RESUMEN
Glycosomal malate dehydrogenase from Trypanosoma cruzi (tcgMDH) catalyzes the oxidation/reduction of malate/oxaloacetate, a crucial step of the glycolytic process occurring in the glycosome of the human parasite. Inhibition of tcgMDH is considered a druggable trait for the development of trypanocidal drugs. Sequence comparison of MDHs from different organisms revealed a distinct insertion of a prolin rich 9-mer (62-KLPPVPRDP-70) in tcgMDH as compared to other eukaryotic MDHs. Crystal structure of tcgMDH is solved here at 2.6 Å resolution with Rwork/Rfree values of 0.206/0.216. The tcgMDH forms homo-dimer with the solvation free energy (ΔGo) gain of -9.77 kcal/mol. The dimeric form is also confirmed in solution by biochemical assays, chemical-crosslinking and dynamic light scattering. The inserted 9-mer adopts a structure of a solvent accessible loop in the vicinity of NAD+ binding site. The distinct sequence and structural feature of tcgMDH, revealed in the present report, provides an anchor point for the development of inhibitors specific for tcgMDH, possible trypanocidal agents of the future.
Asunto(s)
Malato Deshidrogenasa/química , Trypanosoma cruzi/metabolismo , Secuencia de Aminoácidos , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Dispersión Dinámica de Luz , Escherichia/metabolismo , Malato Deshidrogenasa/metabolismo , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Proteínas Recombinantes , Alineación de Secuencia , Trypanosoma cruzi/química , Trypanosoma cruzi/enzimologíaRESUMEN
Recombinant proteins play an important role in medicine and have diverse applications in industrial biotechnology. Lactoglobulin has shown great potential for use in targeted drug delivery and body fluid detoxification because of its ability to bind a variety of molecules. In order to modify the biophysical properties of ß-lactoglobulin, a series of single-site mutations were designed using a structure-based approach. A 3-dimensional structure alignment of homologous molecules led to the design of nine ß-lactoglobulin variants with mutations introduced in the binding pocket region. Seven stable and correctly folded variants (L39Y, I56F, L58F, V92F, V92Y, F105L, M107L) were thoroughly characterized by fluorescence, circular dichroism, isothermal titration calorimetry, size-exclusion chromatography, and X-ray structural investigations. The effects of the amino acid substitutions were observed as slight rearrangements of the binding pocket geometry, but they also significantly influenced the global properties of the protein. Most of the mutations increased the thermal/chemical stability without altering the dimerization constant or pH-dependent conformational behavior. The crystal structures reveal that the I56F and F105L mutations reduced the depth of the binding pocket, which is advantageous since it can reduce the affinity to endogenous fatty acids. The F105L mutant created a unique binding mode for a fatty acid, supporting the idea that lactoglobulin can be altered to bind unique molecules. Selected variants possessing a unique combination of their individual properties can be used for further, more advanced mutagenesis, and the presented results support further research using ß-lactoglobulin as a therapeutic delivery agent or a blood detoxifying molecule.
Asunto(s)
Lactoglobulinas/genética , Mutagénesis Sitio-Dirigida/métodos , Animales , Humanos , Lipocalinas/genética , Ingeniería de ProteínasRESUMEN
The ß-d-glucuronidase DtGlcA from Dictyoglomus thermophilum was engineered to generate an active thioglycoligase that is able to catalyse the formation of numerous S-glucuronides. Its X-ray structure analysis indicated the ability of the biocatalyst to bind aromatic thiol acceptors for S-glycosylation. Noteworthily, the DtGlcA mutant was found to be the first thioligase that is able to use a natural sugar donor different from the widely used synthetic para-nitrophenyl glycosides.
RESUMEN
Molybdenum is an essential nutrient for metabolism in plant, bacteria, and animals. Molybdoenzymes are involved in nitrogen assimilation and oxidoreductive detoxification, and bioconversion reactions of environmental, industrial, and pharmaceutical interest. Molybdoenzymes contain a molybdenum cofactor (Moco), which is a pyranopterin heterocyclic compound that binds a molybdenum atom via a dithiolene group. Because Moco is a large and complex compound deeply buried within the protein, molybdoenzymes are accompanied by private chaperone proteins responsible for the cofactor's insertion into the enzyme and the enzyme's maturation. An efficient recombinant expression and purification of both Moco-free and Moco-containing molybdoenzymes and their chaperones is of paramount importance for fundamental and applied research related to molybdoenzymes. In this work, we focused on a D1 protein annotated as a chaperone of steroid C25 dehydrogenase (S25DH) from Sterolibacterium denitrificans Chol-1S. The D1 protein is presumably involved in the maturation of S25DH engaged in oxygen-independent oxidation of sterols. As this chaperone is thought to be a crucial element that ensures the insertion of Moco into the enzyme and consequently, proper folding of S25DH optimization of the chaperon's expression is the first step toward the development of recombinant expression and purification methods for S25DH. We have identified common E. coli strains and conditions for both expression and purification that allow us to selectively produce Moco-containing and Moco-free chaperones. We have also characterized the Moco-containing chaperone by EXAFS and HPLC analysis and identified conditions that stabilize both forms of the protein. The protocols presented here are efficient and result in protein quantities sufficient for biochemical studies.
Asunto(s)
Proteínas Bacterianas , Coenzimas , Escherichia coli/metabolismo , Expresión Génica , Metaloproteínas , Chaperonas Moleculares , Nitrosomonadaceae/genética , Pteridinas , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Coenzimas/biosíntesis , Coenzimas/química , Coenzimas/genética , Coenzimas/aislamiento & purificación , Escherichia coli/química , Escherichia coli/genética , Metaloproteínas/biosíntesis , Metaloproteínas/química , Metaloproteínas/genética , Metaloproteínas/aislamiento & purificación , Chaperonas Moleculares/biosíntesis , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/aislamiento & purificación , Cofactores de Molibdeno , Nitrosomonadaceae/metabolismo , Pteridinas/química , Pteridinas/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificaciónRESUMEN
Ovine ß-lactoglobulin has been isolated from whey fraction of sheep milk and crystallized. The high-resolution structures of two crystal forms (triclinic and trigonal) obtained at pH 7.0 have been determined revealing that ovine protein, similarly to its bovine analog, is dimeric. Access to the binding site located in the eight-stranded antiparallel ß-barrel in both structures is blocked by the EF loop that has been found in closed conformation. Similarly to bovine lactoglobulin (BLG), conformation of the EF loop is stabilized by hydrogen bond between Glu89 and Ser116 indicating that Tanford transition might occur with the same mechanism. The substitution at six positions in relation to the most abundant isoform B of BLG also affects the distribution of electrostatic potentials and the total charge.
Asunto(s)
Lactoglobulinas/química , Alérgenos/química , Alérgenos/inmunología , Secuencia de Aminoácidos , Animales , Cromatografía en Gel , Cristalización , Cristalografía por Rayos X , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , Alineación de Secuencia , Ovinos , Electricidad EstáticaRESUMEN
The structure of onconase C30A/C75A double mutant has been determined at 1.12Å resolution. The structure has high structural homology to other onconase structures. The changes being results of mutation are relatively small, distributed asymmetrically around the two mutated positions, and they are observed not only in the mutation region but expanded to entire molecule. Different conformation of Lys31 side chain that influences the hydrogen bonding network around catalytic triad is probably responsible for lower catalytic efficiency of double mutant. The decrease in thermal stability observed for the onconase variant might be explained by a less dense packing as manifested by the increase of the molecular volume and the solvent accessible surface area.
Asunto(s)
Modelos Moleculares , Mutación/genética , Ribonucleasas/química , Animales , Dominio Catalítico , Cristalografía por Rayos X , Electricidad EstáticaRESUMEN
Isoforms A (LGB-A) and B (LGB-B) of bovine lactoglobulin, the milk protein, differ in positions 64 (DâG) and 118 (VâA). Interactions of LGB-A and LGB-B with sodium dodecyl sulfate (SDS), dodecyltrimethylammonium chloride (DTAC) and lauric acid (LA), 12-carbon ligands possessing differently charged polar groups, were investigated using isothermal titration calorimetry and X-ray crystallography, to study the proton linkage phenomenon and to distinguish between effects related to different isoforms and different ligand properties. The determined values of ΔS and ΔH revealed that for all ligands, binding is entropically driven. The contribution from enthalpy change is lower and shows strong dependence on type of buffer that indicates proton release from the protein varying with protein isoform and ligand type and involvement of LA and Asp64 (in isoform A) in this process. The ligand affinities for both isoforms were arranged in the same order, DTAC < LA < SDS, and were systematically lower for variant B. The entropy change of the complexation process was always higher for isoform A, but these values were compensated by changes in enthalpy, resulting in almost identical ΔG for complexes of both isoforms. The determined crystal structures showed that substitution in positions 64 and 118 did not influence the overall structure of LGB complexes. The chemical character of the ligand polar group did not affect the position of its aliphatic chain in protein ß-barrel, indicating a major role of hydrophobic interactions in ligand binding that prevailed even with the repulsion between positively charged DTAC and lysine residues located at binding site entrance.
Asunto(s)
Calorimetría/métodos , Lactoglobulinas/química , Lactoglobulinas/metabolismo , Animales , Carbono , Bovinos , Cristalografía por Rayos X , Ácidos Láuricos/química , Ligandos , Unión Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Compuestos de Amonio Cuaternario/química , Dodecil Sulfato de Sodio/química , TermodinámicaRESUMEN
New and original heterocyclic α-enamido phosphine chiral solutes were prepared: four structurally similar racemates with the chirality center placed on the phosphorus atom, and four other related pairs of enantiomers with chirality borne by the carbon atoms of the phospholane ring. The structural variations were placed on an aliphatic heterocycle (six- or seven-member rings) and on the carbamate function (methyl or t-butyl). Their separation was achieved on a commercial cellulose tris-(3,5-dimethylphenylcarbamate) stationary phase (Lux Cellulose-1, Phenomenex) in supercritical fluid chromatography (SFC). The effects of molecular structure on SFC retention and enantioresolution were studied. Among these eight pairs of enantiomers, some reversal of elution order between similar compounds was observed. The effect of changing the organic solvent (methanol and ethanol) and its proportion (between 5 and 40%) in the mobile phase was investigated. Retention data were collected over the temperature range 0-50 °C, and the results interpreted from thermodynamic aspects.
RESUMEN
The homodimeric ß-lactoglobulin belongs to the lipocalin family of proteins that transport a wide range of hydrophobic molecules and can be modified by mutagenesis to develop specificity for novel groups of ligands. In this work, new lactoglobulin variants, FAF (I56F/L39A/M107F) and FAW (I56F/L39A/M107W), were produced and their interactions with the tricyclic drug desipramine (DSM) were studied using X-ray crystallography, calorimetry (ITC) and circular dichroism (CD). The ITC and CD data showed micromolar affinity of the mutants for DSM and interactions according to the classical one-site binding model. However, the crystal structures unambiguously showed that the FAF and FAW dimers are capable of binding DSM not only inside the ß-barrel as expected, but also at the dimer interface and at the entrance to the binding pocket. The presented high-resolution crystal structures therefore provide important evidence of the existence of alternative ligand-binding sites in the ß-lactoglobulin molecule. Analysis of the crystal structures highlighted the importance of shape complementarity for ligand recognition and selectivity. The binding sites identified in the crystal structures of the FAF-DSM and FAW-DSM complexes together with data from the existing literature are used to establish a systematic classification of the ligand-binding sites in the ß-lactoglobulin molecule.
RESUMEN
Lactoglobulin is a natural protein present in bovine milk and common component of human diet, known for binding with high affinity wide range of hydrophobic compounds, among them fatty acids 12-20 carbon atoms long. Shorter fatty acids were reported as not binding to ß-lactoglobulin. We used X-ray crystallography and fluorescence spectroscopy to show that lactoglobulin binds also 8- and 10-carbon caprylic and capric acids, however with lower affinity. The determined apparent association constant for lactoglobulin complex with caprylic acid is 10.8 ± 1.7 × 10(3) M(-1), while for capric acid is 6.0 ± 0.5 × 10(3) M(-1). In crystal structures determined with resolution 1.9 Å the caprylic acid is bound in upper part of central calyx near polar residues located at CD loop, while the capric acid is buried deeper in the calyx bottom and does not interact with polar residues at CD loop. In both structures, water molecule hydrogen-bonded to carboxyl group of fatty acid is observed. Different location of ligands in the binding site indicates that competition between polar and hydrophobic interactions is an important factor determining position of the ligand in ß-barrel.
Asunto(s)
Caprilatos/metabolismo , Ácidos Decanoicos/metabolismo , Lactoglobulinas/química , Lactoglobulinas/metabolismo , Animales , Caprilatos/química , Bovinos , Cristalografía por Rayos X , Ácidos Decanoicos/química , Humanos , Cinética , Modelos Moleculares , Unión Proteica , Estructura Secundaria de Proteína , Espectrometría de Fluorescencia , Electricidad EstáticaRESUMEN
ß-Lactoglobulin (BLG) like other lipocalins can be modified by mutagenesis to re-direct its ligand binding properties. Local site-directed mutagenesis was used to change the geometry of the BLG ligand binding pocket and therefore change BLG ligand preferences. The presented studies are focused on previously described mutants L39Y, I56F, L58F, F105L, and M107L and two new BLG variants, L39K and F105A, and their interactions with local anesthetic drug tetracaine. Binding of tetracaine to BLG mutants was investigated by X-ray crystallography. Structural analysis revealed that for tetracaine binding, the shape of the binding pocket seems to be a more important factor than the substitutions influencing the number of interactions. Analyzed BLG mutants can be classified according to their binding properties to variants: capable of binding tetracaine in the ß-barrel (L58F, M107L); capable of accommodating tetracaine on the protein surface (I56F) and unable to bind tetracaine (F105L). Variants L39K, L39Y, and F105A, had a binding pocket blocked by endogenous fatty acids. The new tetracaine binding site was found in the I56F variant. The site localized on the surface near Arg124 and Trp19 was previously predicted by in silico studies and was confirmed in the crystal structure.
Asunto(s)
Anestésicos Locales/metabolismo , Lactoglobulinas/genética , Lactoglobulinas/metabolismo , Proteínas Mutantes/metabolismo , Tetracaína/metabolismo , Sitios de Unión , Cristalización , Cristalografía por Rayos X/métodos , Ácidos Grasos/metabolismo , Ligandos , Modelos Moleculares , Mutagénesis , Mutación , Unión Proteica , Conformación Proteica en Lámina beta , Multimerización de Proteína , Estructura Terciaria de ProteínaRESUMEN
In this paper we report the synthesis and the structural and magnetic properties of the series of ionic compounds with general formula: M(I)[Co(bpy)(3)][Mo(CN)(8)] x nH(2)O (M(I) = Li, n = 8 (1), M(I) = K, n = 8 (2), M(I) = Rb, n = 8 (3), M(I) = Cs, n = 7.5 (4)). Solids 1-4 are characterized by the optical outer-sphere metal-to-metal charge transfer (MMCT) transition from Mo(IV) center to Co(III) center in the visible region and the Co(III)Mo(IV) <==> Co(II)Mo(V) spin equilibrium strongly dominated by the Co(III)Mo(IV) form. We show a gentle thermal treatment of diamagnetic compounds 1-4 leading to the dehydrated forms 1a-4a, which reveal a significant increase of paramagnetic contribution (from 0.5 to 2% to 30-40%). The rehydration allows to recover the diamagnetic phases 1b-4b of compositions and properties similar to those of 1-4. The irradiation of the dehydrated form 2a within the MMCT band in the Superconducting Quantum Interference Device (SQUID) cavity at T = 10 K causes further increase of the Co(II)Mo(V) contribution giving the metastable phase annealed back to the 2a phase after heating above T = 290 K. The IR, electron paramagnetic resonance (EPR), and X-ray photoelectron spectroscopy (XPS) spectroscopic data along with the magnetic data are interpreted in terms of strong modification of the Co(III)Mo(IV) <==> Co(II)Mo(V) equilibrium occurring in these systems.
RESUMEN
To investigate the structural origin of decreased pressure and temperature stability, the crystal structure of bovine pancreatic ribonuclease A variants V47A, V54A, V57A, I81A, I106A, and V108A was solved at 1.4-2.0 A resolution and compared with the structure of wild-type protein. The introduced mutations had only minor influence on the global structure of ribonuclease A. The structural changes had individual character that depends on the localization of mutated residue, however, they seemed to expand from mutation site to the rest of the structure. Several different parameters have been evaluated to find correlation with decrease of free energy of unfolding DeltaDeltaG(T), and the most significant correlation was found for main cavity volume change. Analysis of the difference distance matrices revealed that the ribonuclease A molecule is organized into five relatively rigid subdomains with individual response to mutation. This behavior consistent with results of unfolding experiments is an intrinsic feature of ribonuclease A that might be surviving remnants of folding intermediates and reflects the dynamic nature of the molecule.
Asunto(s)
Ribonucleasa Pancreática/química , Animales , Bovinos , Cristalografía por Rayos X/métodos , Escherichia coli/metabolismo , Modelos Moleculares , Mutagénesis , Mutagénesis Sitio-Dirigida , Mutación , Páncreas/enzimología , Pliegue de Proteína , Estructura Secundaria de Proteína , Ribonucleasas/química , Temperatura , TermodinámicaRESUMEN
A rapid and stereodivergent access to polyhydroxylated 10-azabicyclo[4.3.1]decanes as new calystegine analogues by way of a double benzotriazolyl/carbon nucleophile exchange followed by a ring-closing metathesis was achieved. Preliminary evaluation of the new compounds as glucocerebrosidase inhibitors was also performed.
Asunto(s)
Compuestos Bicíclicos Heterocíclicos con Puentes/química , Compuestos Bicíclicos Heterocíclicos con Puentes/síntesis química , Tropanos/química , Tropanos/síntesis química , Ciclización , Modelos Moleculares , Estructura Molecular , EstereoisomerismoRESUMEN
Ovine ßlactoglobulin was characterized by spectroscopic (CD), calorimetric (ITC) and X-ray structural studies. The structure of ovine ßlactoglobulin complex with decanol showed that tight packing of molecules in the crystalline phase enforces a distortion of protein flexible loops resulting in the formation of an asymmetric dimer. The loops surrounding ß-barrel in ovine lactoglobulin possessed the same conformational flexibility as observed previously in other lactoglobulins and the change of their conformation regulates the access to the binding pocket. The structure of asymmetric dimer revealed a new region in ß-barrel where ligand polar group can be located. These findings indicated protein adaptability to ligand dimensions and inter- and intramolecular interactions in the crystalline phase. Calorimetric and crystallographic studies provided the experimental evidence that ovine lactoglobulin is able to bind aliphatic ligands. Thermodynamic parameters of sodium dodecyl sulfate binding determined by ITC at pH 7.5 had Ka, ΔH, TΔS and ΔG values similar to those observed for bovine and caprine protein indicating the same mechanism of ligand binding.
Asunto(s)
Sitios de Unión/genética , Lactoglobulinas/química , Ligandos , Unión Proteica/genética , Animales , Calorimetría , Cristalografía por Rayos X , Lactoglobulinas/genética , Modelos Moleculares , Conformación Molecular/efectos de los fármacos , Pliegue de Proteína , Multimerización de Proteína , Ovinos , TermodinámicaRESUMEN
A number of studies were devoted to understanding an immunological effect of pressure-treated ß-lactoglobulin. In our previous work we have proved that high pressure significantly modifies ß-lactoglobulin conformation and consequently its physicochemical properties. Here, structure of ß-lactoglobulin complex with myristic acid determined at the highest accepted by the crystal pressure value of 550â¯MPa is reported. Our results structurally prove that pressure noticeably modifies positions of the major ß-lactoglobulin epitopes. Considering the biological impact of observed changes in epitope regions, high pressure ß-lactoglobulin structure presents a step forward in understanding the pressure modification of food protein allergenicity. The conformational changes of pressurized ß-lactoglobulin did not support the hypothesis that proteolytic digestion facilitated by pressure is caused by an exposure of the digestive sites. Our findings demonstrate that high pressure protein crystallography can potentially identify the most pressure-sensitive fragments in allergens, and can therefore support development of hypoallergenic food products.
Asunto(s)
Alérgenos/inmunología , Hipersensibilidad a los Alimentos/etiología , Lactoglobulinas/química , Presión , Epítopos , HumanosRESUMEN
The de novo pyrimidine biosynthesis pathway is essential for the proliferation of many pathogens. One of the pathway enzymes, dihydroorotase (DHO), catalyzes the reversible interconversion of N-carbamoyl-l-aspartate to 4,5-dihydroorotate. The substantial difference between bacterial and mammalian DHOs makes it a promising drug target for disrupting bacterial growth and thus an important candidate to evaluate as a response to antimicrobial resistance on a molecular level. Here, we present two novel three-dimensional structures of DHOs from Yersinia pestis (YpDHO), the plague-causing pathogen, and Vibrio cholerae (VcDHO), the causative agent of cholera. The evaluations of these two structures led to an analysis of all available DHO structures and their classification into known DHO types. Comparison of all the DHO active sites containing ligands that are listed in DrugBank was facilitated by a new interactive, structure-comparison and presentation platform. In addition, we examined the genetic context of characterized DHOs, which revealed characteristic patterns for different types of DHOs. We also generated a homology model for DHO from Plasmodium falciparum.
Asunto(s)
Dihidroorotasa/química , Dihidroorotasa/metabolismo , Pirimidinas/biosíntesis , Vibrio cholerae/enzimología , Yersinia pestis/enzimología , Secuencia de Aminoácidos , Dominio Catalítico , Dihidroorotasa/genética , Genómica , Malatos/metabolismo , Modelos Moleculares , Homología de Secuencia de Aminoácido , Zinc/metabolismoRESUMEN
Chlorpromazine (CPZ) is a phenothiazine acting as dopamine antagonist. Aside from application in schizophrenia therapy, chlorpromazine is found to be a putative inhibitor of proteins involved in cancers, heritable autism disorder and prion diseases. Four new ß-lactoglobulin variants with double or triple substitutions: I56F/L39A, F105L/L39A, I56F/L39A/M107F or F105L/L39A/M107F changing the shape of the binding pocket were produced and their chlorpromazine binding properties have been investigated by X-ray crystallography, circular dichroism, isothermal titration calorimetry and thermophoresis. The CD spectra and crystal structures revealed that mutations do not affect the protein overall structure but in comparison to WT protein, variants possessing I56F substitution had lower stability while mutation F105L increased melting temperature of the protein. The new variants showed affinity to chlorpromazine in the range 4.2-15.4â¯×â¯103â¯M-1. The CD spectra and crystal structures revealed complementarity of the binding pocket shape, to only one chlorpromazine chiral conformer. The (aR)-CPZ was bonded to variants containing I56F substitution while variants with F105L substitution preferred (aS)-CPZ.
Asunto(s)
Sustitución de Aminoácidos , Clorpromazina/química , Lactoglobulinas/química , Mutación Missense , Sitios de Unión , Dicroismo Circular , Cristalografía por Rayos X , Lactoglobulinas/genéticaRESUMEN
Functional recombinant bovine ß-lactoglobulin has been produced by expression in E. coli using an engineered protein gene and purified to homogeneity by applying a new protocol. Mutations L1A/I2S introduced into the protein sequence greatly facilitate in vivo cleavage of the N-terminal methionine, allowing correctly folded and soluble protein suitable for biochemical, biophysical and structural studies to be obtained. The use of gel filtration on Sephadex G75 at the last purification step enables protein without endogenous ligand to be obtained. The physicochemical properties of recombinant ß-lactoglobulin such as CD spectra, ligand binding (n, K a, ΔH, TΔS, ΔG), chemical and thermal stability (ΔG D, C mid) and crystal structure confirmed that the protein obtained is almost identical to the natural one. The substitutions of N-terminal residues did not influence the binding properties of the recombinant protein so that the lactoglobulin produced and purified according to our protocol is a good candidate for further engineering and potential use in pharmacology and medicine.
Asunto(s)
Lactoglobulinas/química , Lactoglobulinas/aislamiento & purificación , Secuencia de Aminoácidos , Animales , Sitios de Unión , Bovinos , Dicroismo Circular , Escherichia coli/genética , Lactoglobulinas/biosíntesis , Lactoglobulinas/genética , Modelos Moleculares , Mutación , Unión Proteica , Ingeniería de Proteínas , Proteínas Recombinantes/biosíntesis , TermodinámicaRESUMEN
Interactions between bovine and goat ß-lactoglobulin and tetracaine and pramocaine were investigated with isothermal titration calorimetry, X-ray crystallography and molecular modelling. Tetracaine and pramocaine binding to lactoglobulin is an entropy driven endothermic reaction. In this work, we found that determined association constants and thermodynamic parameters indicate that pramocaine has a higher affinity to lactoglobulin than tetracaine. Crystal structures that were determined with resolutions in the range from 1.90 to 2.30 Å revealed in each case the presence of a single drug molecule bound in the ß-barrel in a mode similar to that observed for 14- and 16-carbon fatty acids. The position of the ligand in the ß-barrel indicates the optimal fit of 6-carbon aromatic rings to the binding pocket and the major role of hydrophobic interactions in ligand binding. Calculations of tetracaine and pramocaine docking to lactoglobulin revealed that molecular modelling overestimated the role of polar protein-drug interactions.