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Enrique Querol

Publications and source records attributed to Enrique Querol.

27 records · Page 2Linked to original sources
PubMed2003

Ligand screening by exoproteolysis and mass spectrometry in combination with computer modelling.

Here, we present a new approach for protein ligand screening based on the use of limited exoproteolysis coupled to MALDI-TOF mass spectrometry, combined with computational modelling and prediction of binding energies. As a test for this combined approach, we have screened a combinatorial library containing 8000 peptides (organized in 60 peptide samples) based on positional scanning format. This library is attached to a poly-Pro framework, and screened against the Abl-SH3 domain. The results obtained demonstrated the validity of the experimental and theoretical approaches in identifying better ligands and in rationalizing the changes in affinity. Exoproteolysis coupled to MALDI-TOF mass spectrometry could be used to screen complex libraries in a fast and efficient way.

Algorithms↗
PubMed2003

Identification of protein ligands in complex biological samples using intensity-fading MALDI-TOF mass spectrometry.

The easy detection of biomolecular interactions in complex mixtures using a minimum amount of material is of prime interest in molecular and cellular biology research. In this work, a mass spectrometry MALDI-TOF based approach, which we call intensity-fading (IF MALDI-TOFMS), and which was designed for just such a purpose, is reported. This methodology is based on the use of the MALDI ion intensities to detect quickly the formation of complexes between nonimmobilized biomolecules in which a protein is one of the partners (protein-protein, protein-peptide, protein-organic molecule, and protein-nucleic acid complexes). The complex is detected through the decrease (fading) of the molecular ion intensities of the partners as directly compared to the MALDI mass spectrum of the mixture (problem and control molecules) following the addition of the target molecule. The potential of the approach is examined in several examples of model interactions, mainly involving small nonprotein and protein inhibitors of proteases, at both the qualitative and semiquantitative levels. Using this method, different protein ligands of proteolytic enzymes in total extracts of invertebrate organisms have been identified in a simple way. The proposed procedure should be easily applied to the high-throughput screening of biomolecules, opening a new experimental strategy in functional proteomics.

Animals↗
PubMed2003

Analysis of the effect of potato carboxypeptidase inhibitor pro-sequence on the folding of the mature protein.

Protein folding can be modulated in vivo by many factors. While chaperones act as folding catalysts and show broad substrate specificity, some pro-peptides specifically facilitate the folding of the mature protein to which they are bound. Potato carboxypeptidase inhibitor (PCI), a 39-residue protein carboxypeptidase inhibitor, is synthesized in vivo as a precursor protein that includes a 27-residue N-terminal and a seven-residue C-terminal pro-regions. In this work the disulfide-coupled folding of mature PCI in vitro has been compared with that of the same protein extended with either the N-terminal pro-sequence (ProNtPCI) or both N- and C-terminal pro-sequences (ProPCI), and also with the N-terminal pro-sequence in trans (ProNt + PCI). No significant differences can be observed in the folding kinetics or efficiencies of all these molecules. In addition, in vivo folding studies in Escherichia coli have been performed using wild-type PCI and three PCI mutant forms with and without the N-terminal pro-sequence, the mutations had been previously reported to affect folding of the PCI mature form. The extent to which the 'native-like' form was secreted to the media by each construction was not affected by the presence of the N-terminal pro-sequence. These results indicate that PCI does not depend on the N-terminal pro-sequence for its folding in both, in vitro and in vivo in E. coli. However, structural analysis by spectroscopy, hydrogen exchange and limited proteolysis by mass spectrometry, indicate the capability of such N-terminal pro-sequence to fold within the precursor form.

Amino Acid Sequence↗
PubMed2003

A simple electrostatic criterion for predicting the thermal stability of proteins.

The enhancement of protein thermostability is an important issue for both basic science and biotechnology purposes. We have developed a thermostability criterion for a protein in terms of a quasi-electric dipole moment (contributed by its charged residues) defined for an electric charge distribution whose total charge is not zero. It was found that minimization of the modulus of this dipole moment increased its thermal stability, as demonstrated by surveying these values in pairs of mesostable-thermostable homologous proteins and in mutations described in the literature. The analysis of these observations provides criteria for thermostabilization of a protein, by computing its dipole profile. This profile is obtained by direct substitution of each amino acid of the sequence by either a positive, negative or neutral amino acid, followed by a recalculation of the dipole moment. As an experimental example, these criteria were applied to a beta-glucanase to enhance its thermal stability.

Amino Acid Substitution↗
PubMed2002

Protein secondary structure and stability determined by combining exoproteolysis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

In the post-genomic era, several projects focused on the massive experimental resolution of the three-dimensional structures of all the proteins of different organisms have been initiated. Simultaneously, significant progress has been made in the ab initio prediction of protein three-dimensional structure. One of the keys to the success of such a prediction is the use of local information (i.e. secondary structure). Here we describe a new limited proteolysis methodology, based on the use of unspecific exoproteases coupled with matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), to map quickly secondary structure elements of a protein from both ends, the N- and C-termini. We show that the proteolytic patterns (mass spectra series) obtained can be interpreted in the light of the conformation and local stability of the analyzed proteins, a direct correlation being observed between the predicted and the experimentally derived protein secondary structure. Further, this methodology can be easily applied to check rapidly the folding state of a protein and characterize mutational effects on protein conformation and stability. Moreover, given global stability information, this methodology allows one to locate the protein regions of increased or decreased conformational stability. All of this can be done with a small fraction of the amount of protein required by most of the other methods for conformational analysis. Thus limited exoproteolysis, together with MALDI-TOF MS, can be a useful tool to achieve quickly the elucidation of protein structure and stability.

Carboxypeptidases↗
PubMed2002

Human betacellulin structure modeled from other members of EGF family.

We have modeled betacellulin (BTC) to gain insight into the structural elements that can explain its properties. The epidermal growth factor (EGF) signal transduction pathway, a significant mediator of several cell functions, is based on four closely related tyrosine kinase receptors. The ErbB receptors are transmembrane glycoproteins and signal transduction is initiated by ligand binding that induces receptor homo- or heterodimerization to form a complex containing two molecules of ligand and two molecules of receptor. The EGF family of ligands can be divided into three groups based on their ability to bind and activate distinct ErbB receptor homo- and heterodimers. Each member of the group formed by BTC, heparin binding EGF (HB-EGF) and epiregulin (EP) can interact with both the EGF receptor (EGFR) and heregulin receptors (ErbB-3 and ErbB-4), and are hence called "bispecific" ligands. BTC and EP also present the distinctive feature that they activate all possible heterodimeric ErbB receptors. BTC has been modeled with the program MODELLER, using human EGF, human transforming growth factor alpha (hTGFalpha), human HB-EGF and human heregulin one alpha (hHRG-1alpha) as templates. The structure of the model as well as that of the templates were optimized and a simulation of 100 ps was run for all. The main structural properties of the model and the templates were compared and in conclusion the hBTC conformation was closely similar to that of hTGFalpha.

Amino Acid Sequence↗
PubMed2002

TranScout: prediction of gene expression regulatory proteins from their sequences.

MOTIVATION: The advent of genomics yields thousands of reading frames in search of function. Identification of conserved functional motifs in protein sequences can be helpful for function prediction. RESULTS: A database and a classification of reported DNA-binding protein motifs has been designed. A program ('TranScout') has been developed for the detection and evaluation of conserved motifs in prokaryotic and eukaryotic sequences of proteins with a gene regulatory function. The efficiency of the program is shown in a benchmark against a database obtained from SWISS-PROT without the protein sequences used to train the program. All motifs were detected with a mean average sensitivity of 0.98 and a mean average specificity of 0.92. AVAILABILITY: The program is freely available for use on the internet at http://luz.uab.es/transcout/. The user can find additional information at this site.

Algorithms↗
PubMed2002

Structural similarity to link sequence space: new potential superfamilies and implications for structural genomics.

The current pace of structural biology now means that protein three-dimensional structure can be known before protein function, making methods for assigning homology via structure comparison of growing importance. Previous research has suggested that sequence similarity after structure-based alignment is one of the best discriminators of homology and often functional similarity. Here, we exploit this observation, together with a merger of protein structure and sequence databases, to predict distant homologous relationships. We use the Structural Classification of Proteins (SCOP) database to link sequence alignments from the SMART and Pfam databases. We thus provide new alignments that could not be constructed easily in the absence of known three-dimensional structures. We then extend the method of Murzin (1993b) to assign statistical significance to sequence identities found after structural alignment and thus suggest the best link between diverse sequence families. We find that several distantly related protein sequence families can be linked with confidence, showing the approach to be a means for inferring homologous relationships and thus possible functions when proteins are of known structure but of unknown function. The analysis also finds several new potential superfamilies, where inspection of the associated alignments and superimpositions reveals conservation of unusual structural features or co-location of conserved amino acids and bound substrates. We discuss implications for Structural Genomics initiatives and for improvements to sequence comparison methods.

Amino Acid Sequence↗