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Discovery of local packing motifs in protein structures.

We present a language for describing structural patterns of residues in protein structures and a method for the discovery of such patterns that recur in a set of protein structures. The patterns impose restrictions on the spatial position of each residue, their order along the amino acid chain, and which amino acids are allowed in each position. Unlike other methods for comparing sets of protein structures, our method is not based on the use of pairwise structure comparisons which is often time consuming and can produce inconsistent results. Instead, the method simultaneously takes into account information from all structures in the search for conserved structure patterns which are potential structure motifs. The method is based on describing the spatial neighborhoods of each residue in each structure as a string and applying a sequence pattern discovery method to find patterns common to subsets of these strings. Finally it is checked whether the similarities between the neighborhood strings correspond to spatially similar substructures. We apply the method to analyze sets of very disparate proteins from the four different protein families: serine proteases, cuprodoxins, cysteine proteinases, and ferredoxins. The motifs found by the method correspond well to the site and motif information given in the annotation of these proteins in PDB, Swiss-Prot, and PROSITE. Furthermore, the motifs are confirmed by using the motif data to constrain the structural alignment of the proteins obtained with the program SAP. This gave the best superposition/alignment of the proteins given the motif assignment.

Algorithms↗

The Adh gene promoters of Drosophila melanogaster and Drosophila orena are functionally conserved and share features of sequence structure and nuclease-protected sites.

The sibling species Drosophila melanogaster and D. orena show similar patterns of alcohol dehydrogenase expression, both spatially and temporally. These two species diverged from a common ancestor 6 million to 15 million years ago, and the DNA sequences of the promoter regions of their Adh genes show a mosaic pattern of conservation and change. By interspecific transformation of D. orena sequences into D. melanogaster, we demonstrate a functional equivalence between these sequences. Using both D. melanogaster embryo extracts and purified transcription factor Adf-1, we compare the protection of these promoter sequences from nuclease, demonstrating considerable conservation.

Alcohol Dehydrogenase↗

Multiple cis-acting sequences implicate function diversity in nuclear matrix attachment regions of bovine mammary gland.

Chromosomal DNA in higher eukaryotes is spatially organized into loops by periodic attachment to the nuclear matrix at its base via a specific matrix attachment region (MAR). In order to study the nature of DNA sequences that affixed the loops to the nuclear matrix, we have cloned the MAR DNA from bovine lactating mammary tissues. In vitro binding assay showed that the cloned fragments could be co-complexed with nuclear matrix proteins to form insoluble complex easily removed by centrifugation. Sequences of the two chosen MAR loci are composed of TG-, CA- and GA- blocks, as well as the ATTA motifs. Both the MAR loci show numerous replication/transcription factor binding sites, enhancer motifs, several perfect or imperfect inverted repeats, and sequences sharing the common features of the potential DNA bending core sequence. The possibility that a combination of different elements in the same DNA sequence may function as either positive or negative regulatory elements in controlling a variety of cellular and developmental processes is discussed.

Animals↗

Binding of disparate transcriptional activators to nucleosomal DNA is inherently cooperative.

To investigate mechanisms by which multiple transcription factors access complex promoters and enhancers within cellular chromatin, we have analyzed the binding of disparate factors to nucleosome cores. We used a purified in vitro system to analyze binding of four activator proteins, two GAL4 derivatives, USF, and NF-kappa B (KBF1), to reconstituted nucleosome cores containing different combinations of binding sites. Here we show that binding of any two or all three of these factors to nucleosomal DNA is inherently cooperative. Thus, the binuclear Zn clusters of GAL4, the helix-loop-helix/basic domains of USF, and the rel domain of NF-kappa B all participated in cooperative nucleosome binding, illustrating that this effect is not restricted to a particular DNA-binding domain. Simultaneous binding by two factors increased the affinity of individual factors for nucleosomal DNA by up to 2 orders of magnitude. Importantly, cooperative binding resulted in efficient nucleosome binding by factors (USF and NF-kappa B) which independently possess little nucleosome-binding ability. The participation of GAL4 derivatives in cooperative nucleosome binding required only DNA-binding and dimerization domains, indicating that disruption of histone-DNA contacts by factor binding was responsible for the increased affinity of additional factors. Cooperative nucleosome binding required sequence-specific binding of all transcription factors, appeared to have spatial constraints, and was independent of the orientation of the binding sites on the nucleosome. These results indicate that cooperative nucleosome binding is a general mechanism that may play a significant role in loading complex enhancer and promoter elements with multiple diverse factors in chromatin and contribute to the generation of threshold responses and transcriptional synergy by multiple activator sites in vivo.

Base Sequence↗

Continuous B-epitope maps of cytochrome P450cam (CYP101) obtained by peptide scanning: correlation to spatial structure.

Protein continuous B-epitopes can be revealed using short synthetic peptides that overlap a known protein sequence. Since the whole protein surface is considered to possess antigenic properties, a question that arises is whether a set of linear B-epitopes determined by peptide scanning correlates with a protein spatial structure. We have chosen cytochrome P450cam (CYP101) of Pseudomonas putida, with known 3D structure, as a template. Sera of two rabbits and antibody egg yolk preparations from three chickens were produced against the P450cam molecule. These polyclonals were analyzed separately in ELISA with 409 overlapping P450cam hexapeptides. The whole set of continuous antigenic sites of P450cam covered about 45% of the P450cam sequence. However, immunodominant sites (those revealed with more than 50% antibody preparations), the so-called "antigenic core," represent only 9% of the protein sequence. While the amount of water-accessible residues in the total antigenic map (42%) was close to that in the whole native P450cam molecule (39%), the amount of water-accessible residues in the antigenic core was significantly higher (64%). These results led to the conclusion that antigenic core epitopes can be associated to the molecular surface, whereas epitopes with low detection frequency may partly correspond to unfolded regions of the protein molecule.

Animals↗

Heparin-binding domains in vascular biology.

Heparin is a major anticoagulant with activity mediated primarily through its interaction with antithrombin (AT). Heparan sulfate (HS), structurally related to heparin, binds a wide range of proteins of different functionality, taking part in various physiological and pathological processes. The heparin-AT complex, the most well understood facet of anticoagulation, serves as a prototypical example of the important role of heparin/HS in vascular biology. Extensive studies have identified common structural features in heparin/HS-binding sites of proteins. These include the elucidation of consensus sequences in proteins, patterns of clusters of basic and nonbasic residues, and common spatial arrangements of basic amino acids in the heparin-binding sites. Although these studies have provided valuable information, heparin/HS-binding proteins differ widely in structure. The prediction of heparin/HS-binding proteins from sequence information is not currently possible, and elucidation of protein-binding sites requires the individual study of each glycosaminoglycan-protein complex. Thus, x-ray crystallography and site-directed mutagenesis experiments are among the most powerful tools, providing accurate structural information, facilitating the characterization of heparin-protein complexes. Heparin and structurally related heparan sulfate bind a large number of proteins, taking part in a wide range of biological processes, particularly ones involved in vascular biology. Heparin-binding domains share certain common structural features, but there is no absolute dependency on specific sequences or protein folds.

Amino Acid Sequence↗

Direct least-squares estimation of spatiotemporal distributions from dynamic SPECT projections using a spatial segmentation and temporal B-splines.

Artifacts can result when reconstructing a dynamic image sequence from inconsistent, as well as insufficient and truncated, cone beam single photon emission computed tomography (SPECT) projection data acquired by a slowly rotating gantry. The artifacts can lead to biases in kinetic model parameters estimated from time-activity curves generated by overlaying volumes of interest on the images. However, the biases in time-activity curve estimates and subsequent kinetic parameter estimates can be reduced significantly by first modeling the spatial and temporal distribution of the radiopharmaceutical throughout the projected field of view, and then estimating the time-activity curves directly from the projections. This approach is potentially useful for clinical SPECT studies involving slowly rotating gantries, particularly those using a single-detector system or body contouring orbits with a multidetector system. We have implemented computationally efficient methods for fully four-dimensional (4-D) direct estimation of spatiotemporal distributions from dynamic SPECT projection data. Temporal B-splines providing various orders of temporal continuity, as well as various time samplings, were used to model the time-activity curves for segmented blood pool and tissue volumes in simulated cone beam and parallel beam cardiac data acquisitions. Least-squares estimates of time-activity curves were obtained quickly using a workstation. Given faithful spatial modeling, accurate curve estimates were obtained using cubic, quadratic, or linear B-splines and a relatively rapid time sampling during initial tracer uptake. From these curves, kinetic parameters were estimated accurately for noiseless data and with some bias for noisy data. A preliminary study of spatial segmentation errors showed that spatial model mismatch adversely affected quantitative accuracy, but also resulted in structured errors (projected model versus raw data) that were easily detected in our simulations. This suggests iterative refinement of the spatial model to reduce structured errors as an area of future research.

Algorithms↗

On the role of homologous sequences in chromosomal rearrangements.

Deletions and other chromosomal rearrangements can be generated by recombination between repeated sequences. It has been shown in a number of systems that the probability of exchange or gene conversion decreases with increasing distance between repeats. This paper examines the question of how repeats find each other, using deletion formation in bacteriophage T4 as a model system. Homologous sequences adjacent to the repeats can either stimulate or inhibit recombination, depending on their orientation. I present evidence that the spatial separation between repeats is the key determinant of the distance dependence and conclude that adjacent homologous sequences affect recombination by aligning chromosomes so as to position the recombining sites either closer together or farther apart. Analogous examples of apparent 'targeting' by homologous sequences in eukaryotes and other prokaryotes are noted.

Base Sequence↗

[Shadow optic. An endoscope with optimized ligth].

Modern endoscopes use a single mid-frontal illumination source that yields an unnatural flat image. At the University of Tübingen, in collaboration with Gimmi GmbH (Tuttlingen, Germany), a new concept endoscope has been developed with an additional light source: the shadow optic. With the aid of a secondary light source along the axis of the endoscope, a shadow is obtained which gives an impression of spatial depth. The aim of the study was to assess this new endoscope objectively, focusing on the speed and safety of the surgical act. The shadow optic was used in an experimental setting by 20 probands, each of whom performed five different standardized procedures of increasing difficulty five times, with and without shadow optic in randomized sequence, making a total of 1000 experiments. The procedures consisted in spatial orientation tests, structure-surrounding maneuvers, tissue clamping, needle puncture and suturing. The evaluation criteria were the time required to perform the procedures and the number of predefined errors as determined by electronic control. In each procedure, the time needed to perform the experiments was shorter with the shadow optic, reductions of up to 18% being recorded, and the number of errors was up to 54% lower compared to the experiments conducted with the traditional endoscope. The results of the study demonstrate that, as a result of the more natural spatial view conditions, the procedures were performed faster and with greater precision when the shadow optic was used. The endoscopic imaging with the shadow optic makes for better spatial orientation and a more natural and manageable image.

Endoscopes↗

Two cis elements collaborate to spatially repress transcription from a sea urchin promoter.

The expression pattern of many territory-specific genes in metazoan embryos is maintained by an active process of negative spatial regulation. However, the mechanism of this strategy of gene regulation is not well understood in any system. Here we show that reporter constructs containing regulatory sequence for the SM30-alpha gene of Stronglyocentrotus purpuratus are expressed in a pattern congruent with that of the endogenous SM30 gene(s), largely as a result of active transcriptional repression in cell lineages in which the gene is not normally expressed. Chloramphenicol acetyl transferase assays of deletion constructs from the 2600-bp upstream region showed that repressive elements were present in the region from -1628 to -300. In situ hybridization analysis showed that the spatial fidelity of expression was severely compromised when the region from -1628 to -300 was deleted. Two highly repetitive sequence motifs, (G/A/C)CCCCT and (T/C)(T/A/C)CTTTT(T/A/C), are present in the -1628 to -300 region. Representatives of these elements were analyzed by gel mobility shift experiments and were found to interact specifically with protein in crude nuclear extracts. When oligonucleotides containing either sequence element were co-injected with a correctly regulated reporter as potential competitors, the reporter was expressed in inappropriate cells. When composite oligonucleotides, containing both sequence elements, were fused to a misregulated reporter, the expression of the reporter in inappropriate cells was suppressed. Comparison of composite oligonucleotides with oligonucleotides containing single constituent elements show that both sequence elements are required for effective spatial regulation. Thus, both individual elements are required, but only a composite element containing both elements is sufficient to function as a tissue-specific repressive element.

Animals↗

VH1, a provascular cell-specific receptor kinase that influences leaf cell patterns in Arabidopsis.

The formation of the venation pattern in leaves is ideal for examining signaling pathways that recognize and respond to spatial and temporal information, because the pattern is two-dimensional and heritable and the resulting veins influence the three-dimensional spatial organization of the surrounding differentiating leaf cell types. We identified a provascular/procambial cell-specific gene that encodes a Leu-rich repeat receptor kinase, which we named VASCULAR HIGHWAY1 (VH1). A change in the expression domain and level of VH1 marks the transition from an uncommitted provascular state to a committed procambial state in early vascular development. The coding sequence, expression pattern, and transgenic phenotypes together suggest that VH1 transduces extracellular spatial and temporal signals into downstream cell differentiation responses in provascular/procambial cells.

Amino Acid Sequence↗

Slice-selective fat saturation in MR angiography using spatial-spectral selective prepulses.

Presaturation of fat signals by frequency-selective radiofrequency (RF) pulses is often applied in MR angiography to improve the visualization of the blood vessels. Unfortunately, standard fat saturation methods might cause a considerable reduction of the blood signal in the measured slices. This effect is caused by an attenuation of blood magnetization in remote tissue regions with water protons showing a similar Larmor frequency as the fat protons in the recorded slice. The affected blood water protons subsequently flow into the recorded slice and provide low signal intensity. Suitable spatial-spectral selective methods for slice-selective fat saturation were developed to avoid this unwanted effect. A spatial-spectral fat saturation technique was compared with a corresponding only spectrally selective approach. Both saturation techniques were included in a standard two-dimensional (2D) cine sequence and applied in angiographic examinations of the thighs. The results indicate that spatial-spectral saturation (acting slice selectively) leads to a clearly higher blood signal intensity in fat-suppressed MR angiography compared with standard techniques, especially in measurements performed during the systolic phase of the cardiac cycle.

Adipose Tissue↗

The CArG boxes in the promoter of the Arabidopsis floral organ identity gene APETALA3 mediate diverse regulatory effects.

APETALA3 is a MADS box gene required for normal development of the petals and stamens in the Arabidopsis flower. Studies in yeast, mammals and plants demonstrate that MADS domain transcription factors bind with high affinity to a consensus sequence called the CArG box. The APETALA3 promoter contains three close matches to the consensus CArG box sequence. To gain insights into the APETALA3 regulatory circuitry, we have analyzed the APETALA3 promoter using AP3::uidA(GUS) fusions. 496 base pairs of APETALA3 promoter sequence 5' to the transcriptional start directs GUS activity in the same temporal and spatial expression pattern as the APETALA3 RNA and protein in wild-type flowers. A synthetic promoter consisting of three tandem repeats of a 143 base pair sequence directs reporter gene activity exclusively to petals and stamens in the flower. We have analyzed the role of the CArG boxes by site-specific mutagenesis and find that the three CArG boxes mediate discrete regulatory effects. Mutations in CArG1 result in a decrease in reporter expression suggesting that CArG1 is the binding site for a positively acting factor or factors. Mutations in CArG2 result in a decrease in reporter expression in petals, but the expression pattern in stamens is unchanged. By contrast, mutations in CArG3 result in an increase in the level of reporter gene activity during early floral stages suggesting that CArG3 is the binding site for a negatively acting factor.

Arabidopsis↗

Structural and molecular determinants of intercellular coupling in cardiac myocytes.

Electrical activation of the heart requires intercellular transfer of current through gap junctions connecting individual cardiac myocytes. Using a combination of light and electron microscopic techniques and molecular approaches, we have characterized the number, size, and spatial distribution of intercellular connections at gap junctions in cardiac myocytes and have also cloned, sequenced, and elucidated the subcellular distribution of three physiologically distinct gap junction channel proteins. In this review, we present evidence to suggest that the spatial distribution of myocyte interconnections and the molecular composition of gap junction channels may confer distinct conduction properties on specific tissues of the mammalian heart such as atrial and ventricular myocardium, and the nodes and bundles of the cardiac conduction system.

Animals↗

Metapopulation dynamics and biological invasions: a spatially explicit model applied to a freshwater snail.

The spatial spread of invading organisms is a major contemporary concern. We focus here on invasions in inherently fragmented habitats, such as freshwater systems, and explore the usefulness of metapopulation models in this context. Maximum-likelihood methods allow the estimation of colonization and extinction rates, as functions of habitat patch sizes and positions, from time series of presence/absence data. This framework also provides confidence intervals of these estimates and hypotheses tests. We analyze a previously unpublished 12-year survey of the spread of the introduced snail Tarebia granifera in 47 Martinican rivers. Simple metapopulation models reproduce with reasonable accuracy several quantitative aspects of the invasion, including regional abundance, spatiotemporal structure, and site-by-site colonization dates. Sensitivity analysis reveals that the invasion sequence depended strongly on metapopulation size (number of sites) and spatial structure (distances among sites). The invasion history has also been accelerated by stochastic events, as illustrated by a large, central river that happened to be colonized very early and served as an invasion pool. Finally, we discuss the benefits of this approach for the understanding of invasions in fragmented landscapes.

Animals↗

The calmodulin binding domain of chicken smooth muscle myosin light chain kinase contains a pseudosubstrate sequence.

Smooth muscle myosin light chain kinase contains a 64 residue sequence that binds calmodulin in a Ca2+-dependent manner (Guerriero, V., Jr., Russo, M. A., and Means, A. R. (1987) Biochemistry, in press). Within this region is a sequence with homology to the corresponding sequence reported for the calmodulin binding region of skeletal muscle myosin light chain kinase (Blumenthal, D. K., Takio, K., Edelman, A. M., Charbonneau, H., Titani, L., Walsh, K. A., and Krebs, E. G. (1985) Proc. Natl. Acad. Sci. U.S.A. 82, 3187-3191). Inspection of these sequences reveals that they both share a similar number and spatial arrangement of basic residues with those present in the myosin light chain substrate. We have synthesized a 22-residue peptide corresponding to residues 480-501 (determined from the cDNA) of the smooth muscle myosin light chain kinase. This peptide, Ala-Lys-Lys-Leu-Ser-Lys-Asp-Arg-Met-Lys-Lys-Tyr-Met-Ala-Arg-Arg-Lys-Trp- Gln-Lys-Thr-Gly, inhibited calmodulin-dependent activation of the smooth muscle myosin light chain kinase with an IC50 of 46 nM. At saturating concentrations of calmodulin, the 22-residue peptide inhibited myosin light chain and synthetic peptide substrate phosphorylation competitively with IC50 values of 2.7 and 0.9 microM, respectively. An 11-residue synthetic peptide analog, corresponding to part of the calmodulin-binding sequence in skeletal muscle myosin light chain kinase, Lys-Arg-Arg-Trp-Lys-Lys-Asn-Phe-Ile-Ala-Val, also competitively inhibited synthetic peptide substrate phosphorylation with a Ki of 1 microM. The competitive inhibitory activity of the calmodulin binding regions is similar to the apparent Km of 2.7 microM for phosphorylation of the 23-residue peptide analog of the smooth muscle myosin light chain and raises the possibility that the calmodulin binding region of the myosin light chain kinase may act as a pseudosubstrate inhibitor of the enzyme.

Amino Acid Sequence↗

mRNA stability and localisation of the low-temperature-responsive barley gene family blt14.

Transcription and translation inhibitors have been used to investigate the role of mRNA stability in the low-temperature-regulated expression of the post-transcriptionally controlled low temperature responsive barley gene family, blt14. Genomic clones (blt14.1, blt14.2) representing additional members of the blt14 gene family have been isolated and sequenced. Gene specific probes have been used to analyse the spatial expression of each individual member of the blt14 gene family. Findings indicate that all of the genes are responsive to low temperature, but the organ distribution is different for each gene. The results indicate that blt14.0 mRNA is stabilised by a low-temperature-dependent protein factor. Taken together, the results suggest that organ-specific post-transcriptional mechanisms are important in the low-temperature regulation of blt14 gene expression.

Amino Acid Sequence↗

The two cysteine-rich head domains of minicollagen from Hydra nematocysts differ in their cystine framework and overall fold despite an identical cysteine sequence pattern.

Synthetic replicates of naturally occurring cysteine-rich peptides such as hormones, neurotransmitters, growth factors, enzyme inhibitors, defensins and toxins often can be oxidatively folded in high yields to their native structure in simple redox buffers. Thereby, identical cysteine patterns in the sequence were found to generate identical disulfide connectivities and homologous spatial structures despite significant variability in the non-cysteine positions. Minicollagen-1 from the nematocysts of Hydra is a trimeric protein that contains cysteine-rich domains at the N and C termini, which are involved in the assembly of an intermolecular disulfide network. Determination of the three-dimensional structures of peptides corresponding to the N-terminal and C-terminal domains by NMR spectroscopy revealed a remarkable exception from the general rule. Despite an identical cysteine pattern, the two domains of minicollagen-1 form different disulfide bridges and exhibit distinctly different folds, both of which are not found in the current structural databases. To our knowledge, this is the first case where two relatively short peptides with the abundant cysteine residues in identical sequence positions fold uniquely and with high yields into defined, but differing, structures. Therefore, the cysteine-rich domains of minicollagen constitute ideal model systems for studies of the interplay between folding and oxidation in proteins.

Amino Acid Sequence↗