Roberto Salinas - Academia.edu (original) (raw)
Books by Roberto Salinas
This papers provides description of the need of science and technology policies in Guatemala sui... more This papers provides description of the need of science and technology policies in Guatemala suing a specific case studies on sater literacy.
Papers by Roberto Salinas
Revista Cubana de Educación Superior, Jan 4, 2015
As from the look of the Bolivian average citizen, that which incorporates to his/her every day an... more As from the look of the Bolivian average citizen, that which incorporates to his/her every day and concrete world the tangible and intangible products of scientific and technologic development, what he/she sees is discouraging. The answers to the country’s needs, going from a more effective irrigation system, to political participation and incidence as citizenship’s manifestations, are unstable, circumstantial, and depending. On the other hand, the managers of knowledge –universities and State– have not been able to carry out scientific and technologic development policies that go beyond the required demands because of the historic needs. Moreover, this answer has been conditioned by one of the variables that, in Latin American in general, and in Bolivia in particular, is determining: finance for social science research development.
Revista Cubana de Educación Superior, Apr 1, 2015
RESUMEN Desde la mirada del habitante promedio de Bolivia, aquella que incorpora a su mundo cotid... more RESUMEN Desde la mirada del habitante promedio de Bolivia, aquella que incorpora a su mundo cotidiano y concreto los productos tangibles e intangibles del desarrollo científico y tecnológico, lo que se observa es desalentador. Las respuestas a las necesidades del país, que van desde el desarrollo de sistemas de riego más eficientes hasta estrategias de participación e incidencia política como manifestación de su ciudadanía, son precarias, coyunturales y dependientes. Por otra parte, los gestores del conocimientouniversidades y Estado-no han logrado políticas de desarrollo científico y tecnológico que vayan más allá de las demandas requeridas por la necesidad histórica; además esa respuesta ha estado condicionada por una de las variables que en Latinoamérica en general, y en Bolivia en particular, son determinantes: el financiamiento para el desarrollo de la investigación de la ciencia social.
Resumen Desde la mirada del habitante promedio de Bolivia, aquella que incorpora a su mundo cotid... more Resumen Desde la mirada del habitante promedio de Bolivia, aquella que incorpora a su mundo cotidiano y concreto los productos tangibles e intangibles del desarrollo cientifico y tecnologico, lo que se observa es desalentador. Las respuestas a las necesidades del pais, que van desde el desarrollo de sistemas de riego mas eficientes hasta estrategias de participacion e incidencia politica como manifestacion de su ciudadania, son precarias, coyunturales y dependientes. Por otra parte, los gestores del conocimiento –universidades y Estado– no han logrado politicas de desarrollo cientifico y tecnologico que vayan mas alla de las demandas requeridas por la necesidad historica; ademas esa respuesta ha estado condicionada por una de las variables que en Latinoamerica en general, y en Bolivia en particular, son determinantes: el financiamiento para el desarrollo de la investigacion de la ciencia social. Abstrac t As from the look of the Bolivian average citizen, that which incorporates to h...
Journal of Molecular Biology, 2013
Journal of Molecular Biology, Jul 1, 2009
The PilZ protein was originally identified as necessary for type IV pilus (T4P) biogenesis. Since... more The PilZ protein was originally identified as necessary for type IV pilus (T4P) biogenesis. Since then, a large and diverse family of bacterial PilZ homology domains have been identified, some of which have been implicated in signaling pathways that control important processes, including motility, virulence and biofilm formation. Furthermore, many PilZ homology domains, though not PilZ itself, have been shown to bind the important bacterial second messenger bis(3′→5′)cyclic diGMP (c-diGMP). The crystal structures of the PilZ orthologs from Xanthomonas axonopodis pv citri (PilZ XAC1133 , this work) and from Xanthomonas campestris pv campestris (XC1028) present significant structural differences to other PilZ homologs that explain its failure to bind c-diGMP. NMR analysis of PilZ XAC1133 shows that these structural differences are maintained in solution. In spite of their emerging importance in bacterial signaling, the means by which PilZ proteins regulate specific processes is not clear. In this study, we show that PilZ XAC1133 binds to PilB, an ATPase required for T4P polymerization, and to the EAL domain of FimX XAC2398 , which regulates T4P biogenesis and localization in other bacterial species. These interactions were confirmed in NMR, two-hybrid and far-Western blot assays and are the first interactions observed between any PilZ domain and a target protein. While we were unable to detect phosphodiesterase activity for FimX XAC2398 in vitro, we show that it binds c-diGMP both in the presence and in the absence of PilZ XAC1133 . Site-directed mutagenesis studies for conserved and exposed residues suggest that PilZ XAC1133 interactions with FimX XAC2398 and PilB XAC3239 are mediated through a hydrophobic surface and an unstructured C-terminal extension conserved only in PilZ orthologs. The FimX-PilZ-PilB interactions involve a full set of "degenerate" GGDEF, EAL and PilZ domains and provide the first evidence of the means by which PilZ orthologs and FimX interact directly with the TP4 machinery.
Protein Sci, 2006
repressor headpiece bound to a symmetric variant of the lac operator, as inferred from hydrogen– ... more repressor headpiece bound to a symmetric variant of the lac operator, as inferred from hydrogen– deuterium (H–D) exchange experiments monitored by NMR spectroscopy. In the EX1 limit, observed exchange rates become pH-independent and exclusively sensitive to local structure fluctuations that expose the amide proton HN to exchange. Close to this regime, we measured decay rates of individual backbone HN signals
Peptides: The Wave of the Future, 2001
The EMBO Journal, 2002
The lac repressor±operator system is a model system for understanding protein±DNA interactions an... more The lac repressor±operator system is a model system for understanding protein±DNA interactions and allosteric mechanisms in gene regulation. Despite the wealth of biochemical data provided by extensive mutations of both repressor and operator, the speci®c recognition mechanism of the natural lac operators by lac repressor has remained elusive. Here we present the ®rst high-resolution structure of a dimer of the DNA-binding domain of lac repressor bound to its natural operator O1. The global positioning of the dimer on the operator is dramatically asymmetric, which results in a different pattern of speci®c contacts between the two sites. Speci®c recognition is accomplished by a combination of elongation and twist by 48°of the right lac subunit relative to the left one, signi®cant rearrangement of many side chains as well as sequence-dependent deformability of the DNA. The set of recognition mechanisms involved in the lac repressor±operator system is unique among other protein±DNA complexes and presents a nice example of the adaptability that both proteins and DNA exhibit in the context of their mutual interaction. Keywords: asymmetric DNA binding/DNA deformation/ lac repressor/natural lac operator/NMR structure ã European Molecular Biology Organization
Protein Science, 2006
We present experimental evidence for a cooperative unfolding transition of an a-helix in the lac ... more We present experimental evidence for a cooperative unfolding transition of an a-helix in the lac repressor headpiece bound to a symmetric variant of the lac operator, as inferred from hydrogendeuterium (H-D) exchange experiments monitored by NMR spectroscopy. In the EX1 limit, observed exchange rates become pH-independent and exclusively sensitive to local structure fluctuations that expose the amide proton H N to exchange. Close to this regime, we measured decay rates of individual backbone H N signals in D 2 O, and of their mutual H N -H N NOE by time-resolved two-dimensional (2D) NMR experiments. The data revealed correlated exchange at the center of the lac headpiece recognition helix, Val20-Val23, and suggested that the correlation breaks down at Val24, at the C terminus of the helix. A lower degree of correlation was observed for the exchange of Val9 and Ala10 at the center of helix 1, while no correlation was observed for Val38 and Glu39 at the center of helix 3. We conclude that H N exchange in the recognition helix and, to some extent, in helix 1 is a cooperative event involving the unfolding of these helices, whereas the H N exchange in helix 3 is dominated by random local structure fluctuations.
Journal of Molecular Biology, 2009
The PilZ protein was originally identified as necessary for type IV pilus (T4P) biogenesis. Since... more The PilZ protein was originally identified as necessary for type IV pilus (T4P) biogenesis. Since then, a large and diverse family of bacterial PilZ homology domains have been identified, some of which have been implicated in signaling pathways that control important processes, including motility, virulence and biofilm formation. Furthermore, many PilZ homology domains, though not PilZ itself, have been shown to bind the important bacterial second messenger bis(3′→5′)cyclic diGMP (c-diGMP). The crystal structures of the PilZ orthologs from Xanthomonas axonopodis pv citri (PilZ XAC1133 , this work) and from Xanthomonas campestris pv campestris (XC1028) present significant structural differences to other PilZ homologs that explain its failure to bind c-diGMP. NMR analysis of PilZ XAC1133 shows that these structural differences are maintained in solution. In spite of their emerging importance in bacterial signaling, the means by which PilZ proteins regulate specific processes is not clear. In this study, we show that PilZ XAC1133 binds to PilB, an ATPase required for T4P polymerization, and to the EAL domain of FimX XAC2398 , which regulates T4P biogenesis and localization in other bacterial species. These interactions were confirmed in NMR, two-hybrid and far-Western blot assays and are the first interactions observed between any PilZ domain and a target protein. While we were unable to detect phosphodiesterase activity for FimX XAC2398 in vitro, we show that it binds c-diGMP both in the presence and in the absence of PilZ XAC1133 . Site-directed mutagenesis studies for conserved and exposed residues suggest that PilZ XAC1133 interactions with FimX XAC2398 and PilB XAC3239 are mediated through a hydrophobic surface and an unstructured C-terminal extension conserved only in PilZ orthologs. The FimX-PilZ-PilB interactions involve a full set of "degenerate" GGDEF, EAL and PilZ domains and provide the first evidence of the means by which PilZ orthologs and FimX interact directly with the TP4 machinery.
Journal of Biological Chemistry, 2011
The Na + /Ca 2+ exchanger (NCX) is a membrane protein, which catalyzes the counter transport of N... more The Na + /Ca 2+ exchanger (NCX) is a membrane protein, which catalyzes the counter transport of Na + and Ca 2+ ions across the plasma membrane, playing a key role in the maintenance of the intracellular Ca 2+ homeostasis in various cell types. NCX consists of a transmembrane part and a large intracellular loop. The activation of the NCX transport function requires the binding of Ca 2+ to two tandem C2 domains, CBD1 and CBD2, which are an integral part of the exchanger's intracellular loop. Although high-resolution structures of individual CBD1 and CBD2 are available, their interdomain structure and dynamics and the atomic level mechanism of allosteric Ca 2+ -regulation remains unknown. Here, we use solution NMR spectroscopy to study the interdomain dynamics of CBD12, a 32 kDa construct that contains both the CBD1 and CBD2 domains connected by a short linker. Analysis of NMR residual dipolar couplings shows that CBD12 assumes on average an elongated shape both in the absence and in the presence of Ca 2+ . NMR 15 N relaxation data of the Apo state indicate that the two domains sample a wide range of relative arrangements on the nanosecond time scale. These arrangements comprise significantly non-linear inter-domain orientations. Binding of Ca 2+ to CBD1 significantly restricts the inter-domain flexibility, stabilizing a more rigid elongated conformation. These findings suggest a molecular mechanism for the role of CBD12 in the function of NCX.
Biopolymers, 2002
Homology modeling of the angiotensin II AT(1A) receptor based on rhodopsin&am... more Homology modeling of the angiotensin II AT(1A) receptor based on rhodopsin's crystal structure has assigned the 92-100 (YRWPFGNHL) sequence of the receptor to its first extracellular loop. Solution and membrane-bound conformational properties of a peptide containing this sequence (EL1) were examined by CD, fluorescence, and (1)H-NMR. CD spectra in aqueous solution revealed an equilibrium between less organized and folded conformers. NMR spectra indicated the coexistence of trans and cis isomers of the Trp(3)-Pro(4) bond. A positive band at 226 nm in the CD spectra suggested aromatic ring stacking, modulated by EL1's ionization degree. CD spectra showed that trifluoroethanol (TFE), or binding to detergent micelles and phospholipid bilayers, shifted the equilibrium toward conformers with higher secondary structure content. Different media gave rise to spectra suggestive of different beta-turns. Chemical shift changes in the NMR spectra corroborated the stabilization of different conformations. Thus, environments of lower polarity or binding to interfaces probably favored the formation of hydrogen bonds, stabilizing beta-turns, predicted for this sequence in the whole receptor. Increases in Trp(3) fluorescence intensity and anisotropy, blue shifts of the maximum emission wavelength, and pK changes also evinced the interaction between EL1 and model membranes. Binding was seen to depend on both hydrophobic and electrostatic interactions, as well as lipid phase packing. Studies with water-soluble and membrane-bound fluorescence quenchers demonstrated that Trp(3) is located close to the water-membrane interface. The results are discussed with regard to possible implications in receptor folding and function.
Toxicon, Oct 31, 2001
Sticholysins I and II are two highly hemolytic polypeptides purified from the Caribbean Sea anemo... more Sticholysins I and II are two highly hemolytic polypeptides purified from the Caribbean Sea anemone Stichodactyla helianthus. Their high sequence homology (93%) indicates that they correspond to isoforms of the same hemolysin. The spectroscopic measurements show a close similarity in the secondary structure content, conformation and stability of both toxins. Exposure of the toxins to high pHs (>11), a free radical source (AAPH), urea or temperature produce permanent changes in the toxin that lead to a significant loss of HA. It is significant to note that this loss of hemolytic activity occurs when other indicators, probably with the only exception of near-UV CD spectra, barely detect changes in the protein structure. This emphasizes the sensitivity of the protein function to changes in the macromolecule conformation. The most noticeable difference between both toxins is the considerably higher activity of St II, both measured in terms of erythrocyte internal K(+) exit or hemolysis; which is related to enthalpic factors. This difference is not due to an incomplete association of St I to the membrane. We consider then that the different pore forming capacity of both toxins in erythrocytes can be explained in terms of the difference in charge of the N-terminal fragment, than can considerably reduce the St I insertion rate in the membrane probably due to the negatively charged outer leaflet of the red blood cell, without a significant reduction of its capacity to bind to the cell membrane. This electrostatic effect, together with a slightly more relaxed structure in St II, could explain the higher pore forming capacity of St II in the red blood cell membrane.
Proteins: Structure, Function, and Bioinformatics, 2016
The Na(+) /Ca(2+) exchanger provides a major Ca(2+) extrusion pathway in excitable cells and play... more The Na(+) /Ca(2+) exchanger provides a major Ca(2+) extrusion pathway in excitable cells and plays a key role for the control of intracellular Ca(2+) concentrations. In Canis familiaris, Na(+) /Ca(2+) exchanger (NCX) activity is regulated by the binding of Ca(2+) to two cytosolic Ca(2+) -binding domains, CBD1 and CBD2, such that Ca(2+) -binding activates the exchanger. Despite its physiological importance, little is known about the exchanger's global structure, and the mechanism of allosteric Ca(2+) -regulation remains unclear. It was found previously that for NCX in the absence of Ca(2+) the two domains CBD1 and CBD2 of the cytosolic loop are flexibly linked, while after Ca(2+) -binding they adopt a rigid arrangement that is slightly tilted. A realistic model for the mechanism of the exchanger's allosteric regulation should not only address this property, but it should also explain the distinctive behavior of Drosophila melanogaster's sodium-calcium exchanger, CALX, for which Ca(2+) -binding to CBD1 inhibits Ca(2+) exchange. Here, NMR spin relaxation and residual dipolar couplings were used to show that Ca(2+) modulates CBD1 and CBD2 inter-domain flexibility of CALX in an analogous way as for NCX. A mechanistic model for the allosteric Ca(2+) regulation of the Na(+) /Ca(2+) exchanger is proposed. In this model, the intracellular loop acts as an entropic spring whose strength is modulated by Ca(2+) binding to CBD1 controlling ion transport across the plasma membrane. This article is protected by copyright. All rights reserved.
Biochemistry Usa, Sep 21, 2010
High-resolution nuclear magnetic resonance is used to investigate the conformational dynamics of ... more High-resolution nuclear magnetic resonance is used to investigate the conformational dynamics of human glucokinase, a 52 kDa monomeric enzyme that displays kinetic cooperativity. (1)H-(15)N transverse relaxation optimized spectra of uniformly labeled glucokinase, recorded in the absence and presence of glucose, reveal significant cross-peak overlap and heterogeneous peak intensities that persist over a range of temperatures. (15)N-specific labeling of isoleucines and tryptophans, reporting on backbone and side chain dynamics, respectively, demonstrates that both unliganded and glucose-bound enzymes sample multiple conformations, although glucose stabilizes certain conformations. These results provide the first direct evidence of glucokinase conformational heterogeneity and hence shed light on the molecular basis of cooperativity.
Revista Cubana de Educación Superior, Jan 4, 2015
As from the look of the Bolivian average citizen, that which incorporates to his/her every day an... more As from the look of the Bolivian average citizen, that which incorporates to his/her every day and concrete world the tangible and intangible products of scientific and technologic development, what he/she sees is discouraging. The answers to the country’s needs, going from a more effective irrigation system, to political participation and incidence as citizenship’s manifestations, are unstable, circumstantial, and depending. On the other hand, the managers of knowledge –universities and State– have not been able to carry out scientific and technologic development policies that go beyond the required demands because of the historic needs. Moreover, this answer has been conditioned by one of the variables that, in Latin American in general, and in Bolivia in particular, is determining: finance for social science research development.
Revista Cubana de Educación Superior, Apr 1, 2015
RESUMEN Desde la mirada del habitante promedio de Bolivia, aquella que incorpora a su mundo cotid... more RESUMEN Desde la mirada del habitante promedio de Bolivia, aquella que incorpora a su mundo cotidiano y concreto los productos tangibles e intangibles del desarrollo científico y tecnológico, lo que se observa es desalentador. Las respuestas a las necesidades del país, que van desde el desarrollo de sistemas de riego más eficientes hasta estrategias de participación e incidencia política como manifestación de su ciudadanía, son precarias, coyunturales y dependientes. Por otra parte, los gestores del conocimientouniversidades y Estado-no han logrado políticas de desarrollo científico y tecnológico que vayan más allá de las demandas requeridas por la necesidad histórica; además esa respuesta ha estado condicionada por una de las variables que en Latinoamérica en general, y en Bolivia en particular, son determinantes: el financiamiento para el desarrollo de la investigación de la ciencia social.
Resumen Desde la mirada del habitante promedio de Bolivia, aquella que incorpora a su mundo cotid... more Resumen Desde la mirada del habitante promedio de Bolivia, aquella que incorpora a su mundo cotidiano y concreto los productos tangibles e intangibles del desarrollo cientifico y tecnologico, lo que se observa es desalentador. Las respuestas a las necesidades del pais, que van desde el desarrollo de sistemas de riego mas eficientes hasta estrategias de participacion e incidencia politica como manifestacion de su ciudadania, son precarias, coyunturales y dependientes. Por otra parte, los gestores del conocimiento –universidades y Estado– no han logrado politicas de desarrollo cientifico y tecnologico que vayan mas alla de las demandas requeridas por la necesidad historica; ademas esa respuesta ha estado condicionada por una de las variables que en Latinoamerica en general, y en Bolivia en particular, son determinantes: el financiamiento para el desarrollo de la investigacion de la ciencia social. Abstrac t As from the look of the Bolivian average citizen, that which incorporates to h...
Journal of Molecular Biology, 2013
Journal of Molecular Biology, Jul 1, 2009
The PilZ protein was originally identified as necessary for type IV pilus (T4P) biogenesis. Since... more The PilZ protein was originally identified as necessary for type IV pilus (T4P) biogenesis. Since then, a large and diverse family of bacterial PilZ homology domains have been identified, some of which have been implicated in signaling pathways that control important processes, including motility, virulence and biofilm formation. Furthermore, many PilZ homology domains, though not PilZ itself, have been shown to bind the important bacterial second messenger bis(3′→5′)cyclic diGMP (c-diGMP). The crystal structures of the PilZ orthologs from Xanthomonas axonopodis pv citri (PilZ XAC1133 , this work) and from Xanthomonas campestris pv campestris (XC1028) present significant structural differences to other PilZ homologs that explain its failure to bind c-diGMP. NMR analysis of PilZ XAC1133 shows that these structural differences are maintained in solution. In spite of their emerging importance in bacterial signaling, the means by which PilZ proteins regulate specific processes is not clear. In this study, we show that PilZ XAC1133 binds to PilB, an ATPase required for T4P polymerization, and to the EAL domain of FimX XAC2398 , which regulates T4P biogenesis and localization in other bacterial species. These interactions were confirmed in NMR, two-hybrid and far-Western blot assays and are the first interactions observed between any PilZ domain and a target protein. While we were unable to detect phosphodiesterase activity for FimX XAC2398 in vitro, we show that it binds c-diGMP both in the presence and in the absence of PilZ XAC1133 . Site-directed mutagenesis studies for conserved and exposed residues suggest that PilZ XAC1133 interactions with FimX XAC2398 and PilB XAC3239 are mediated through a hydrophobic surface and an unstructured C-terminal extension conserved only in PilZ orthologs. The FimX-PilZ-PilB interactions involve a full set of "degenerate" GGDEF, EAL and PilZ domains and provide the first evidence of the means by which PilZ orthologs and FimX interact directly with the TP4 machinery.
Protein Sci, 2006
repressor headpiece bound to a symmetric variant of the lac operator, as inferred from hydrogen– ... more repressor headpiece bound to a symmetric variant of the lac operator, as inferred from hydrogen– deuterium (H–D) exchange experiments monitored by NMR spectroscopy. In the EX1 limit, observed exchange rates become pH-independent and exclusively sensitive to local structure fluctuations that expose the amide proton HN to exchange. Close to this regime, we measured decay rates of individual backbone HN signals
Peptides: The Wave of the Future, 2001
The EMBO Journal, 2002
The lac repressor±operator system is a model system for understanding protein±DNA interactions an... more The lac repressor±operator system is a model system for understanding protein±DNA interactions and allosteric mechanisms in gene regulation. Despite the wealth of biochemical data provided by extensive mutations of both repressor and operator, the speci®c recognition mechanism of the natural lac operators by lac repressor has remained elusive. Here we present the ®rst high-resolution structure of a dimer of the DNA-binding domain of lac repressor bound to its natural operator O1. The global positioning of the dimer on the operator is dramatically asymmetric, which results in a different pattern of speci®c contacts between the two sites. Speci®c recognition is accomplished by a combination of elongation and twist by 48°of the right lac subunit relative to the left one, signi®cant rearrangement of many side chains as well as sequence-dependent deformability of the DNA. The set of recognition mechanisms involved in the lac repressor±operator system is unique among other protein±DNA complexes and presents a nice example of the adaptability that both proteins and DNA exhibit in the context of their mutual interaction. Keywords: asymmetric DNA binding/DNA deformation/ lac repressor/natural lac operator/NMR structure ã European Molecular Biology Organization
Protein Science, 2006
We present experimental evidence for a cooperative unfolding transition of an a-helix in the lac ... more We present experimental evidence for a cooperative unfolding transition of an a-helix in the lac repressor headpiece bound to a symmetric variant of the lac operator, as inferred from hydrogendeuterium (H-D) exchange experiments monitored by NMR spectroscopy. In the EX1 limit, observed exchange rates become pH-independent and exclusively sensitive to local structure fluctuations that expose the amide proton H N to exchange. Close to this regime, we measured decay rates of individual backbone H N signals in D 2 O, and of their mutual H N -H N NOE by time-resolved two-dimensional (2D) NMR experiments. The data revealed correlated exchange at the center of the lac headpiece recognition helix, Val20-Val23, and suggested that the correlation breaks down at Val24, at the C terminus of the helix. A lower degree of correlation was observed for the exchange of Val9 and Ala10 at the center of helix 1, while no correlation was observed for Val38 and Glu39 at the center of helix 3. We conclude that H N exchange in the recognition helix and, to some extent, in helix 1 is a cooperative event involving the unfolding of these helices, whereas the H N exchange in helix 3 is dominated by random local structure fluctuations.
Journal of Molecular Biology, 2009
The PilZ protein was originally identified as necessary for type IV pilus (T4P) biogenesis. Since... more The PilZ protein was originally identified as necessary for type IV pilus (T4P) biogenesis. Since then, a large and diverse family of bacterial PilZ homology domains have been identified, some of which have been implicated in signaling pathways that control important processes, including motility, virulence and biofilm formation. Furthermore, many PilZ homology domains, though not PilZ itself, have been shown to bind the important bacterial second messenger bis(3′→5′)cyclic diGMP (c-diGMP). The crystal structures of the PilZ orthologs from Xanthomonas axonopodis pv citri (PilZ XAC1133 , this work) and from Xanthomonas campestris pv campestris (XC1028) present significant structural differences to other PilZ homologs that explain its failure to bind c-diGMP. NMR analysis of PilZ XAC1133 shows that these structural differences are maintained in solution. In spite of their emerging importance in bacterial signaling, the means by which PilZ proteins regulate specific processes is not clear. In this study, we show that PilZ XAC1133 binds to PilB, an ATPase required for T4P polymerization, and to the EAL domain of FimX XAC2398 , which regulates T4P biogenesis and localization in other bacterial species. These interactions were confirmed in NMR, two-hybrid and far-Western blot assays and are the first interactions observed between any PilZ domain and a target protein. While we were unable to detect phosphodiesterase activity for FimX XAC2398 in vitro, we show that it binds c-diGMP both in the presence and in the absence of PilZ XAC1133 . Site-directed mutagenesis studies for conserved and exposed residues suggest that PilZ XAC1133 interactions with FimX XAC2398 and PilB XAC3239 are mediated through a hydrophobic surface and an unstructured C-terminal extension conserved only in PilZ orthologs. The FimX-PilZ-PilB interactions involve a full set of "degenerate" GGDEF, EAL and PilZ domains and provide the first evidence of the means by which PilZ orthologs and FimX interact directly with the TP4 machinery.
Journal of Biological Chemistry, 2011
The Na + /Ca 2+ exchanger (NCX) is a membrane protein, which catalyzes the counter transport of N... more The Na + /Ca 2+ exchanger (NCX) is a membrane protein, which catalyzes the counter transport of Na + and Ca 2+ ions across the plasma membrane, playing a key role in the maintenance of the intracellular Ca 2+ homeostasis in various cell types. NCX consists of a transmembrane part and a large intracellular loop. The activation of the NCX transport function requires the binding of Ca 2+ to two tandem C2 domains, CBD1 and CBD2, which are an integral part of the exchanger's intracellular loop. Although high-resolution structures of individual CBD1 and CBD2 are available, their interdomain structure and dynamics and the atomic level mechanism of allosteric Ca 2+ -regulation remains unknown. Here, we use solution NMR spectroscopy to study the interdomain dynamics of CBD12, a 32 kDa construct that contains both the CBD1 and CBD2 domains connected by a short linker. Analysis of NMR residual dipolar couplings shows that CBD12 assumes on average an elongated shape both in the absence and in the presence of Ca 2+ . NMR 15 N relaxation data of the Apo state indicate that the two domains sample a wide range of relative arrangements on the nanosecond time scale. These arrangements comprise significantly non-linear inter-domain orientations. Binding of Ca 2+ to CBD1 significantly restricts the inter-domain flexibility, stabilizing a more rigid elongated conformation. These findings suggest a molecular mechanism for the role of CBD12 in the function of NCX.
Biopolymers, 2002
Homology modeling of the angiotensin II AT(1A) receptor based on rhodopsin&am... more Homology modeling of the angiotensin II AT(1A) receptor based on rhodopsin's crystal structure has assigned the 92-100 (YRWPFGNHL) sequence of the receptor to its first extracellular loop. Solution and membrane-bound conformational properties of a peptide containing this sequence (EL1) were examined by CD, fluorescence, and (1)H-NMR. CD spectra in aqueous solution revealed an equilibrium between less organized and folded conformers. NMR spectra indicated the coexistence of trans and cis isomers of the Trp(3)-Pro(4) bond. A positive band at 226 nm in the CD spectra suggested aromatic ring stacking, modulated by EL1's ionization degree. CD spectra showed that trifluoroethanol (TFE), or binding to detergent micelles and phospholipid bilayers, shifted the equilibrium toward conformers with higher secondary structure content. Different media gave rise to spectra suggestive of different beta-turns. Chemical shift changes in the NMR spectra corroborated the stabilization of different conformations. Thus, environments of lower polarity or binding to interfaces probably favored the formation of hydrogen bonds, stabilizing beta-turns, predicted for this sequence in the whole receptor. Increases in Trp(3) fluorescence intensity and anisotropy, blue shifts of the maximum emission wavelength, and pK changes also evinced the interaction between EL1 and model membranes. Binding was seen to depend on both hydrophobic and electrostatic interactions, as well as lipid phase packing. Studies with water-soluble and membrane-bound fluorescence quenchers demonstrated that Trp(3) is located close to the water-membrane interface. The results are discussed with regard to possible implications in receptor folding and function.
Toxicon, Oct 31, 2001
Sticholysins I and II are two highly hemolytic polypeptides purified from the Caribbean Sea anemo... more Sticholysins I and II are two highly hemolytic polypeptides purified from the Caribbean Sea anemone Stichodactyla helianthus. Their high sequence homology (93%) indicates that they correspond to isoforms of the same hemolysin. The spectroscopic measurements show a close similarity in the secondary structure content, conformation and stability of both toxins. Exposure of the toxins to high pHs (>11), a free radical source (AAPH), urea or temperature produce permanent changes in the toxin that lead to a significant loss of HA. It is significant to note that this loss of hemolytic activity occurs when other indicators, probably with the only exception of near-UV CD spectra, barely detect changes in the protein structure. This emphasizes the sensitivity of the protein function to changes in the macromolecule conformation. The most noticeable difference between both toxins is the considerably higher activity of St II, both measured in terms of erythrocyte internal K(+) exit or hemolysis; which is related to enthalpic factors. This difference is not due to an incomplete association of St I to the membrane. We consider then that the different pore forming capacity of both toxins in erythrocytes can be explained in terms of the difference in charge of the N-terminal fragment, than can considerably reduce the St I insertion rate in the membrane probably due to the negatively charged outer leaflet of the red blood cell, without a significant reduction of its capacity to bind to the cell membrane. This electrostatic effect, together with a slightly more relaxed structure in St II, could explain the higher pore forming capacity of St II in the red blood cell membrane.
Proteins: Structure, Function, and Bioinformatics, 2016
The Na(+) /Ca(2+) exchanger provides a major Ca(2+) extrusion pathway in excitable cells and play... more The Na(+) /Ca(2+) exchanger provides a major Ca(2+) extrusion pathway in excitable cells and plays a key role for the control of intracellular Ca(2+) concentrations. In Canis familiaris, Na(+) /Ca(2+) exchanger (NCX) activity is regulated by the binding of Ca(2+) to two cytosolic Ca(2+) -binding domains, CBD1 and CBD2, such that Ca(2+) -binding activates the exchanger. Despite its physiological importance, little is known about the exchanger's global structure, and the mechanism of allosteric Ca(2+) -regulation remains unclear. It was found previously that for NCX in the absence of Ca(2+) the two domains CBD1 and CBD2 of the cytosolic loop are flexibly linked, while after Ca(2+) -binding they adopt a rigid arrangement that is slightly tilted. A realistic model for the mechanism of the exchanger's allosteric regulation should not only address this property, but it should also explain the distinctive behavior of Drosophila melanogaster's sodium-calcium exchanger, CALX, for which Ca(2+) -binding to CBD1 inhibits Ca(2+) exchange. Here, NMR spin relaxation and residual dipolar couplings were used to show that Ca(2+) modulates CBD1 and CBD2 inter-domain flexibility of CALX in an analogous way as for NCX. A mechanistic model for the allosteric Ca(2+) regulation of the Na(+) /Ca(2+) exchanger is proposed. In this model, the intracellular loop acts as an entropic spring whose strength is modulated by Ca(2+) binding to CBD1 controlling ion transport across the plasma membrane. This article is protected by copyright. All rights reserved.
Biochemistry Usa, Sep 21, 2010
High-resolution nuclear magnetic resonance is used to investigate the conformational dynamics of ... more High-resolution nuclear magnetic resonance is used to investigate the conformational dynamics of human glucokinase, a 52 kDa monomeric enzyme that displays kinetic cooperativity. (1)H-(15)N transverse relaxation optimized spectra of uniformly labeled glucokinase, recorded in the absence and presence of glucose, reveal significant cross-peak overlap and heterogeneous peak intensities that persist over a range of temperatures. (15)N-specific labeling of isoleucines and tryptophans, reporting on backbone and side chain dynamics, respectively, demonstrates that both unliganded and glucose-bound enzymes sample multiple conformations, although glucose stabilizes certain conformations. These results provide the first direct evidence of glucokinase conformational heterogeneity and hence shed light on the molecular basis of cooperativity.
Nature Communications, 2015
Proteins: Structure, Function, and Bioinformatics, 2009