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Francisco Conejero-Lara

Bio: Francisco Conejero-Lara is an academic researcher from University of Granada. The author has contributed to research in topics: Protein folding & Heptad repeat. The author has an hindex of 19, co-authored 60 publications receiving 1128 citations. Previous affiliations of Francisco Conejero-Lara include Spanish National Research Council & University of Oxford.


Papers
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Journal ArticleDOI
TL;DR: The results suggest that the influence of environmental variables on protein solvation is crucial in determining the nucleation kinetics, the pathway of assembly, and the final fibril morphology.

107 citations

Journal ArticleDOI
TL;DR: Equilibrium sedimentation studies show that malate, the chemoattractant that causes the strongest chemotactic response, stabilizes the dimeric state of McpS-LBD, the only chemoreceptor of TCA cycle intermediates in the strain under study.

84 citations

Journal ArticleDOI
TL;DR: The results demonstrate that under native conditions the SH3 domain needs to be considered as an ensemble of conformations and that the hydrogen exchange data obtained under those conditions cannot be interpreted by a two-state equilibrium.
Abstract: The folding/unfolding equilibrium of the α-spectrin SH3 domain has been measured by NMR-detected hydrogen/deuterium exchange and by differential scanning calorimetry. Protection factors against exchange have been obtained under native conditions for more than half of the residues in the domain. Most protected residues are located at the β-strands, the short 310 helix, and part of the long RT loop, whereas the loops connecting secondary structure elements show no measurable protection. Apparent stability constants per residue and their corresponding Gibbs energies have been calculated from the exchange experiments. The most stable region of the SH3 domain is defined by the central portions of the β-strands. The peptide binding region, on the other hand, is composed of a highly stable region (residues 53−57) and a highly unstable region, the loop between residues 34−41 (n-Src loop). All residues in the domain have apparent Gibbs energies lower than the global unfolding Gibbs energy measured by differential ...

74 citations

Journal ArticleDOI
TL;DR: This work analyzed the thermal melting of the amyloid fibrils of the N47A mutant of the alpha-spectrin SH3 domain by differential scanning calorimetry (DSC) and found that with the use of appropriate models of analysis DSC has an extraordinary potential to analyze the thermodynamic determinants of amyloids fibril stability.
Abstract: In contrast to the thermal unfolding of native proteins, very few studies of the thermally induced melting of amyloid fibrils have been reported to date due to the complex nature of these protein aggregates and the lack of theoretical formalisms to rationalize the data. In this work, we analyzed the thermal melting of the amyloid fibrils of the N47A mutant of the alpha-spectrin SH3 domain by differential scanning calorimetry (DSC). The thermal melting of the isolated fibrils occurred in single endothermic transitions, yielding the fully unfolded protein. The enthalpy and heat capacity changes of fibril melting were significantly lower than those of the unfolding of the native protein, indicating a lower density of interactions and a higher solvent-exposed surface area for the protein within the fibrils relative to the native state. In addition, these magnitudes did not change significantly between fibrils showing different morphology. The independence of the transitions with the scan rate and the observation of a considerable mass-action-like effect upon the melting temperatures indicated that the fibril melting is not separated significantly from equilibrium and could be considered in good approximation as a reversible process. A simple equilibrium model of polymerization coupled to monomer unfolding allowed us for the first time to interpret quantitatively the thermal melting of amyloid fibrils. The model captured very well the general features of the thermal behavior of amyloid fibrils and allowed us to estimate the partitioning of the energy of overall melting into the unfolding of monomers and fibril elongation. We conclude that with the use of appropriate models of analysis DSC has an extraordinary potential to analyze the thermodynamic determinants of amyloid fibril stability.

73 citations

Journal ArticleDOI
TL;DR: Results indicate that each structural domain of SK behaves as a single cooperative unfolding unit under equilibrium conditions, and that in the aggregates the N-terminal segment 1-63 and the whole of SK domain C are at least partially structured, while domain B is highly unstructured.
Abstract: The thermal denaturation of streptokinase from Streptococcus equisimilis (SK) together with that of a set of fragments encompassing each of its three domains has been investigated using differential scanning calorimetry (DSC). Analysis of the effects of pH, sample concentration and heating rates on the DSC thermograms has allowed us to find conditions where thermal unfolding occurs unequivocally under equilibrium. Under these conditions, pH 7.0 and a sample concentration of less than approximately 1.5 mg x mL(-1), or pH 8.0, the heat capacity curves of intact SK can be quantitatively described by three independent two-state transitions, each of which compares well with the two-state transition observed for the corresponding isolated SK domain. The results indicate that each structural domain of SK behaves as a single cooperative unfolding unit under equilibrium conditions. At pH 7.0 and high sample concentration, or at pH 6.0 at any concentration investigated, the thermal unfolding of domain A was accompanied by the time-dependent formation of aggregates of SK. This produces a severe deformation of the DSC curves, which become concentration dependent and kinetically controlled, and thus precludes their proper analysis by standard deconvolution methods. A simple model involving time-dependent, high-order aggregation may account for the observed effects. Limited-proteolysis experiments suggest that in the aggregates the N-terminal segment 1-63 and the whole of SK domain C are at least partially structured, while domain B is highly unstructured. Unfolding of domain A, under conditions where the N-terminal segment 1-63 has a high propensity for beta sheet structure and a partially formed hydrophobic core, gives rise to rapid aggregation. It is likely that this region is able to act as a nucleus for the aggregation of the full-length protein.

63 citations


Cited by
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01 May 2005

2,648 citations

Journal ArticleDOI
TL;DR: The purpose of the current review is to provide a fundamental understanding of the mechanisms by which proteins aggregate and by which varying solution conditions, such as temperature, pH, salt type, salt concentration, cosolutes, preservatives, and surfactants, affect this process.
Abstract: Irreversible protein aggregation is problematic in the biotechnology industry, where aggregation is encountered throughout the lifetime of a therapeutic protein, including during refolding, purification, sterilization, shipping, and storage processes. The purpose of the current review is to provide a fundamental understanding of the mechanisms by which proteins aggregate and by which varying solution conditions, such as temperature, pH, salt type, salt concentration, cosolutes, preservatives, and surfactants, affect this process.

1,359 citations

Journal ArticleDOI
TL;DR: This review deals with the molecular physiology of spectrin, ankyrin, which links spectrin to the anion exchanger, and two spectrin-associated proteins that promote spectrin interactions with actin: adducin and protein 4.1.
Abstract: The spectrin-based membrane skeleton of the humble mammalian erythrocyte has provided biologists with a set of interacting proteins with diverse roles in organization and survival of cells in metazoan organisms. This review deals with the molecular physiology of spectrin, ankyrin, which links spectrin to the anion exchanger, and two spectrin-associated proteins that promote spectrin interactions with actin: adducin and protein 4.1. The lack of essential functions for these proteins in generic cells grown in culture and the absence of their genes in the yeast genome have, until recently, limited advances in understanding their roles outside of erythrocytes. However, completion of the genomes of simple metazoans and application of homologous recombination in mice now are providing the first glimpses of the full scope of physiological roles for spectrin, ankyrin, and their associated proteins. These functions now include targeting of ion channels and cell adhesion molecules to specialized compartments within the plasma membrane and endoplasmic reticulum of striated muscle and the nervous system, mechanical stabilization at the tissue level based on transcellular protein assemblies, participation in epithelial morphogenesis, and orientation of mitotic spindles in asymmetric cell divisions. These studies, in addition to stretching the erythrocyte paradigm beyond recognition, also are revealing novel cellular pathways essential for metazoan life. Examples are ankyrin-dependent targeting of proteins to excitable membrane domains in the plasma membrane and the Ca(2+) homeostasis compartment of the endoplasmic reticulum. Exciting questions for the future relate to the molecular basis for these pathways and their roles in a clinical context, either as the basis for disease or more positively as therapeutic targets.

942 citations

Journal ArticleDOI
TL;DR: The algorithm is shown to identify a series of protein fragments involved in the aggregation of disease-related proteins and to predict the effect of genetic mutations on their deposition propensities, which shall facilitate the identification of possible therapeutic targets for anti-depositional strategies in conformational diseases.
Abstract: Protein aggregation correlates with the development of several debilitating human disorders of growing incidence, such as Alzheimer's and Parkinson's diseases. On the biotechnological side, protein production is often hampered by the accumulation of recombinant proteins into aggregates. Thus, the development of methods to anticipate the aggregation properties of polypeptides is receiving increasing attention. AGGRESCAN is a web-based software for the prediction of aggregation-prone segments in protein sequences, the analysis of the effect of mutations on protein aggregation propensities and the comparison of the aggregation properties of different proteins or protein sets. AGGRESCAN is based on an aggregation-propensity scale for natural amino acids derived from in vivo experiments and on the assumption that short and specific sequence stretches modulate protein aggregation. The algorithm is shown to identify a series of protein fragments involved in the aggregation of disease-related proteins and to predict the effect of genetic mutations on their deposition propensities. It also provides new insights into the differential aggregation properties displayed by globular proteins, natively unfolded polypeptides, amyloidogenic proteins and proteins found in bacterial inclusion bodies. By identifying aggregation-prone segments in proteins, AGGRESCAN http://bioinf.uab.es/aggrescan/ shall facilitate (i) the identification of possible therapeutic targets for anti-depositional strategies in conformational diseases and (ii) the anticipation of aggregation phenomena during storage or recombinant production of bioactive polypeptides or polypeptide sets.

844 citations

Journal ArticleDOI
TL;DR: Although ITC is particularly suitable to follow the energetics of an association reaction between biomolecules, the combination of ITC and DSC provides a more comprehensive description of the thermodynamics of an associating system.
Abstract: The principles of isothermal titration calorimetry (ITC) and differential scanning calorimetry (DSC) are reviewed together with the basic thermodynamic formalism on which the two techniques are based. Although ITC is particularly suitable to follow the energetics of an association reaction between biomolecules, the combination of ITC and DSC provides a more comprehensive description of the thermodynamics of an associating system. The reason is that the parameters DeltaG, DeltaH, DeltaS, and DeltaCp obtained from ITC are global properties of the system under study. They may be composed to varying degrees of contributions from the binding reaction proper, from conformational changes of the component molecules during association, and from changes in molecule/solvent interactions and in the state of protonation.

739 citations