Structural studies of new β-lactoglobulin variants possessing tryptophan mutations in the binding site

Lipokaliny to niewielkie białka zdolne do transportowania małych, hydrofobowych molekuł. Białka z rodziny lipokalin posiadają wysoce konserwatywny motyw β-baryłki tworzący sztywny rdzeń. Do tej rodziny należy β-laktoglobulina, która jest białkiem globularnym, występującym w serwatkowej frakcji mleka wielu ssaków. Białko to wykazuje wysokie powinowactwo do hydrofobowych ligandów, których centralnym miejscem wiązania jest β-baryłka. Nowe warianty β-laktoglobuliny, dla których prowadzono badania strukturalne w ramach tej pracy, zawierały wielokrotne mutacje w rejonie kieszeni wiążącej, w tym substytucje wybranej reszty aminokwasowej na tryptofan. Zaprojektowane mutanty białka posiadały zmienioną geometrię miejsca wiążącego, co powodowało zwiększenie powinowactwa do trójcyklicznych leków. Celem pracy było znalezienie odpowiednich warunków krystalizacji i otrzymanie monokryształów kompleksów nowych wariantów β-laktoglobuliny z lekami o trójcyklicznej geometrii. Podjęto próby krystalizacji dwóch wariantów β-laktoglobuliny: I56F/L39A/M107W oraz F105L/L39A/M107W. Krystalizację prowadzono metodą dyfuzji przez fazę gazową, techniką wiszącej kropli. Warunki krystalizacji optymalizowano, zmieniając stężenie roztworów białka i precypitanta oraz skład kropli.Lipocalins are small proteins which can transport hydrophobic molecules. Proteins from the lipocalin family have a well conserved β-barrel motif, forming a rigid protein core. β-lactoglobulin, found in the whey of many mammals is the globular protein belonging to the lipocalin family,. This protein has a high affinity to hydrophobic ligands which are bound in the β-barrel. Structural investigations were made for the new β-lactoglobulin variants containing multiple mutations introduced into the binding pocket, including tryptophan substitution of the selected residues. These substitutions altered the geometry of the binding pocket in order to increase protein affinity to drugs of tricyclic geometry. The aim of the studies was to find optimal conditions for crystallization of new β-lactoglobulin variants with drugs of tricyclic geometry. Crystallization of two lactoglobulin variants: I56F/L39A/M107W, F105L/L39A/M107W and their complexes with ligands were performed. Protein-ligand complexes were crystallized by the vapour diffusion method, hanging drop technique. In order to obtain high quality crystals, the crystallization conditions were optimized by modifying the concentration of protein, precipitant and the composition of the drop

Na drodze do odkrycia oryginalnych NeoLektyn

Ze względu na ogromną różnorodność węglowodanów, które pełnią kluczową rolę w wielu procesach biologicznych, istnieje równie duża liczba enzymów na nie działających. Wśród nich, szczególnie interesujące są hydrolazy glikozydowe, które zdolne są do hydrolizy wiązań glikozydowych, jak również ich tworzenia w określonych warunkach. Co więcej, poprzez pozbawienie ich aktywności hydrolitycznej, hydrolazy glikozydowe mogą zostać przekształcone do białek wykazujących właściwości lektyn, zdolnych do rozpoznawania i wiązania glikozydów. W ostatnich latach użycie katalitycznie nieaktywnych hydrolaz glikozydowych wytworzonych poprzez losową lub ukierunkowaną mutagenezę było opisywane w literaturze. W tej pracy wygenerowano mutanty β-ksylozydazy z Dictyoglomus thermophilum, w których reszta katalityczna E161 została podstawiona w sposób losowy, tym samym tworząc potencjalne neolektyny. Zastosowano dwa różne podejścia do scharakteryzowania wyprodukowanych wariantów β-ksylozydazy w odniesieniu do ich powinowactwa do ksylozydów. Pierwsze podejście oparte było na aktywności mutantów, która powinna być przesunięta w kierunku reakcji tioglikozylacji zamiast hydrolizy, natomiast drugie wykorzystywało chromatografię powinowactwa oraz fakt, iż wyprodukowane neolektyny powinny się wiązać specyficznie do ksylozydów immobilizowanych na złożu. Celem tego projektu było zidentyfikowanie neolektyn otrzymanych z β-ksylozydazy pochodzącej z Dictyoglomus thermophilum oraz scharakteryzowanie tych z interesującymi właściwościami wiązania ksylozydów.Due to the large diversity of carbohydrates and to the fact that they play a crucial role in many biological processes, there is a great variety of enzymes acting upon these substrates. Among them, glycoside hydrolases are of particular interest since they are capable of both cleaving glycosidic bonds and forming them depending on the experimental conditions. Moreover, by inactivation of their hydrolytic activity, glycoside hydrolases can be converted to lectin-like proteins able to recognize and bind glycosides. In recent years, the use of catalytically inactive glycosidases generated through random or site-directed mutagenesis has been reported in the literature. In this study, β-D-xylosidase from Dictyoglomus thermophilum was used to produce mutant enzymes in which the catalytic acid/base residue E161 has been substituted in a random manner, thereby creating potential neolectins. Two different approaches were applied to characterize generated β-xylosidase variants with regard to their affinity towards xylosides. One approach employed screening of enzyme mutants activity that should be shifted towards thioglycosylation instead of hydrolysis, while the other was based on affinity chromatography and the fact that produced neolectins should bind specifically to xylosides immobilized on the resin. The aim of this project was to identify original neolectins from Dictyoglomus thermophilum β-D-xylosidase and to characterize those with interesting xyloside binding properties

Biochemical characterization of L-asparaginase isoforms from Rhizobium etli—the boosting effect of zinc

L-Asparaginases, divided into three structural Classes, catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia. The members of Class 3, ReAIV and ReAV, encoded in the genome of the nitrogen fixing Rhizobium etli, have the same fold, active site, and quaternary structure, despite low sequence identity. In the present work we examined the biochemical consequences of this difference. ReAIV is almost twice as efficient as ReAV in asparagine hydrolysis at 37°C, with the kinetic KM, kcat parameters (measured in optimal buffering agent) of 1.5 mM, 770 s-1 and 2.1 mM, 603 s-1, respectively. The activity of ReAIV has a temperature optimum at 45°C–55°C, whereas the activity of ReAV, after reaching its optimum at 37°C, decreases dramatically at 45°C. The activity of both isoforms is boosted by 32 or 56%, by low and optimal concentration of zinc, which is bound three times more strongly by ReAIV then by ReAV, as reflected by the KD values of 1.2 and 3.3 μM, respectively. We also demonstrate that perturbation of zinc binding by Lys→Ala point mutagenesis drastically decreases the enzyme activity but also changes the mode of response to zinc. We also examined the impact of different divalent cations on the activity, kinetics, and stability of both isoforms. It appeared that Ni2+, Cu2+, Hg2+, and Cd2+ have the potential to inhibit both isoforms in the following order (from the strongest to weakest inhibitors) Hg2+ > Cu2+ > Cd2+ > Ni2+. ReAIV is more sensitive to Cu2+ and Cd2+, while ReAV is more sensitive to Hg2+ and Ni2+, as revealed by IC50 values, melting scans, and influence on substrate specificity. Low concentration of Cd2+ improves substrate specificity of both isoforms, suggesting its role in substrate recognition. The same observation was made for Hg2+ in the case of ReAIV. The activity of the ReAV isoform is less sensitive to Cl− anions, as reflected by the IC50 value for NaCl, which is eightfold higher for ReAV relative to ReAIV. The uncovered complementary properties of the two isoforms help us better understand the inducibility of the ReAV enzyme

DataSheet1_Biochemical characterization of L-asparaginase isoforms from Rhizobium etli—the boosting effect of zinc.pdf

L-Asparaginases, divided into three structural Classes, catalyze the hydrolysis of L-asparagine to L-aspartic acid and ammonia. The members of Class 3, ReAIV and ReAV, encoded in the genome of the nitrogen fixing Rhizobium etli, have the same fold, active site, and quaternary structure, despite low sequence identity. In the present work we examined the biochemical consequences of this difference. ReAIV is almost twice as efficient as ReAV in asparagine hydrolysis at 37°C, with the kinetic KM, kcat parameters (measured in optimal buffering agent) of 1.5 mM, 770 s-1 and 2.1 mM, 603 s-1, respectively. The activity of ReAIV has a temperature optimum at 45°C–55°C, whereas the activity of ReAV, after reaching its optimum at 37°C, decreases dramatically at 45°C. The activity of both isoforms is boosted by 32 or 56%, by low and optimal concentration of zinc, which is bound three times more strongly by ReAIV then by ReAV, as reflected by the KD values of 1.2 and 3.3 μM, respectively. We also demonstrate that perturbation of zinc binding by Lys→Ala point mutagenesis drastically decreases the enzyme activity but also changes the mode of response to zinc. We also examined the impact of different divalent cations on the activity, kinetics, and stability of both isoforms. It appeared that Ni2+, Cu2+, Hg2+, and Cd2+ have the potential to inhibit both isoforms in the following order (from the strongest to weakest inhibitors) Hg2+ > Cu2+ > Cd2+ > Ni2+. ReAIV is more sensitive to Cu2+ and Cd2+, while ReAV is more sensitive to Hg2+ and Ni2+, as revealed by IC50 values, melting scans, and influence on substrate specificity. Low concentration of Cd2+ improves substrate specificity of both isoforms, suggesting its role in substrate recognition. The same observation was made for Hg2+ in the case of ReAIV. The activity of the ReAV isoform is less sensitive to Cl− anions, as reflected by the IC50 value for NaCl, which is eightfold higher for ReAV relative to ReAIV. The uncovered complementary properties of the two isoforms help us better understand the inducibility of the ReAV enzyme.</p

New ligand-binding sites identified in the crystal structures of β\beta-lactoglobulin complexes with desipramine

The homodimeric β-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 β-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 β-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 β-lactoglobulin molecule