Enrique García-Hernández

Structural energetics of protein–carbohydrate interactions: Insights derived from the study of lysozyme binding to its natural saccharide inhibitors

High‐sensitivity isothermal titration calorimetry was used to characterize the binding of the glycohydrolitic enzyme hen egg‐white lysozyme to its natural saccharide inhibitors, chitobiose and chitrotriose. Measurements were done at a pH of 4.7, in the 15°C −45°C temperature range. Using a structural‐energetic parameterization derived previously for lectin‐carbohydrate associations, both binding enthalpies and entropies for the present systems and for the complex of chitobiose with turkey egg‐white lysozyme from the literature were correctly accounted for. These observations suggest that both lysozymes and lectins follow the same structural‐energetic behavior in the binding to their ligands. From the analysis of lysozyme data in conjunction with other binding data reported in the literature, an ad hoc parameterization of ΔCp for protein–carbohydrate complexes was derived for the first time. The novel parameters for both polar and apolar surface areas differed significantly from correlations obtained previously from model compounds and protein‐folding data. As ΔCp is extremely sensitive to changes in solvent structure, this finding indicates that protein–carbohydrate complexes have distinctive hydration properties. According to our analysis, the dehydration of polar groups is the major cause for the observed decrease in ΔCp, which implies that these groups behave hydrophobically. The contribution of apolar surface areas was found of the expected sign, but their specific weight is much smaller than those obtained in other correlations. This small contribution to ΔCp is consistent with Lemieuxs hypothesis of a low degree of hydration of apolar surfaces on carbohydrates.

Energetics of protein homodimerization: Effects of water sequestering on the formation of β-lactoglobulin dimer

Transient protein–protein interactions are functionally relevant as a control mechanism in a variety of biological processes. Analysis of the 3D structure of protein–protein complexes indicates that water molecules trapped at the interface are very common; however, their role in the stability and specificity of protein homodimer interactions has been not addressed yet. To provide new insights into the energetic bases that govern the formation of highly hydrated interfaces, the dissociation process of bovine βlg variant A at a neutral pH was characterized here thermodynamically by conducting dilution experiments with an isothermal titration calorimeter. Association was enthalpically driven throughout the temperature range spanned. ΔH and ΔCp were significantly more negative than estimates based on surface area changes, suggesting the occurrence of effects additional to the dehydration of the contact surfaces between subunits. Near‐UV CD spectra proved to be independent of protein concentration, indicating a rigid body‐like association. Furthermore, the process proved not to be coupled to significant changes in the protonation state of ionizable groups or counterion exchange. In contrast, both osmotic stress experiments and a computational analysis of the dimers 3D structure indicated that a large number of water molecules are incorporated into the interface upon association. Numerical estimates considering the contributions of interface area desolvation and water immobilization accounted satisfactorily for the experimental ΔCp. Thus, our study highlights the importance of explicitly considering the effects of water sequestering to perform a proper quantitative analysis of the formation of homodimers with highly hydrated interfaces. Proteins 2008.

Role of vitamin B12 on methylmalonyl-CoA mutase activity

Vitamin B12 is an organometallic compound with important metabolic derivatives that act as cofactors of certain enzymes, which have been grouped into three subfamilies depending on their cofactors. Among them, methylmalonyl-CoA mutase (MCM) has been extensively studied. This enzyme catalyzes the reversible isomerization of L-methylmalonyl-CoA to succinyl-CoA using adenosylcobalamin (AdoCbl) as a cofactor participating in the generation of radicals that allow isomerization of the substrate. The crystal structure of MCM determined in Propionibacterium freudenreichii var. shermanii has helped to elucidate the role of this cofactor AdoCbl in the reaction to specify the mechanism by which radicals are generated from the coenzyme and to clarify the interactions between the enzyme, coenzyme, and substrate. The existence of human methylmalonic acidemia (MMA) due to the presence of mutations in MCM shows the importance of its role in metabolism. The recent crystallization of the human MCM has shown that despite being similar to the bacterial protein, there are significant differences in the structural organization of the two proteins. Recent studies have identified the involvement of an accessory protein called MMAA, which interacts with MCM to prevent MCM’s inactivation or acts as a chaperone to promote regeneration of inactivated enzyme. The interdisciplinary studies using this protein as a model in different organisms have helped to elucidate the mechanism of action of this isomerase, the impact of mutations at a functional level and their repercussion in the development and progression of MMA in humans. It is still necessary to study the mechanisms involved in more detail using new methods.

Ligand binding and self-association cooperativity of β-lactoglobulin.

Unlike most small globular proteins, lipocalins lack a compact hydrophobic core. Instead, they present a large central cavity that functions as the primary binding site for hydrophobic molecules. Not surprisingly, these proteins typically exhibit complex structural dynamics in solution, which is intricately modified by intermolecular recognition events. Although many lipocalins are monomeric, an increasing number of them have been proven to form oligomers. The coupling effects between self‐association and ligand binding in these proteins are largely unknown. To address this issue, we have calorimetrically characterized the recognition of dodecyl sulfate by bovine β‐lactoglobulin, which forms weak homodimers at neutral pH. A thermodynamic analysis based on coupled‐equilibria revealed that dimerization exerts disparate effects on the ligand‐binding capacity of β‐lactoglobulin. Protein dimerization decreases ligand affinity (or, reciprocally, ligand binding promotes dimer dissociation). The two subunits in the dimer exhibit a positive, entropically driven cooperativity. To investigate the structural determinants of the interaction, the crystal structure of β‐lactoglobulin bound to dodecyl sulfate was solved at 1.64 Å resolution. Copyright

Structure and dynamics of β-lactoglobulin in complex with dodecyl sulfate and laurate: A molecular dynamics study

Bovine β-lactoglobulin (βlg) is able to recognize a wide variety of hydrophobic ligands. Although binding promiscuity is characteristic of highly hydrophobic interactions, the structural plasticity of the βlg binding cavity entrance seems to be crucial for the interaction with polar moieties of different ligands. On the other hand, thermodynamic studies have shown that βlg can associate to cognate ligands with distinctly different binding energetics, as in the case of the closely related molecules lauric acid (LA) and dodecyl sulfate (DS). In the recognition of LA, βlg shows a classical hydrophobic signature (entropically driven), whereas the interaction of βlg with DS exhibits a nonclassical hydrophobic signature (enthalpically driven). To gain insights into these opposed binding behaviors, MD simulations were carried out on βlg in apo-form and bound to DS or LA. Overall, the results suggested that the distinct energetic signatures of these ligands come from distinct optimizations of both hydrophilic and hydrophobic contacts with the protein.

Influence of pH control in the formation of inclusion bodies during production of recombinant sphingomyelinase-D in Escherichia coli

BackgroundInclusion bodies (IBs) are aggregated proteins that form clusters when protein is overexpressed in heterologous expression systems. IBs have been considered as non-usable proteins, but recently they are being used as functional materials, catalytic particles, drug delivery agents, immunogenic structures, and as a raw material in recombinant therapeutic protein purification. However, few studies have been made to understand how culture conditions affect the protein aggregation and the physicochemical characteristics that lead them to cluster. The objective of our research was to understand how pH affects the physicochemical properties of IBs formed by the recombinant sphingomyelinase-D of tick expressed in E. coli BL21-Gold (DE3) by evaluating two pH culture strategies.ResultsUncontrolled pH culture conditions favored recombinant sphingomyelinase-D aggregation and IB formation. The IBs of sphingomyelinase-D produced under controlled pH at 7.5 and after 24 h were smaller (<500 nm) than those produced under uncontrolled pH conditions (>500 nm). Furthermore, the composition, conformation and β-structure formation of the aggregates were different. Under controlled pH conditions in comparison to uncontrolled conditions, the produced IBs presented higher resistance to denaturants and proteinase-K degradation, presented β-structure, but apparently as time passes the IBs become compacted and less sensitive to amyloid dye binding.ConclusionsThe manipulation of the pH has an impact on IB formation and their physicochemical characteristics. Particularly, uncontrolled pH conditions favored the protein aggregation and sphingomyelinase-D IB formation. The evidence may lead to find methodologies for bioprocesses to obtain biomaterials with particular characteristics, extending the application possibilities of the inclusion bodies.

Amino acid sequence, biochemical characterization, and comparative modeling of a nonspecific lipid transfer protein from Amaranthus hypochondriacus

Plant nonspecific lipid transfer proteins (nsLTPs) are characterized by their ability to bind a broad range of hydrophobic ligands in vitro. Their biological function has not yet been elucidated, but they could play a major role in plant defense to physical and biological stress. An nsLTP was isolated from Amaranthus hypochondriacus seeds and purified by gel filtration and reversed-phase high-performance liquid chromatography techniques. The molecular mass of the protein as determined by mass spectrometry is 9747.29 Da. Data from amino acid sequence, circular dichroism and binding/displacement of a fluorescent lipid revealed that it belongs to the nsLTP1 family. The protein shows the alpha-helical secondary structure typical for plant nsLTPs 1 and shares 40 to 57% sequence identity with nsLTPs 1 from other plant species and 100% identity with an nsLTP1 from Amaranthus caudatus. A model structure of the protein in complex with stearate based on known structures of maize and rice nsLTPs 1 suggests a protein fold complexed with lipids closely related to that of maize nsLTP1.

A Group 6 Late Embryogenesis Abundant Protein from Common Bean Is a Disordered Protein with Extended Helical Structure and Oligomer-forming Properties

Background: Late embryogenesis-abundant proteins accumulate under water-deficit and are widely distributed in plants and anhydrobiotic organisms. Results: A common bean group-6 LEA protein shows structural disorder, adopts helicity under water deficit or high molecular crowding, and presents oligomeric forms. Conclusion: PvLEA6 protein adopts different conformations and/or a quaternary structure depending on its environment. Significance: Similar characteristics might be present in different LEA proteins, which could be relevant to their function. 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 dichroism and nuclear magnetic resonance showing that it is a disordered protein in aqueous solution. Using the same techniques, we show that despite its unstructured nature, the addition of trifluoroethanol exhibited an intrinsic potential in this protein to gain helicity. This property was also promoted by high osmotic potentials or molecular crowding. Furthermore, we demonstrate that PvLEA6 protein is able to form soluble homo-oligomeric complexes that also show high levels of structural disorder. The association between PvLEA6 monomers to form dimers was shown to occur in plant cells by bimolecular fluorescence complementation, pointing to the in vivo functional relevance of this association.

Energetic effects of magnesium in the recognition of adenosine nucleotides by the F(1)-ATPase beta subunit.

Nucleotide-induced conformational changes of the catalytic beta 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 beta subunit from thermophilic Bacillus PS3 (Tbeta), 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 Tbeta with Mg.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 Tbeta-Mg.ATP complex from calorimetric data. This analysis yielded significantly different DeltaH(b) and DeltaS(b) values in relation to those obtained using a single-binding site model, while DeltaG(b) was almost unchanged. Published calorimetric data for the titration of Tbeta with Mg.ADP [Perez-Hernandez, G., et al. (2002) Arch. Biochem. Biophys. 408, 177-183] were reanalyzed with the ternary model to determine the corresponding true binding parameters. Interactions of Tbeta with Mg.ATP, ATP, Mg.ADP, or ADP were enthalpically driven. Larger differences in thermodynamic properties were observed between Tbeta-Mg.ATP and Tbeta-ATP complexes than between Tbeta-Mg.ADP and Tbeta-ADP complexes or between Tbeta-Mg.ATP and Tbeta-Mg.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 Tbeta [i.e., Tbeta.AT(D)P + Mg(II) right harpoon over left harpoon Tbeta.AT(D)P-Mg]. Because of a more favorable binding enthalpy, Mg(II) is recognized more avidly by the Tbeta.ATP complex, indicating better stereochemical complementarity than in the Tbeta.ADP complex. Furthermore, a structural-energetic analysis suggests that Tbeta adopts a more closed conformation when it is bound to Mg.ATP than to ATP or Mg.ADP, in agreement with recently published NMR data [Yagi, H., et al. (2009) 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.

Ligand entry into the calyx of β‐lactoglobulin

Although the thermodynamic principles that control the binding of drug molecules to their protein targets are well understood, the detailed process of how a ligand reaches a protein binding site has been an intriguing question over decades. The short time interval between the encounter between a ligand and its receptor to the formation of the stable complex has prevented experimental observations. Bovine β‐lactoglobulin (βlg) is a lipocalin member that carries fatty acids (FAs) and other lipids in the cellular environment. Βlg accommodates a FA molecule in its highly hydrophobic cavity and exhibits the capability of recognizing a wide variety of hydrophobic ligands. To elucidate the ligand entry process on βlg, we report molecular dynamics simulations of the encounter between palmitate (PA) or laurate (LA) and βlg. Our results show that residues localized in loops at the cavity entrance play an important role in the ligand penetration process. Analysis of the short‐term interaction energies show that the forces operating on the systems lead to average conformations very close to the crystallographic holo‐forms. Whereas the binding free energy analysis using the molecular mechanics Generalized Born surface area method shows that these conformations were thermodynamically favorable.