Goran Neshich

STING Millennium: a web-based suite of programs for comprehensive and simultaneous analysis of protein structure and sequence

STING Millennium Suite (SMS) is a new web-based suite of programs and databases providing visualization and a complex analysis of molecular sequence and structure for the data deposited at the Protein Data Bank (PDB). SMS operates with a collection of both publicly available data (PDB, HSSP, Prosite) and its own data (contacts, interface contacts, surface accessibility). Biologists find SMS useful because it provides a variety of algorithms and validated data, wrapped-up in a user friendly web interface. Using SMS it is now possible to analyze sequence to structure relationships, the quality of the structure, nature and volume of atomic contacts of intra and inter chain type, relative conservation of amino acids at the specific sequence position based on multiple sequence alignment, indications of folding essential residue (FER) based on the relationship of the residue conservation to the intra-chain contacts and Calpha-Calpha and Cbeta-Cbeta distance geometry. Specific emphasis in SMS is given to interface forming residues (IFR)-amino acids that define the interactive portion of the protein surfaces. SMS may simultaneously display and analyze previously superimposed structures. PDB updates trigger SMS updates in a synchronized fashion. SMS is freely accessible for public data at http://www.cbi.cnptia.embrapa.br, http://mirrors.rcsb.org/SMS and http://trantor.bioc.columbia.edu/SMS.

Protein cutoff scanning: A comparative analysis of cutoff dependent and cutoff free methods for prospecting contacts in proteins

In this study, we carried out a comparative analysis between two classical methodologies to prospect residue contacts in proteins: the traditional cutoff dependent (CD) approach and cutoff free Delaunay tessellation (DT). In addition, two alternative coarse‐grained forms to represent residues were tested: using alpha carbon (CA) and side chain geometric center (GC). A database was built, comprising three top classes: all alpha, all beta, and alpha/beta. We found that the cutoff value at about 7.0 Å emerges as an important distance parameter. Up to 7.0 Å, CD and DT properties are unified, which implies that at this distance all contacts are complete and legitimate (not occluded). We also have shown that DT has an intrinsic missing edges problem when mapping the first layer of neighbors. In proteins, it may produce systematic errors affecting mainly the contact network in beta chains with CA. The almost‐Delaunay (AD) approach has been proposed to solve this DT problem. We found that even AD may not be an advantageous solution. As a consequence, in the strict range up to 7.0 Å, the CD approach revealed to be a simpler, more complete, and reliable technique than DT or AD. Finally, we have shown that coarse‐grained residue representations may introduce bias in the analysis of neighbors in cutoffs up to 6.8 Å, with CA favoring alpha proteins and GC favoring beta proteins. This provides an additional argument pointing to the value of 7.0 Å as an important lower bound cutoff to be used in contact analysis of proteins. Proteins 2009.

The Diamond STING server

Diamond STING is a new version of the STING suite of programs for a comprehensive analysis of a relationship between protein sequence, structure, function and stability. We have added a number of new functionalities by both providing more structure parameters to the STING Database and by improving/expanding the interface for enhanced data handling. The integration among the STING components has also been improved. A new key feature is the ability of the STING server to handle local files containing protein structures (either modeled or not yet deposited to the Protein Data Bank) so that they can be used by the principal STING components: JavaProtein Dossier (JPD) and STING Report. The current capabilities of the new STING version and a couple of biologically relevant applications are described here. We have provided an example where Diamond STING identifies the active site amino acids and folding essential amino acids (both previously determined by experiments) by filtering out all but those residues by selecting the numerical values/ranges for a set of corresponding parameters. This is the fundamental step toward a more interesting endeavor—the prediction of such residues. Diamond STING is freely accessible at and .

ACGT and vicilin core sequences in a promoter domain required for seed-specific expression of a 2S storage protein gene are recognized by the opaque-2 regulatory protein

The expression of Brazil nut storage albumin genes is highly regulated during seed development. Several sequences in the promoter of one of these genes show homologies with the target sites of the maize O2 bZIP regulatory protein. We therefore asked whether the O2 protein would recognize these promoter sequences. We show that the O2 protein binds to three different sequences (F1, F2 and F3). F1 and F3 are hybrid C/G and A/G boxes, respectively, that are homologous to the O2-binding site of a maize α-zein gene. F2 is a new O2-binding sequence related to the O2 target sites of the Coix α-coxin, the maize b-32 genes and the AP-1 pseudopalindrome. Molecular modelling showed that an Asn and a Ser in the 02 DNA binding domain make different base-specific contacts with each operator. 5′ Promoter deletions of the be2S1 gene showed that the domain containing the O2 target sites F1 and F2 is required for detectable reporter gene expression in transgenic tobacco seeds. Moreover, the homologous coix O2 protein was shown to in situ transactivate the promoter region encompassing the three O2-binding sites F1, F2 and F3. Thus, these sites may be in vivo regulatory sequences mediating activation by bZIP regulatory proteins.

Analysis of the black-eyed pea trypsin and chymotrypsin inhibitor-α-chymotrypsin complex

The black‐eyed pea trypsin and chymotrypsin inhibitor (BTCI) is a member of the Bowman‐Birk protease inhibitor (BBI) family. The three‐dimensional model of the BTCI‐chymotrypsin complex was built based on the homology to Bowman‐Birk inhibitors with known structures. An extensive theoretical and experimental study of these known structures has been performed. The model confirms the ideas about Bowman‐Birk inhibitor structure‐function relations and agrees well with our experimental data (circular dichroism, IR and light scattering). The electrostatic potentials at the enzyme‐inhibitor contact surface reveal a pattern of complementary electrostatic potentials from which mutations can be inferred that could give these inhibitors an altered specificity.

A corm-specific gene encodes tarin, a major globulin of taro (Colocasia esculenta L. Schott)

A gene encoding a globulin from a major taro (Colocasia esculenta L. Schott) corm protein family, tarin (G1, ca. 28 kDa) was isolated from a λ Charon 35 library, using a cDNA derived from a highly abundant corm-specific mRNA, as probe. The gene, named tar1, and the corresponding cDNA were characterized and compared. No introns were found. The major transcription start site was determined by primer extension analysis. The gene has an open reading frame (ORF) of 765 bp, and the deduced amino acid sequence indicated a precursor polypeptide of 255 residues that is post-translationally processed into two subunits of about 12.5 kDa each. The deduced protein is 45% homologous to curculin, a sweet-tasting protein found in the fruit pulp of Curculigo latifolia and 40% homologous to a mannose-binding lectin from Galanthus nivalis. Significant similarity was also found at the nucleic acid sequence level with genes encoding lectins from plant species of the Amaryllidaceae and Lilliaceae families.

Modified 2S albumins with improved tryptophan content are correctly expressed in transgenic tobacco plants

Brazil nut 2S albumins lack the essential amino acid tryptophan. In order to improve the proteins nutritional value and create a basis for structural investigations, three separate modified Brazil nut 2S albumin genes were constructed. The first mutant contains five consecutive tryptophan codons, while the other two modified genes encode proteins carrying single tryptophan residues at sites that will allow confirmation of the predicted protein structure through fluorescence quenching techniques. The modified genes, under the regulation of the CaMV 35S promoter, were introduced into Nicotiana tabacum. All three modified genes were correctly transcribed and the 2S albumin accumulated in the seeds of transgenic plants.

Structural basis of the lisinopril-binding specificity in N- and C-domains of human somatic ACE.

Angiotensin I-converting enzyme (ACE) is a dipeptidyl carboxypeptidase which converts angiotensin I into the vasopressor peptide angiotensin II and also inactivates the hypotensive peptide bradykinin, playing an important role in blood pressure regulation. The present work describes the molecular modeling of the N-terminal human somatic ACE in complex with the inhibitor lisinopril, identifying the residues involved in the inhibitor-binding pocket. The obtained results identify differences in the lisinopril lysine moiety-binding residues for N- and C-terminals of sACE domains and an important carboxy-terminal proline hydrophobic accommodations mediated by the aromatic ring of Tyr532 and Tyr1128 residues, respectively. The present model will be useful for the development of a new inhibitor family based on the natural BPP peptides and derivatives, or even to improve the binding capacities and the domain specificity of the already known inhibitors.

STING Contacts: a web-based application for identification and analysis of amino acid contacts within protein structure and across protein interfaces

UNLABELLED Amino acid contacts in terms of atomic interactions are essential factors to be considered in the analysis of the structure of a protein and its complexes. Consequently, molecular biologists do require specific tools for the identification and visualization of all such contacts. Graphical contacts (GC) and interface forming residue graphical contacts (IFRgc) presented here, calculate atomic contacts among amino acids based on a table of predefined pairs of the atom types and their distances, and then display them using number of different forms. The inventory of currently listed contact types by GC and IFRgc include hydrogen bonds (in nine different flavors), hydrophobic interactions, charge-charge interactions, aromatic stacking and disulfide bonds. Such extensive catalog of the interactions, representing the forces that govern protein folding, stability and binding, is the key feature of these two applications. GC and IFRgc are part of STING Millennium Suite. AVAILABILITY http://sms.cbi.cnptia.embrapa.br/SMS, http://trantor.bioc.columbia.edu/SMS, http://mirrors.rcsb.org//SMS, http://www.es.embnet.org/SMS and http://www.ar.embnet.org/SMS (Options: Graphical Contacts and IFR Graphical Contacts).

STING Report: convenient web-based application for graphic and tabular presentations of protein sequence, structure and function descriptors from the STING database

The Sting Report is a versatile web-based application for extraction and presentation of detailed information about any individual amino acid of a protein structure stored in the STING Database. The extracted information is presented as a series of GIF images and tables, containing the values of up to 125 sequence/structure/function descriptors/parameters. The GIF images are generated by the Gold STING modules. The HTML page resulting from the STING Report query can be printed and, most importantly, it can be composed and visualized on a computer platform with an elementary configuration. Using the STING Report, a user can generate a collection of customized reports for amino acids of specific interest. Such a collection comes as an ideal match for a demand for the rapid and detailed consultation and documentation of data about structure/function. The inclusion of information generated with STING Report in a research report or even a textbook, allows for the increased density of its contents. STING Report is freely accessible within the Gold STING Suite at http://www.cbi.cnptia.embrapa.br, http://www.es.embnet.org/SMS/, http://gibk26.bse.kyutech.ac.jp/SMS/ and http://trantor.bioc.columbia.edu/SMS (option: STING Report).