Nancy Pulido - Academia.edu (original) (raw)

Papers by Nancy Pulido

Research paper thumbnail of Thermodynamic and kinetic analysis of the LAO binding protein and its isolated domains reveal non-additivity in stability, folding, and function

Substrate-binding proteins (SBP) are used by organisms from the three domains of life for transpo... more Substrate-binding proteins (SBP) are used by organisms from the three domains of life for transport and signaling. SBPs are composed of two domains that collectively trap ligands with high affinity and selectivity. To explore the role of the domains and the integrity of the hinge region between them in the function and conformation of SBPs, here we describe the ligand binding, conformational stability, and folding kinetics of the Lysine Arginine Ornithine binding protein (LAO) fromSalmonella thiphimuriumand constructs corresponding to its two independent domains. LAO is a class II SBP formed by a continuous and a discontinuous domain. Contrary to the expected behavior based on their connectivity, the discontinuous domain shows a stable native-like structure that binds L-arginine with moderate affinity, whereas the continuous domain is barely stable and shows no detectable ligand binding. Regarding folding kinetics, studies of the entire protein revealed the presence of at least two ...

Research paper thumbnail of On the molecular basis of the high affinity binding of basic amino acids to LAOBP, a periplasmic binding protein from Salmonella typhimurium

Journal of Molecular Recognition, 2015

The rational designing of binding abilities in proteins requires an understanding of the relation... more The rational designing of binding abilities in proteins requires an understanding of the relationship between structure and thermodynamics. However, our knowledge of the molecular origin of high-affinity binding of ligands to proteins is still limited; such is the case for l-lysine-l-arginine-l-ornithine periplasmic binding protein (LAOBP), a periplasmic binding protein from Salmonella typhimurium that binds to l-arginine, l-lysine, and l-ornithine with nanomolar affinity and to l-histidine with micromolar affinity. Structural studies indicate that ligand binding induces a large conformational change in LAOBP. In this work, we studied the thermodynamics of l-histidine and l-arginine binding to LAOBP by isothermal titration calorimetry. For both ligands, the affinity is enthalpically driven, with a binding ΔCp of ~-300 cal mol(-1) K(-1) , most of which arises from the burial of protein nonpolar surfaces that accompanies the conformational change. Osmotic stress measurements revealed that several water molecules become sequestered upon complex formation. In addition, LAOBP prefers positively charged ligands in their side chain. An energetic analysis shows that the protein acquires a thermodynamically equivalent state with both ligands. The 1000-fold higher affinity of LAOBP for l-arginine as compared with l-histidine is mainly of enthalpic origin and can be ascribed to the formation of an extra pair of hydrogen bonds. Periplasmic binding proteins have evolved diverse energetic strategies for ligand recognition. STM4351, another arginine binding protein from Salmonella, shows an entropy-driven micromolar affinity toward l-arginine. In contrast, our data show that LAOBP achieves nanomolar affinity for the same ligand through enthalpy optimization. Copyright © 2015 John Wiley & Sons, Ltd.

Research paper thumbnail of The interplay of protein‐ligand and water‐mediated interactions shape affinity and selectivity in the LAO binding protein

The FEBS Journal

The study of binding thermodynamics is essential to understand how affinity and selectivity are a... more The study of binding thermodynamics is essential to understand how affinity and selectivity are acquired in molecular complexes. Periplasmic binding proteins (PBPs) are macromolecules of biotechnological interest that bind a broad number of ligands and have been used to design biosensors. The lysine‐arginine‐ornithine binding protein (LAO) is a PBP of 238 residues that binds the basic amino acids l‐arginine and l‐histidine with nm and μm affinity, respectively. It has been shown that the affinity difference for arginine and histidine binding is caused by enthalpy, this correlates with the higher number of protein–ligand contacts formed with arginine. In order to elucidate the structural bases that determine binding affinity and selectivity in LAO, the contribution of protein–ligand contacts to binding energetics was assessed. To this end, an alanine scanning of the LAO‐binding site residues was performed and arginine and histidine binding were characterized by isothermal titration calorimetry and X‐ray crystallography. Although unexpected enthalpy and entropy changes were observed in some mutants, thermodynamic data correlated with structural information, especially, the binding heat capacity change. We found that selectivity is conferred by several residues rather than exclusive arginine–protein interactions. Furthermore, crystallographic structures revealed that protein–ligand contributions to binding thermodynamics are highly influenced by the solvent. Finally, we found a similar backbone conformation in all the closed structures obtained, but different structures in the open state, suggesting that the binding site residues of LAO play an important role in stabilizing not only the holo conformation, but also the apo state.

Research paper thumbnail of The role of conserved non-aromatic residues in the Lactobacillus amylovorus α-amylase CBM26-starch interaction

International journal of biological macromolecules, Jan 15, 2018

It is generally accepted that carbohydrate binding modules (CBMs) recognize their carbohydrate li... more It is generally accepted that carbohydrate binding modules (CBMs) recognize their carbohydrate ligands by hydrophobic and CH-π interactions. Point mutations of one CBM26 of the Lactobacillus amylovorus α-amylase starch-binding domain (LaCBM26) showed that conserved non-aromatic residue are essential in the starch recognition function of the domain, as the mutation of a single glutamine (Q68L) eliminates binding to starch and β-cyclodextrin, even in the presence of aromatic amino acids necessary for ligand binding. The secondary structure of mutated proteins was verified and showed no differences from the wild-type domain. However, random mutations of five residues involved in binding (Y18, Y20, Q68, E74, and F77) did cause change in the secondary structure of the protein, which also causes loss of function. Much of the diversity introduced in the LaCBM26 was probably incompatible with the appropriate folding of these proteins, suggesting that the domain has little tolerance to change.

Research paper thumbnail of Expression of recombinant SnRK1 in E. coli. Characterization of adenine nucleotide binding to the SnRK1.1/AKINβγ-β3 complex

Plant science : an international journal of experimental plant biology, 2017

The SnRK1 complexes in plants belong to the family of AMPK/SNF1 kinases, which have been associat... more The SnRK1 complexes in plants belong to the family of AMPK/SNF1 kinases, which have been associated with the control of energy balance, in addition to being involved in the regulation of other aspects of plant growth and development. Analysis of complex formation indicates that increased activity is achieved when the catalytic subunit is phosphorylated and bound to regulatory subunits. SnRK1.1 subunit activity is higher than that of SnRK1.2, which also exhibits reduced activation due to the regulatory subunits. The catalytic phosphomimetic subunits (T175/176D) do not exhibit high activity levels, which indicate that the amino acid change does not produce the same effect as phosphorylation. Based on the mammalian AMPK X-ray structure, the plant SnRK1.1/AKINβγ-β3 was modeled by homology modeling and Molecular Dynamics simulations (MD). The model predicted an intimate and extensive contact between a hydrophobic region of AKINβγ and the β3 subunit. While the AKINβγ prediction retains th...

Research paper thumbnail of Article Energetic Effects of Magnesium in the Recognition of Adenosine Nucleotides by the F1-ATPase β Subunit

Research paper thumbnail of Current applications of isothermal titration calorimetry to the study of protein complexes

The absorption or liberation of heat has proven a widely spread property in biomolecular processe... more The absorption or liberation of heat has proven a widely spread property in biomolecular processes. Isothermal titration calorimetry (ITC) measures this property directly. This feature not only implies high precision in determining the binding enthalpy, but also allows us to infer the reaction mechanism in a more objective way than many non-calorimetric techniques. In this chapter, the principles of ITC are reviewed together with the basic thermodynamic formalism on which the technique is based. In addition, the current state of the art in calorimetry in protein recognition is described, with particular emphasis on advances in the last few years.

Research paper thumbnail of A group 6 late embryogenesis abundant protein from common bean is a disordered protein with extended helical structure and oligomer-forming properties

The Journal of biological chemistry, Jan 14, 2014

Late embryogenesis-abundant proteins accumulate to high levels in dry seeds. Some of them also ac... more Late embryogenesis-abundant proteins accumulate to high levels in dry seeds. Some of them also accumulate in response to water deficit in vegetative tissues, which leads to a remarkable association between their presence and low water availability conditions. A major sub-group of these proteins, also known as typical LEA proteins, shows high hydrophilicity and a high percentage of glycine and other small amino acid residues, distinctive physicochemical properties that predict a high content of structural disorder. Although all typical LEA proteins share these characteristics, seven groups can be distinguished by sequence similarity, indicating structural and functional diversity among them. Some of these groups have been extensively studied; however, others require a more detailed analysis to advance in their functional understanding. In this work, we report the structural characterization of a group 6 LEA protein from a common bean (Phaseolus vulgaris L.) (PvLEA6) by circular dichr...

Research paper thumbnail of Phases Dispersion and Oxygen Transfer in a Simulated Fermentation Broth Containing Castor Oil and Proteins

Biotechnology Progress, 2004

The sizes of air bubbles and castor oil drops were studied by image analysis as a function of the... more The sizes of air bubbles and castor oil drops were studied by image analysis as a function of the concentration of soluble protein (bovine serum albumin [BSA] and lipase, as model proteins) in a three-phase system using a simulated fermentation medium (aqueous salt solution, castor oil, and air). Small amounts of proteins (<0.02 g/L) caused an important decrease in oil drops and bubbles sizes, together with a pronounced decrease in surface tension. The extent and profiles of this decrease seem to be determined by the conformation of the protein at the interface. The k L a value increased considerably for increasing concentration (up to 0.02 g/L) of the two proteins but was very different (2-fold higher for the lipase) at the highest concentrations tested (0.5 g/L), a phenomenon that can be caused by the extent to which bubbles are trapped within oil drops.

Research paper thumbnail of Multithermal titration calorimetry: A rapid method to determine binding heat capacities

Biophysical Chemistry, 2006

Herein a new method that allows binding DCp to be determined with a single experiment is presente... more Herein a new method that allows binding DCp to be determined with a single experiment is presented. Multithermal titration calorimetry (MTC) is a simple extension of isothermal titration calorimetry (ITC) that explicitly takes into account the thermal dependences of DH and the binding constant. Experimentally, this is accomplished by performing a single stepwise titration with ITC equipment, allowing temperature readjustments of the system at intermediate states of the titration process. Thus, from the resulting multitherm, DCp can also be determined. The experimental feasibility of MTC was tested by using the well-characterized lysozyme -chitotriose complex as a model system. D

Research paper thumbnail of Energetic Effects of Magnesium in the Recognition of Adenosine Nucleotides by the F 1 -ATPase β Subunit

Biochemistry, 2010

Nucleotide-induced conformational changes of the catalytic β subunits play a crucial role in the ... more Nucleotide-induced conformational changes of the catalytic β subunits play a crucial role in the rotary mechanism of F 1 -ATPase. To gain insights into the energetic bases that govern the recognition of nucleotides by the isolated β subunit from thermophilic Bacillus PS3 (Tβ), the binding of this monomer to Mg(II)-free and Mg(II)bound adenosine nucleotides was characterized using high-precision isothermal titration calorimetry. The interactions of Mg(II) with free ATP or ADP were also measured calorimetrically. A model that considers simultaneously the interactions of Tβ with Mg 3 ATP or with ATP and in which ATP is able to bind two Mg(II) atoms sequentially was used to determine the formation parameters of the Tβ-Mg 3 ATP complex from calorimetric data. This analysis yielded significantly different ΔH b and ΔS b values in relation to those obtained using a single-binding site model, while ΔG b was almost unchanged. Published calorimetric data for the titration of Tβ with Mg 3 ADP [P erez-Hern andez, G., et al. Arch. Biochem. Biophys. 408, 177-183] were reanalyzed with the ternary model to determine the corresponding true binding parameters. Interactions of Tβ with Mg 3 ATP, ATP, Mg 3 ADP, or ADP were enthalpically driven. Larger differences in thermodynamic properties were observed between Tβ-Mg 3 ATP and Tβ-ATP complexes than between Tβ-Mg 3 ADP and Tβ-ADP complexes or between Tβ-Mg 3 ATP and Tβ-Mg 3 ADP complexes. These binding data, in conjunction with those for the association of Mg(II) with free nucleotides, allowed for a determination of the energetic effects of the metal ion on the recognition of adenosine nucleotides by Tβ [i.e., Tβ 3 AT(D)P þ Mg(II) h Tβ 3 AT(D)P-Mg]. Because of a more favorable binding enthalpy, Mg(II) is recognized more avidly by the Tβ 3 ATP complex, indicating better stereochemical complementarity than in the Tβ 3 ADP complex. Furthermore, a structural-energetic analysis suggests that Tβ adopts a more closed conformation when it is bound to Mg 3 ATP than to ATP or Mg 3 ADP, in agreement with recently published NMR data ) J. Biol. Chem. 284, 2374-2382. Using published binding data, a similar analysis of Mg(II) energetic effects was performed for the free energy change of F 1 catalytic sites, in the framework of bi-or tri-site binding models. 1 Abbreviations: Mg(II), free magnesium ion; AMPPNP, adenylyl 5 0 -imidodiphosphate; P i , inorganic phosphate; EDTA, ethylenediaminetetraacetic acid; Tris, 2-amino-2-(hydroxymethyl)propane-1,3diol; MgCl 2 , magnesium chloride; NaOH, sodium hydroxide; ATPase, adenosine 5 0 -triphosphatase; Tβ, isolated β subunit from thermophilic Bacillus PS3; TF 1 , F 1 sector from Bacillus PS3; EF 1 , F 1 sector from Escherichia coli; S1-S3, high-, medium-, and low-affinity β subunit sites in F 1 , respectively; β DP and β TP , β subunits in the crystal structure of F 1 bound to Mg 3 ADP and Mg 3 AMPPNP, respectively; ITC, isothermal titration calorimetry; NMR, nuclear magnetic resonance; K b , equilibrium binding constant; ΔH b , binding enthalpy; ΔG b , Gibbs binding free energy; ΔS b , binding entropy; ΔC pb , binding heat capacity; K b1 and K b2 , stepwise association constants for the interaction of ATP with Mg(II); K P1 and K P2 , association constants for ATP and Mg 3 ATP, respectively, binding to Tβ; ΔH int , intrinsic binding enthalpy; ΔH dsolv , desolvation enthalpy; ΔS conf , ΔS solv , and ΔS r-t , conformational, solvation, and roto-translational entropy changes, respectively; ΔA, surface area change; Δc pi , specific heat capacity for the surface area of the type i; R, cooperative heterotropic association constant; Δh, cooperative enthalpy; Δs, cooperative entropy.

Research paper thumbnail of On the molecular basis of the high affinity binding of basic amino acids to LAOBP, a periplasmic binding protein from Salmonella typhimurium

Journal of Molecular Recognition, 2015

The rational designing of binding abilities in proteins requires an understanding of the relation... more The rational designing of binding abilities in proteins requires an understanding of the relationship between structure and thermodynamics. However, our knowledge of the molecular origin of high-affinity binding of ligands to proteins is still limited; such is the case for L-lysine-L-arginine-L-ornithine periplasmic binding protein (LAOBP), a periplasmic binding protein from Salmonella typhimurium that binds to L-arginine, L-lysine, and L-ornithine with nanomolar affinity and to L-histidine with micromolar affinity. Structural studies indicate that ligand binding induces a large conformational change in LAOBP. In this work, we studied the thermodynamics of L-histidine and L-arginine binding to LAOBP by isothermal titration calorimetry. For both ligands, the affinity is enthalpically driven, with a binding ΔCp of~À300 cal mol À1 K À1 , most of which arises from the burial of protein nonpolar surfaces that accompanies the conformational change. Osmotic stress measurements revealed that several water molecules become sequestered upon complex formation. In addition, LAOBP prefers positively charged ligands in their side chain. An energetic analysis shows that the protein acquires a thermodynamically equivalent state with both ligands. The 1000-fold higher affinity of LAOBP for L-arginine as compared with L-histidine is mainly of enthalpic origin and can be ascribed to the formation of an extra pair of hydrogen bonds. Periplasmic binding proteins have evolved diverse energetic strategies for ligand recognition. STM4351, another arginine binding protein from Salmonella, shows an entropy-driven micromolar affinity toward L-arginine. In contrast, our data show that LAOBP achieves nanomolar affinity for the same ligand through enthalpy optimization.

Research paper thumbnail of Thermodynamic and kinetic analysis of the LAO binding protein and its isolated domains reveal non-additivity in stability, folding, and function

Substrate-binding proteins (SBP) are used by organisms from the three domains of life for transpo... more Substrate-binding proteins (SBP) are used by organisms from the three domains of life for transport and signaling. SBPs are composed of two domains that collectively trap ligands with high affinity and selectivity. To explore the role of the domains and the integrity of the hinge region between them in the function and conformation of SBPs, here we describe the ligand binding, conformational stability, and folding kinetics of the Lysine Arginine Ornithine binding protein (LAO) fromSalmonella thiphimuriumand constructs corresponding to its two independent domains. LAO is a class II SBP formed by a continuous and a discontinuous domain. Contrary to the expected behavior based on their connectivity, the discontinuous domain shows a stable native-like structure that binds L-arginine with moderate affinity, whereas the continuous domain is barely stable and shows no detectable ligand binding. Regarding folding kinetics, studies of the entire protein revealed the presence of at least two ...

Research paper thumbnail of On the molecular basis of the high affinity binding of basic amino acids to LAOBP, a periplasmic binding protein from Salmonella typhimurium

Journal of Molecular Recognition, 2015

The rational designing of binding abilities in proteins requires an understanding of the relation... more The rational designing of binding abilities in proteins requires an understanding of the relationship between structure and thermodynamics. However, our knowledge of the molecular origin of high-affinity binding of ligands to proteins is still limited; such is the case for l-lysine-l-arginine-l-ornithine periplasmic binding protein (LAOBP), a periplasmic binding protein from Salmonella typhimurium that binds to l-arginine, l-lysine, and l-ornithine with nanomolar affinity and to l-histidine with micromolar affinity. Structural studies indicate that ligand binding induces a large conformational change in LAOBP. In this work, we studied the thermodynamics of l-histidine and l-arginine binding to LAOBP by isothermal titration calorimetry. For both ligands, the affinity is enthalpically driven, with a binding ΔCp of ~-300 cal mol(-1) K(-1) , most of which arises from the burial of protein nonpolar surfaces that accompanies the conformational change. Osmotic stress measurements revealed that several water molecules become sequestered upon complex formation. In addition, LAOBP prefers positively charged ligands in their side chain. An energetic analysis shows that the protein acquires a thermodynamically equivalent state with both ligands. The 1000-fold higher affinity of LAOBP for l-arginine as compared with l-histidine is mainly of enthalpic origin and can be ascribed to the formation of an extra pair of hydrogen bonds. Periplasmic binding proteins have evolved diverse energetic strategies for ligand recognition. STM4351, another arginine binding protein from Salmonella, shows an entropy-driven micromolar affinity toward l-arginine. In contrast, our data show that LAOBP achieves nanomolar affinity for the same ligand through enthalpy optimization. Copyright © 2015 John Wiley &amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp;amp; Sons, Ltd.

Research paper thumbnail of The interplay of protein‐ligand and water‐mediated interactions shape affinity and selectivity in the LAO binding protein

The FEBS Journal

The study of binding thermodynamics is essential to understand how affinity and selectivity are a... more The study of binding thermodynamics is essential to understand how affinity and selectivity are acquired in molecular complexes. Periplasmic binding proteins (PBPs) are macromolecules of biotechnological interest that bind a broad number of ligands and have been used to design biosensors. The lysine‐arginine‐ornithine binding protein (LAO) is a PBP of 238 residues that binds the basic amino acids l‐arginine and l‐histidine with nm and μm affinity, respectively. It has been shown that the affinity difference for arginine and histidine binding is caused by enthalpy, this correlates with the higher number of protein–ligand contacts formed with arginine. In order to elucidate the structural bases that determine binding affinity and selectivity in LAO, the contribution of protein–ligand contacts to binding energetics was assessed. To this end, an alanine scanning of the LAO‐binding site residues was performed and arginine and histidine binding were characterized by isothermal titration calorimetry and X‐ray crystallography. Although unexpected enthalpy and entropy changes were observed in some mutants, thermodynamic data correlated with structural information, especially, the binding heat capacity change. We found that selectivity is conferred by several residues rather than exclusive arginine–protein interactions. Furthermore, crystallographic structures revealed that protein–ligand contributions to binding thermodynamics are highly influenced by the solvent. Finally, we found a similar backbone conformation in all the closed structures obtained, but different structures in the open state, suggesting that the binding site residues of LAO play an important role in stabilizing not only the holo conformation, but also the apo state.

Research paper thumbnail of The role of conserved non-aromatic residues in the Lactobacillus amylovorus α-amylase CBM26-starch interaction

International journal of biological macromolecules, Jan 15, 2018

It is generally accepted that carbohydrate binding modules (CBMs) recognize their carbohydrate li... more It is generally accepted that carbohydrate binding modules (CBMs) recognize their carbohydrate ligands by hydrophobic and CH-π interactions. Point mutations of one CBM26 of the Lactobacillus amylovorus α-amylase starch-binding domain (LaCBM26) showed that conserved non-aromatic residue are essential in the starch recognition function of the domain, as the mutation of a single glutamine (Q68L) eliminates binding to starch and β-cyclodextrin, even in the presence of aromatic amino acids necessary for ligand binding. The secondary structure of mutated proteins was verified and showed no differences from the wild-type domain. However, random mutations of five residues involved in binding (Y18, Y20, Q68, E74, and F77) did cause change in the secondary structure of the protein, which also causes loss of function. Much of the diversity introduced in the LaCBM26 was probably incompatible with the appropriate folding of these proteins, suggesting that the domain has little tolerance to change.

Research paper thumbnail of Expression of recombinant SnRK1 in E. coli. Characterization of adenine nucleotide binding to the SnRK1.1/AKINβγ-β3 complex

Plant science : an international journal of experimental plant biology, 2017

The SnRK1 complexes in plants belong to the family of AMPK/SNF1 kinases, which have been associat... more The SnRK1 complexes in plants belong to the family of AMPK/SNF1 kinases, which have been associated with the control of energy balance, in addition to being involved in the regulation of other aspects of plant growth and development. Analysis of complex formation indicates that increased activity is achieved when the catalytic subunit is phosphorylated and bound to regulatory subunits. SnRK1.1 subunit activity is higher than that of SnRK1.2, which also exhibits reduced activation due to the regulatory subunits. The catalytic phosphomimetic subunits (T175/176D) do not exhibit high activity levels, which indicate that the amino acid change does not produce the same effect as phosphorylation. Based on the mammalian AMPK X-ray structure, the plant SnRK1.1/AKINβγ-β3 was modeled by homology modeling and Molecular Dynamics simulations (MD). The model predicted an intimate and extensive contact between a hydrophobic region of AKINβγ and the β3 subunit. While the AKINβγ prediction retains th...

Research paper thumbnail of Article Energetic Effects of Magnesium in the Recognition of Adenosine Nucleotides by the F1-ATPase β Subunit

Research paper thumbnail of Current applications of isothermal titration calorimetry to the study of protein complexes

The absorption or liberation of heat has proven a widely spread property in biomolecular processe... more The absorption or liberation of heat has proven a widely spread property in biomolecular processes. Isothermal titration calorimetry (ITC) measures this property directly. This feature not only implies high precision in determining the binding enthalpy, but also allows us to infer the reaction mechanism in a more objective way than many non-calorimetric techniques. In this chapter, the principles of ITC are reviewed together with the basic thermodynamic formalism on which the technique is based. In addition, the current state of the art in calorimetry in protein recognition is described, with particular emphasis on advances in the last few years.

Research paper thumbnail of A group 6 late embryogenesis abundant protein from common bean is a disordered protein with extended helical structure and oligomer-forming properties

The Journal of biological chemistry, Jan 14, 2014

Late embryogenesis-abundant proteins accumulate to high levels in dry seeds. Some of them also ac... more Late embryogenesis-abundant proteins accumulate to high levels in dry seeds. Some of them also accumulate in response to water deficit in vegetative tissues, which leads to a remarkable association between their presence and low water availability conditions. A major sub-group of these proteins, also known as typical LEA proteins, shows high hydrophilicity and a high percentage of glycine and other small amino acid residues, distinctive physicochemical properties that predict a high content of structural disorder. Although all typical LEA proteins share these characteristics, seven groups can be distinguished by sequence similarity, indicating structural and functional diversity among them. Some of these groups have been extensively studied; however, others require a more detailed analysis to advance in their functional understanding. In this work, we report the structural characterization of a group 6 LEA protein from a common bean (Phaseolus vulgaris L.) (PvLEA6) by circular dichr...

Research paper thumbnail of Phases Dispersion and Oxygen Transfer in a Simulated Fermentation Broth Containing Castor Oil and Proteins

Biotechnology Progress, 2004

The sizes of air bubbles and castor oil drops were studied by image analysis as a function of the... more The sizes of air bubbles and castor oil drops were studied by image analysis as a function of the concentration of soluble protein (bovine serum albumin [BSA] and lipase, as model proteins) in a three-phase system using a simulated fermentation medium (aqueous salt solution, castor oil, and air). Small amounts of proteins (<0.02 g/L) caused an important decrease in oil drops and bubbles sizes, together with a pronounced decrease in surface tension. The extent and profiles of this decrease seem to be determined by the conformation of the protein at the interface. The k L a value increased considerably for increasing concentration (up to 0.02 g/L) of the two proteins but was very different (2-fold higher for the lipase) at the highest concentrations tested (0.5 g/L), a phenomenon that can be caused by the extent to which bubbles are trapped within oil drops.

Research paper thumbnail of Multithermal titration calorimetry: A rapid method to determine binding heat capacities

Biophysical Chemistry, 2006

Herein a new method that allows binding DCp to be determined with a single experiment is presente... more Herein a new method that allows binding DCp to be determined with a single experiment is presented. Multithermal titration calorimetry (MTC) is a simple extension of isothermal titration calorimetry (ITC) that explicitly takes into account the thermal dependences of DH and the binding constant. Experimentally, this is accomplished by performing a single stepwise titration with ITC equipment, allowing temperature readjustments of the system at intermediate states of the titration process. Thus, from the resulting multitherm, DCp can also be determined. The experimental feasibility of MTC was tested by using the well-characterized lysozyme -chitotriose complex as a model system. D

Research paper thumbnail of Energetic Effects of Magnesium in the Recognition of Adenosine Nucleotides by the F 1 -ATPase β Subunit

Biochemistry, 2010

Nucleotide-induced conformational changes of the catalytic β subunits play a crucial role in the ... more Nucleotide-induced conformational changes of the catalytic β subunits play a crucial role in the rotary mechanism of F 1 -ATPase. To gain insights into the energetic bases that govern the recognition of nucleotides by the isolated β subunit from thermophilic Bacillus PS3 (Tβ), the binding of this monomer to Mg(II)-free and Mg(II)bound adenosine nucleotides was characterized using high-precision isothermal titration calorimetry. The interactions of Mg(II) with free ATP or ADP were also measured calorimetrically. A model that considers simultaneously the interactions of Tβ with Mg 3 ATP or with ATP and in which ATP is able to bind two Mg(II) atoms sequentially was used to determine the formation parameters of the Tβ-Mg 3 ATP complex from calorimetric data. This analysis yielded significantly different ΔH b and ΔS b values in relation to those obtained using a single-binding site model, while ΔG b was almost unchanged. Published calorimetric data for the titration of Tβ with Mg 3 ADP [P erez-Hern andez, G., et al. Arch. Biochem. Biophys. 408, 177-183] were reanalyzed with the ternary model to determine the corresponding true binding parameters. Interactions of Tβ with Mg 3 ATP, ATP, Mg 3 ADP, or ADP were enthalpically driven. Larger differences in thermodynamic properties were observed between Tβ-Mg 3 ATP and Tβ-ATP complexes than between Tβ-Mg 3 ADP and Tβ-ADP complexes or between Tβ-Mg 3 ATP and Tβ-Mg 3 ADP complexes. These binding data, in conjunction with those for the association of Mg(II) with free nucleotides, allowed for a determination of the energetic effects of the metal ion on the recognition of adenosine nucleotides by Tβ [i.e., Tβ 3 AT(D)P þ Mg(II) h Tβ 3 AT(D)P-Mg]. Because of a more favorable binding enthalpy, Mg(II) is recognized more avidly by the Tβ 3 ATP complex, indicating better stereochemical complementarity than in the Tβ 3 ADP complex. Furthermore, a structural-energetic analysis suggests that Tβ adopts a more closed conformation when it is bound to Mg 3 ATP than to ATP or Mg 3 ADP, in agreement with recently published NMR data ) J. Biol. Chem. 284, 2374-2382. Using published binding data, a similar analysis of Mg(II) energetic effects was performed for the free energy change of F 1 catalytic sites, in the framework of bi-or tri-site binding models. 1 Abbreviations: Mg(II), free magnesium ion; AMPPNP, adenylyl 5 0 -imidodiphosphate; P i , inorganic phosphate; EDTA, ethylenediaminetetraacetic acid; Tris, 2-amino-2-(hydroxymethyl)propane-1,3diol; MgCl 2 , magnesium chloride; NaOH, sodium hydroxide; ATPase, adenosine 5 0 -triphosphatase; Tβ, isolated β subunit from thermophilic Bacillus PS3; TF 1 , F 1 sector from Bacillus PS3; EF 1 , F 1 sector from Escherichia coli; S1-S3, high-, medium-, and low-affinity β subunit sites in F 1 , respectively; β DP and β TP , β subunits in the crystal structure of F 1 bound to Mg 3 ADP and Mg 3 AMPPNP, respectively; ITC, isothermal titration calorimetry; NMR, nuclear magnetic resonance; K b , equilibrium binding constant; ΔH b , binding enthalpy; ΔG b , Gibbs binding free energy; ΔS b , binding entropy; ΔC pb , binding heat capacity; K b1 and K b2 , stepwise association constants for the interaction of ATP with Mg(II); K P1 and K P2 , association constants for ATP and Mg 3 ATP, respectively, binding to Tβ; ΔH int , intrinsic binding enthalpy; ΔH dsolv , desolvation enthalpy; ΔS conf , ΔS solv , and ΔS r-t , conformational, solvation, and roto-translational entropy changes, respectively; ΔA, surface area change; Δc pi , specific heat capacity for the surface area of the type i; R, cooperative heterotropic association constant; Δh, cooperative enthalpy; Δs, cooperative entropy.

Research paper thumbnail of On the molecular basis of the high affinity binding of basic amino acids to LAOBP, a periplasmic binding protein from Salmonella typhimurium

Journal of Molecular Recognition, 2015

The rational designing of binding abilities in proteins requires an understanding of the relation... more The rational designing of binding abilities in proteins requires an understanding of the relationship between structure and thermodynamics. However, our knowledge of the molecular origin of high-affinity binding of ligands to proteins is still limited; such is the case for L-lysine-L-arginine-L-ornithine periplasmic binding protein (LAOBP), a periplasmic binding protein from Salmonella typhimurium that binds to L-arginine, L-lysine, and L-ornithine with nanomolar affinity and to L-histidine with micromolar affinity. Structural studies indicate that ligand binding induces a large conformational change in LAOBP. In this work, we studied the thermodynamics of L-histidine and L-arginine binding to LAOBP by isothermal titration calorimetry. For both ligands, the affinity is enthalpically driven, with a binding ΔCp of~À300 cal mol À1 K À1 , most of which arises from the burial of protein nonpolar surfaces that accompanies the conformational change. Osmotic stress measurements revealed that several water molecules become sequestered upon complex formation. In addition, LAOBP prefers positively charged ligands in their side chain. An energetic analysis shows that the protein acquires a thermodynamically equivalent state with both ligands. The 1000-fold higher affinity of LAOBP for L-arginine as compared with L-histidine is mainly of enthalpic origin and can be ascribed to the formation of an extra pair of hydrogen bonds. Periplasmic binding proteins have evolved diverse energetic strategies for ligand recognition. STM4351, another arginine binding protein from Salmonella, shows an entropy-driven micromolar affinity toward L-arginine. In contrast, our data show that LAOBP achieves nanomolar affinity for the same ligand through enthalpy optimization.