MRS: a fast and compact retrieval system for biological data

The biological data explosion of the ‘omics’ era requires fast access to many data types in rapidly growing data banks. The MRS server allows for very rapid queries in a large number of flat-file data banks, such as EMBL, UniProt, OMIM, dbEST, PDB, KEGG, etc. This server combines a fast and reliable backend with a very user-friendly implementation of all the commonly used information retrieval facilities. The MRS server is freely accessible at . Moreover, the MRS software is freely available at for those interested in making their own data banks available via a web-based server

On the complexity of Engh and Huber refinement restraints: the angle τ as example

The angle τ (backbone N—Cα—C) is the most contested Engh and Huber refinement target parameter. It is shown that this parameter is ‘correct’ as a PDB-wide average, but can be improved by taking into account residue types, secondary structures and many other aspects of our knowledge of the biophysical relations between residue type and protein structure

In search of excellence - Innovation contests to foster innovation and entrepreneurship in Portugal

Innovation contests are one of numerous initiatives of different nature which have taken place in Portugal over the recent years aiming at raising consciousness of the importance and advantages of innovation and entrepreneurship. From sporadic and unpretentious events before 2000, the phenomenon gained unprecedented dimension and became a popular means that many different organizations use to uncovering novel business ideas and promoting innovation and entrepreneurship. Based on a large data base purposefully built for this research by the author, this paper characterizes the phenomenon of innovation contests in Portugal over the period 2000-2008. Findings show an increasing trend in the number of innovation contests launched annually in Portugal, high rates of rotation of the innovation contests launched annually and growing diversification of promoters

Conformational Transitions Accompanying Oligomerization of Yeast Alcohol Oxidase, a Peroxisomal Flavoenzyme

Alcohol oxidase (AO) is a homo-octameric flavoenzyme which catalyzes methanol oxidation in methylotrophic yeasts. AO protein is synthesized in the cytosol and subsequently sorted to peroxisomes where the active enzyme is formed. To gain further insight in the molecular mechanisms involved in AO activation, we studied spectroscopically native AO from Hansenula polymorpha and Pichia pastoris and three putative assembly intermediates. Fluorescence studies revealed that both Trp and FAD are suitable intramolecular markers of the conformation and oligomeric state of AO. A direct relationship between dissociation of AO octamers and increase in Trp fluorescence quantum yield and average fluorescence lifetime was found. The time-resolved fluorescence of the FAD cofactor showed a rapid decay component which reflects dynamic quenching due to the presence of aromatic amino acids in the FAD-binding pocket. The analysis of FAD fluorescence lifetime profiles showed a remarkable resemblance of pattern for purified AO and AO present in intact yeast cells. Native AO contains a high content of ordered secondary structure which was reduced upon FAD-removal. Dissociation of octamers into monomers resulted in a conversion of β-sheets into α-helices. Our results are explained in relation to a 3D model of AO, which was built based on the crystallographic data of the homologous enzyme glucose oxidase from Aspergillus niger. The implications of our results for the current model of the in vivo AO assembly pathway are discussed.

Molecular interactions during the assembly of cowpea chlorotic mottle virus studied by magnetic resonance

This thesis describes the application of 1 H- and 13 C- NMR, EPR, ST-EPR and calculational methods to study cowpea chlorotic mottle virus. This virus consists of RNA encapsidated by 180 identical protein subunits, arranged icosahedrally. The isolated coat protein of cowpea chlorotic mottle virus can be brougth into several well defined states of aggregation. This study could be carried out, because these stages can be produced in quantities sufficient to allow magnetic resonance measurements. All the results obtained are combined in the following model for the assembly proces of the virus:In this model a rigid protein core with a highly mobile, basic, N- terminal arm is invoked. This arm is the RNA binding part of the protein. The high mobility of this arm enhances the probability of interaction with the RNA and enables the protein to exhibit different modes of bonding to different local structures of the RNA. Upon binding the RNA, the N-terminal arm adopts a rigid a-helical conformation.In chapter two it is shown that virtually no mobility on a timescale faster than 10 -7 s can be observed in the virus.In chapter three an EPR and ST-EPR study is presented on spin-labelled virus, From the results it is concluded that no anisotropic subunit mobility is present in the virus on the 10 -5 -10 -7 s timescale. Also in this chapter it is suggested that many ST-EPR results on maleimide- labelled proteins, in which anisotropic protein mobility was invoked for the interpretation of the spectra, are based on artefacts.Chapter four shows that the N-terminal arm is the RNA binding part of the protein. This arm is found to be very mobile in the absence of RNA, whereas immobilization occurs upon binding RNA.Chapter five is a small excursion to other plant viruses. It is shown that brome mosaic virus and belladonna mottle virus show the same behaviour as cowpea chlorotic mottle virus, whereas cowpea mosaic virus behaves completely different.Chapter six deals with the molecular interactions during the assembly process. In this chapter it is shown that the arginine and lysine containing part of the N-terminal arm is responsible for the binding of oligo-nucleotides. It is suggested that a double stranded nucleotide conformation is required for proper interaction.In chapter 7 a secondary structure prediction of the coat protein of CCMV is presented. The results indicate that the N-terminal arm shows no structural preference when the positive charges of arginine and lysine are retained, whereas in the absence of these positive charges there is a tendency towards α-helix formation. Also it is concluded that cowpea chlorotic mottle virus possesses the same β-role topology as southern bean mosaic virus, sattelite tobacco necrosis virus and tomato bushy stunt virus.Chapter eight presents the results of an energy calculation study on the N-terminal arm. Although the method used contains too many approximations to allow detailed conclusions, the results of chapter seven are fully confirmed by these calculations.In the model for the protein-RNA interaction in CCMV which is presented in chapter five, a random coil conformation for the N-terminal protein arm is invoked. In chapter nine an extension of this part of the model is presented. It is shown that the N-terminal possesses some time-averaged secondary structure (presumably an α-helix) in the absence of RNA. A conformational exchange process is observed in the N-terminal arm, in which about half 'of the residues participate. In empty capsids not all the N-terminal arms turn out to be mobile. 20-55% of the N-terminal arms is immobile on the NMR timescale in the empty capsids of the coat protein of CCMV.<p/