Browsing by Subject "Peptides and proteins"
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- PublicationEmbargoContribution of ion binding affinity to ion selectivity and permeation in KcsA, a model potassium channel(2012-04-17) Renart Pérez, María Lourdes; Montoya Díaz, Estefanía; Fernández Carvajal, Asia María; Molina Gallego, María Luisa; Poveda Larrosa, José Antonio; Encinar Hidalgo, José Antonio; Ayala Torres, Antonio Vicente; Gómez Pérez, Francisco Javier; Morales Calderón, Andrés; González Ros, José Manuel; Bioquímica y Biología Molecular B e InmunologíaIon permeation and selectivity, key features in ion channel function, are believed to arise from a complex ensemble of energetic and kinetic variables. Here we evaluate the contribution of pore cation binding to ion permeation and selectivity features of KcsA, a model potassium channel. For this, we used E71A and M96V KcsA mutants in which the equilibrium between conductive and nonconductive conformations of the channel is differently shifted. E71A KcsA is a noninactivating channel mutant. Binding of K+ to this mutant reveals a single set of low-affinity K+ binding sites, similar to that seen in the binding of K+ to wild-type KcsA that produces a conductive, low-affinity complex. This seems consistent with the observed K+ permeation in E71A. Nonetheless, the E71A mutant retains K+ selectivity, which cannot be explained on the basis of just its low affinity for this ion. At variance, M96V KcsA is a rapidly inactivating mutant that has lost selectivity for K+ and also conducts Na+. Here, low-affinity binding and high-affinity binding of both cations are detected, seemingly in agreement with both being permeating species in this mutant channel. In conclusion, binding of the ion to the channel protein seemingly explains certain gating, ion selectivity, and permeation properties. Ion binding stabilizes greatly the channel and, depending upon ion type and concentration, leads to different conformations and ion binding affinities. High-affinity states guarantee binding of specific ions and mediate ion selectivity but are nonconductive. Conversely, low-affinity states would not discriminate well among different ions but allow permeation to occur.
- PublicationEmbargoInfluence of C-terminal protein domains and protein-lipid interactions on tetramerization and stability of the potassium channel KcsA(American Chemical Society, 2004-11-05) Molina Gallego, María Luisa; Encinar Hidalgo, José Antonio; Barrera Olivares, Francisco Nicolás; Fernández Ballester, Gregorio; Riquelme Pino, Gloria; González Ros, José Manuel; Bioquímica y Biología Molecular B e InmunologíaKcsA is a prokaryotic potassium channel formed by the assembly of four identical subunits around a central aqueous pore. Although the high-resolution X-ray structure of the transmembrane portion of KcsA is known [Doyle, D. A., Morais, C. J., Pfuetzner, R. A., Kuo, A., Gulbis, J. M., Cohen, S. L., Chait, B. T., and MacKinnon, R. (1998) Science280, 69−77], the identification of the molecular determinant(s) involved in promoting subunit tetramerization remains to be determined. Here, C-terminal deletion channel mutants, KcsA Δ125−160 and Δ120−160, as well as 1−125 KcsA obtained from chymotrypsin cleavage of full-length 1−160 KcsA, have been used to evaluate the role of the C-terminal segment on the stability and tetrameric assembly of the channel protein. We found that the lack of the cytoplasmic C-terminal domain of KcsA, and most critically the 120−124 sequence stretch, impairs tetrameric assembly of channel subunits in a heterologous E. coli expression system. Molecular modeling of KcsA predicts that, indeed, such sequence stretch provides intersubunit interaction sites by hydrogen bonding to amino acid residues in N- and C-terminal segments of adjacent subunits. However, once the KcsA tetramer is assembled, its remarkable in vitro stability to detergent or to heat-induced dissociation into subunits is not greatly influenced by whether the entire C-terminal domain continues being part of the protein. Finally and most interestingly, it is observed that, even in the absence of the C-terminal domain involved in tetramerization, reconstitution into membrane lipids promotes in vitro KcsA tetramerization very efficiently, an event which is likely mediated by allowing proper hydrophobic interactions involving intramembrane protein domains.
- PublicationEmbargoIntraresidual correlated motions in peptide chains(American Chemical Society, 2019-10-31) Bastida, Adolfo; Carmona García, Javier; Zúñiga, José; Requena, Alberto; Cerezo, Javier; Química FísicaWe investigate the interresidual and intraresidual correlations between dihedral displacements of adjacent residues within model polyalanine peptides by analyzing extensive molecular dynamics trajectories. Correlations are evaluated individually at different residue conformations covering the whole (ϕi ,ψi )-space. From these, we draw maps that unveil an unprecedented strong intramolecular correlation displaying opposite (correlated/anticorrelated) behaviors at different conformations. Both interresidual and intraresidual correlations arise from the propensity of the peptide to minimize the overall atomic displacements.
- PublicationEmbargoOccupancy of nonannular lipid binding sites on KcsA greatly increases the stability of the tetrameric protein(American Chemical Society, 2010-05-19) Triano García, Irene; Barrera Olivares, Francisco Nicolás; Renart Pérez, María Lourdes; Molina Gallego, María Luisa; Fernández Ballester, Gregorio; Poveda Larrosa, José Antonio; Fernández Carvajal, Asia María; Encinar Hidalgo, José Antonio; Ferrer Montiel, Antonio Vicente; Otzen, Daniel; González Ros, José Manuel; Bioquímica y Biología Molecular B e InmunologíaKcsA, a homotetrameric potassium channel from prokaryotes, contains noncovalently bound lipids appearing in the X-ray crystallographic structure of the protein. The binding sites for such high-affinity lipids are referred to as “nonannular” sites, correspond to intersubunit protein domains, and bind preferentially anionic phospholipids. Here we used a thermal denaturation assay and detergent−phospholipid mixed micelles containing KcsA to study the effects of different phospholipids on protein stability. We found that anionic phospholipids stabilize greatly the tetrameric protein against irreversible, heat-induced unfolding and dissociation into subunits. This occurs in a phospholipid concentration-dependent manner, and phosphatidic acid species with acyl chain lengths ranging 14 to 18 carbon atoms are more efficient than similar phosphatidylglycerols in protecting the protein. A docking model of the KcsA−phospholipid complex suggests that the increased protein stability originates from the intersubunit nature of the binding sites and, thus, interaction of the phospholipid with such sites holds together adjacent subunits within the tetrameric protein. We also found that simpler amphiphiles, such as alkyl sulfates longer than 10 carbon atoms, also increase the protein stability to the same extent as anionic phospholipids, although at higher concentrations than the latter. Modeling the interaction of these simpler amphiphiles with KcsA and comparing it with that of anionic phospholipids serve to delineate the features of a hydrophobic pocket in the nonannular sites. Such pocket is predicted to comprise residues from the M2 transmembrane segment of a subunit and from the pore helix of the adjacent subunit and seems most relevant to protein stabilization.
- PublicationEmbargoProbing the channel-bound shaker B inactivating peptide by stereoisomeric substitution at a strategic tyrosine residue(American Chemical Society, 2003-07-01) Encinar Hidalgo, José Antonio; Fernández Carvajal, Asia María; Poveda Larrosa, José Antonio; Molina Gallego, María Luisa; Albar, J.P.; Gavilanes Franco, Francisco; González Ros, José Manuel; Bioquímica y Biología Molecular B e InmunologíaA synthetic peptide patterned after the sequence of the inactivating ball domain of the Shaker B K+ channel, the ShB peptide, fully restores fast inactivation in the deletion Shaker BΔ6−46 K+ channel, which lacks the constitutive ball domains. On the contrary, a similar peptide in which tyrosine 8 is substituted by the secondary structure-disrupting d-tyrosine stereoisomer does not. This suggests that the stereoisomeric substitution prevents the peptide from adopting a structured conformation when bound to the channel during inactivation. Moreover, characteristic in vitro features of the wild-type ShB peptide such as the marked propensity to adopt an intramolecular β-hairpin structure when challenged by anionic phospholipid vesicles, a model target mimicking features of the inactivation site in the channel protein, or to insert into their hydrophobic bilayers, are lost in the d-tyrosine-containing peptide, whose behavior is practically identical to that of noninactivating peptide mutants. In the absence of high resolution crystallographic data on the inactivated channel/peptide complex, these latter findings suggest that the structured conformation required for the peptide to promote channel inactivation, as referred to above, is likely to be β-hairpin.
- PublicationOpen AccessStructure and Enzymatic Properties of an Unusual Cysteine Tryptophylquinone-Dependent Glycine Oxidase from Pseudoalteromonas luteoviolacea(ACS Publications, 2018) Andreo-Vidal, Andres; Mamounis, Kyle J.; Sehanobish, Esha; Avalos, Dante; Campillo-Brocal, Jonatan C.; Sanchez-Amat, Antonio; Yukl, Erik T.; Davidson, Victor L.; Genética y MicrobiologíaGlycine oxidase from Pseudoalteromonas luteoviolacea (PlGoxA) is a cysteine tryptophylquinone (CTQ)-dependent enzyme. Sequence and phylogenetic analysis place it in a newly designated subgroup (Group IID) of a recently identified family of LodA-like proteins, which are predicted to possess CTQ. The crystal structure of PlGoxA reveals that it is a homo-tetramer. It possesses an N-terminal domain with no close structural homologues in the Protein Data Bank. The active site is quite small due to intersubunit interactions, which may account for the observed cooperativy towards glycine. Steady-state kinetic analysis yielded values of kcat=6.0±0.2 s−1, K0.5=187±18 μM and h=1.77±0.27. In contrast to other quinoprotein amine dehydrogenases and oxidases that exhibit anomalously large primary kinetic isotope effects on the rate of reduction of the quinone cofactor by the amine substrate, no significant primary kinetic isotope effect was observed for this reaction of PlGoxA. The absorbance spectrum of the glycine-reduced PlGoxA exhibits features in the 400-650 nm range that have not previously been seen in other quinoproteins. Thus, in addition to the unusual structural features of PlGoxA, the kinetic and chemical reaction mechanisms of the reductive half-reaction of PlGoxA appear to be distinct from those of other amine dehydrogenases and amine oxidases that use tryptophylquinone and tyrosylquinone cofactors.
- PublicationEmbargoTyrosine phosphorylation of the inactivating peptide of the Shaker B potassium channel: a structural-functional correlate(American Chemical Society, 2002-09-12) Encinar Hidalgo, José Antonio; Fernández Carvajal, Asia María; Molina Gallego, María Luisa; Molina, A.; Poveda Larrosa, José Antonio; Albar, J.P.; López Barneo, José; Gavilanes Franco, Francisco; Ferrer Montiel, Antonio Vicente; González Ros, José Manuel; Bioquímica y Biología Molecular B e InmunologíaA synthetic peptide patterned after the sequence of the inactivating “ball” domain of the Shaker B K+ channel restores fast (N-type) inactivation in mutant deletion channels lacking their constitutive ball domains, as well as in K+ channels that do not normally inactivate. We now report on the effect of phosphorylation at a single tyrosine in position 8 of the inactivating peptide both on its ability to restore fast channel inactivation in deletion mutant channels and on the conformation adopted by the phosphorylated peptide when challenged by anionic lipid vesicles, a model target mimicking features of the inactivation site in the channel protein. We find that the inactivating peptide phosphorylated at Y8 behaves functionally as well as structurally as the noninactivating mutant carrying the mutation L7E. Moreover, it is observed that the inactivating peptide can be phosphorylated by the Src tyrosine kinase either as a free peptide in solution or when forming part of the membrane-bound protein channel as the constitutive inactivating domain. These findings suggest that tyrosine phosphorylation−dephosphorylation of this inactivating ball domain could be of physiological relevance to rapidly interconvert fast-inactivating channels into delayed rectifiers and vice versa.
- PublicationEmbargoUnfolding and refolding in vitro of a tetrameric, a-helical membrane protein: the prokaryotic potassium channel KcsA(American Chemical Society, 2005-10-06) Barrera Olivares, Francisco Nicolás; Renart Pérez, María Lourdes; Molina Gallego, María Luisa; Poveda Larrosa, José Antonio; Encinar Hidalgo, José Antonio; Fernández Carvajal, Asia María; Neira Faleiro, José Luis; González Ros, José Manuel; Bioquímica y Biología Molecular B e Inmunología2,2,2-Trifluoroethanol (TFE) effectively destabilizes the otherwise highly stable tetrameric structure of the potassium channel KcsA, a predominantly α-helical membrane protein [Valiyaveetil, F. I., Zhou, Y., and MacKinnon, R. (2002) Biochemistry 41, 10771−10777]. Here, we report that the effects on the protein structure of increasing concentrations of TFE in detergent solution include two successive protein concentration-dependent, cooperative transitions. In the first of such transitions, occurring at lower TFE concentrations, the tetrameric KcsA simultaneously increases the exposure of tryptophan residues to the solvent, partly loses its secondary structure, and dissociates into its constituent subunits. Under these conditions, simple dilution of the TFE permits a highly efficient refolding and tetramerization of the protein in the detergent solution. Moreover, following reconstitution into asolectin giant liposomes, the refolded protein exhibits nativelike potassium channel activity, as assessed by patch-clamp methods. Conversely, the second cooperative transition occurring at higher TFE concentrations results in the irreversible denaturation of the protein. These results are interpreted in terms of a protein and TFE concentration-dependent reversible equilibrium between the folded tetrameric protein and partly unfolded monomeric subunits, in which folding and oligomerization (or unfolding and dissociation in the other direction of the equilibrium process) are seemingly coupled processes. At higher TFE concentrations this is followed by the irreversible conversion of the unfolded monomers into a denatured protein form.