecules form only one strong transannular hydrogen bond. The conformational differences between the two independent molecules (A and B) are much larger than the differences between the corresponding molecules of the two structures (A and A, and B and B). The crystal structures of the two peptides are very similar and consist of parallel bands of hydrophobic side chains and polar peptide regions. In each structure, molecules are stacked one over another with the hexapeptide ring lying perpendicular to the axis of the stack. The water molecules form well delimited solvent channels sandwiched between the layers of peptide molecules, and bridge the peptides through extensive hydrogen bonding.

Structure and molecular mechanics of ferrirhodin.

C41H64FeN9O17.7 1/2H2O, Mr = 1146.0, orthorhombic, P2(1)2(1)2(1), a = 9.740 (7), b = 16.764 (10), c = 32.632 (17) A, V = 5328 (6) A3, Z = 4, D chi = 1.43 g cm-3, Mo K alpha, lambda = 0.71069 A, mu = 3.26 cm-1, F(000) = 2428, T = 138 (2) K, R = 0.0986 for 3543 observed reflections. Ferrirhodin, a ferrichrome siderophore (iron transport agent) was isolated from low-iron cultures of Aspergillus versicolor and A. nidulans. The compound is isomeric with another microbial siderophore, ferrirubin, but is different in having cis, rather than trans, anhydromevalonic acid as acyl groups. The conformation of the molecular backbone and iron coordination geometry compares well with ferrirubin and other ferrichrome structures. The differences between the acyl groups of ferrirubin and ferrirhodin are explored using molecular-mechanics modeling.

New jaeschkeanadiol derivatives from Ferula jaeschkeana.

Two new jaeschkeanadiol derivatives have been isolated from Ferula jaeschkeana rhizomes. These have been identified as a dichloro compound, jaeschkenol [1], and 2 alpha,3 alpha-dihydroxy-4-keto-5 alpha-p-hydroxybenzoyl-jaeschkeanadiol [2], on the basis of spectral data and the X-ray analysis of the former.

Crystallographic, molecular modeling, and biophysical characterization of the valine beta 67 (E11)-->threonine variant of hemoglobin.

The crystal structure of the mutant deoxyhemoglobin in which the beta-globin Val67(E11) has been replaced with threonine [Fronticelli et al. (1993) Biochemistry 32, 1235-1242] has been determined at 2.2 A resolution. Prior to the crystal structure determination, molecular modeling indicated that the Thr67(E11) side chain hydroxyl group in the distal beta-heme pocket forms a hydrogen bond with the backbone carbonyl of His63(E7) and is within hydrogen-bonding distance of the N delta of His63(E7). The mutant crystal structure indicates only small changes in conformation in the vicinity of the E11 mutation confirming the molecular modeling predictions. Comparison of the structures of the mutant beta-subunits and recombinant porcine myoglobin with the identical mutation [Cameron et al. (1993) Biochemistry 32, 13061-13070] indicates similar conformations of residues in the distal heme pocket, but there is no water molecule associated with either of the threonines of the beta-subunits. The introduction of threonine into the distal heme pocket, despite having only small perturbations in the local structure, has a marked affect on the interaction with ligands. In the oxy derivative there is a 2-fold decrease in O2 affinity [Fronticelli et al. (1993) Biochemistry 32, 1235-1242], and the rate of autoxidation is increased by 2 orders of magnitude. In the CO derivative the IR spectrum shows modifications with respect to that of normal human hemoglobin, suggesting the presence of multiple CO conformers. In the nitrosyl derivative an interaction with the O gamma atom of Thr67(E11) is probably responsible for the 10-fold increase in the rate of NO release from the beta-subunits. In the aquomet derivative there is a 6-fold decrease in the rate of hemin dissociation suggesting an interaction of the Fe-coordinated water with the O gamma of Thr67(E11).