Influence of pH on the Structure and Oleic Acid Binding Ability of Bovine α-Lactalbumin

The biological function of ??-lactalbumin (??-LA) depends on its conformation. ??-LA can adopt a stable intermediate state induced by heating or pH change. This intermediate state associates with oleic acid (OA) to form an anti-tumor complex. The effect of temperature on the formation the complex has been studied, whereas the effect of pH on complex formation remains unresolved. The effect of pH on tryptophan residues, hydrophobic clusters and secondary structure of Ca²⁺-depleted bovine ??-LA (BLA) was studied by fluorescence spectroscopy and circular dichroism. BLA was found to adopt a more flexible conformation between pH 7.0 and 9.0 with buried hydrophobic clusters. The binding ability of ??-LA towards OA and the anti-tumor activity of the corresponding complex were also studied. BLA was found to bind more OA over the pH range of 7.0?C9.0 and the corresponding complexes showed a higher anti-tumor activity than those complexes formed under acidic conditions. Our study indicates that alkaline pH aided the formation of the complex as well as its anti-tumor activity. We also propose a possible characteristic structure that facilitates binding of OA.     

Multiple Interactions of Unsaturated Fatty Acids with Opiate and Ouabain Binding Sites and β-Adrenergic Sensitive Adenylate Cyclase System

Abstract

The unsaturated fatty acids oleic, linoleic and arachidonic inhibited binding of ligands to the ouabain, opiate, and β-adrenergic plasma membrane receptors. Low concentrations of fatty acids slightly increased the binding of ouabain to its binding sites. The effect of these fatty acids on β-adrenergic sensitive adenylate cyclase was more complex. 0.2–0.3 mM fatty acids increased adenylate cyclase activity, while higher concentrations of arachidonic and linoleic acids, but not oleic acid     

Structure and Properties of a Complex of α-Synuclein and a Single-Domain Camelid Antibody

The aggregation of the intrinsically disordered protein α-synuclein to form fibrillar amyloid structures is intimately associated with a variety of neurological disorders, most notably Parkinson's disease. The molecular mechanism of α-synuclein aggregation and toxicity is not yet understood in any detail, not least because of the paucity of structural probes through which to study the behavior of such a disordered system. Here, we describe an investigation involving a single-domain camelid antibody, NbSyn2, selected by phage display techniques to bind to α-synuclein, including the exploration of its effects on the in vitro aggregation of the protein under a variety of conditions. We show using isothermal calorimetric methods that NbSyn2 binds specifically to monomeric α-synuclein with nanomolar affinity and by means of NMR spectroscopy that it interacts with the four C-terminal residues of the protein. This latter finding is confirmed by the determination of a crystal structure of NbSyn2 bound to a peptide encompassing the nine C-terminal residues of α-synuclein. The NbSyn2:α-synuclein interaction is mediated mainly by side-chain interactions while water molecules cross-link the main-chain atoms of α-synuclein to atoms of NbSyn2, a feature we believe could be important in intrinsically disordered protein interactions more generally. The aggregation behavior of α-synuclein at physiological pH, including the morphology of the resulting fibrillar structures, is remarkably unaffected by the presence of NbSyn2 and indeed we show that NbSyn2 binds strongly to the aggregated as well as to the soluble forms of α-synuclein. These results give strong support to the conjecture that the C-terminal region of the protein is not directly involved in the mechanism of aggregation and suggest that binding of NbSyn2 could be a useful probe for the identification of α-synuclein aggregation in vitro and possibly in vivo.     

Effect of Methylglyoxal Modification of Human α-Crystallin on the Structure,Stability and Chaperone Function

α-Crystallin functions as a molecular chaperone and maintains transparency of eye lens by protecting other lens-proteins. Non-enzymatic glycation of α-crystallin by methylglyoxal, plays a crucial role on its chaperone function and structural stability. Our studies showed that methylglyoxal modification even in lower concentration caused significant decrease in chaperone function of α-crystallin as reflected both in thermal aggregation assay and enzyme refolding assay. Thermal denaturation studies showed drastic reduction of denaturation temperature with increase in the degree of modification. Thermodynamic stability studies by urea denaturation assay reflected a decrease of transition midpoint. Quantitatively we found that ΔG° of native α-crystallin decreased from 21.6 kJ/mol to 10.4 kJ/mol due to 72 h modification by 10 mM methylglyoxal. The surface hydrophobicity of α-crystallin after MG modification, was found to be decreased. Circular dichroism spectroscopy revealed conversion of β-sheet structure to random coil structure. Significant cross-linking was also observed due to methylglyoxal modification of human α-crystallin.     

Effect of Polyglycerol Esters of Fatty Acids on the Physicochemical Properties and Stability of β-Carotene Emulsions during Digestion in Simulated Gastric Fluid

The effects of six different polyglycerol esters of fatty acids (PGEs) and two different particle sizes produced using various processing parameters on the physicochemical properties and stability of the β-carotene emulsions during digestion in simulated gastric fluid (SGF) were investigated. β-Carotene emulsions were prepared by high-pressure homogenization using β-carotene (0.1% w/w) in soybean oil as the oil phase and 1% (w/w) PGE in Milli-Q water as the water phase. The particle size of β-carotene emulsions was measured by a laser diffraction technique, and the stability of emulsions was interpreted in terms of the increase in particle size and span value of emulsion droplets and the retention of β-carotene during digestion in SGF. The average particle size ranges of emulsions were 0.17 to 0.27 μm for fine emulsions and 1.16 to 1.59 μm for coarse emulsions. In the prepared β-carotene emulsions, the particle size decreased with increasing polymerization of the glycerol in PGEs, and the higher polymerization of the glycerol also increased the stability of emulsions during digestion in SGF. Although the β-carotene content in the emulsions significantly decreased with increasing digestion period, loss of β-carotene was more severe in unstable emulsions than in stable emulsions, suggesting that the particles incorporated into droplets could provide some protective barrier for decreasing the β-carotene degradation. Therefore, β-carotene emulsions stabilized by PGEs with high polymerization of the glycerol may be useful for further applications in food and drug formulations. Decaglycerol monooleate (MO750) was demonstrated to be the most effective emulsifier in stabilizing β-carotene emulsions in this study.     

Carbohydrate and Protein-based Inhibitors of Porcine Pancreatic α-Amylase: Structure Analysis and Comparison of Their Binding Characteristics

The crystal structures of porcine pancreatic α-amylase isozyme II (PPA II) in its free form and complexed with the trestatin A derived pseudo-octasaccharide V-1532 have been determined using Patterson search techniques at resolutions of 2.3 and 2.2 Å, respectively. Seven rings of the competitive inhibitor V-1532 could be detected in the active site region as well as two maltose units in secondary binding sites on the surface.V-1532 occupies the five central sugar binding subsites similar to the PPA/acarbose structure. A sixth ring exists at the reducing end, connecting two symmetry related PPA molecules. The seventh moiety, a 6-hydroxymethylconduritol ring, is located at the non-reducing end. The electron density for this ring is relatively weak, indicating considerable disorder.This study shows that PPA is able to accommodate more than five rings in the active site region, but that additional rings would increase the binding affinity only slightly, which is in accordance with kinetic experiments.A comparison of the structures of free PPA, PPA/V-1532 and PPA/Tendamistat shows the characteristic conformational changes that accompany inhibitor binding and distinguish pseudo-oligosaccharide inhibitors from proteinaceous inhibitors. Although both classes of inhibitors block the sugar binding subsites in the active site region, the extreme specificity and binding affinity of the proteinaceous inhibitors is probably due to an intricate interaction pattern involving areas further away from the catalytic center.     

Refinement and Analysis of the Mature Zika Virus Cryo-EM Structure at 3.1 Å Resolution

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Studies of α- and βL-Crystallin Complex Formation in Solution at 60°C

Studies of molecular mechanisms of chaperone-like activity of -crystallin became an active field of research over last years. However, fine interactions between -crystallin and the damaged protein and their complex organization remain largely uncovered. Complexation between - and _L-crystallins was studied during thermal denaturation of _L-crystallin at 60°C using small-angle X-ray scattering (SAXS), light scattering, gel-permeation chromatography, and electrophoresis. A mixed solution of - and _L-crystallins at concentrations about 10 mg/ml incubated at 60°C was found to contain their soluble complexes with a mean radius of gyration 14 nm, mean molecular mass 4 MDa and maximal size over 40 nm. In pure _L-crystallin solution, no complexes were observed at 60°C. In SAXS studies, transitions in the -crystallin quaternary structure at 60°C were shown to occur and result in doubling of the molecular weight. This suggests that during the temperature-induced denaturation of _L-crystallin it binds with modified -crystallin or, alternatively, _L-crystallin complexation and -crystallin modifications are concurrent. Estimates of the -_L-crystallin complex size and relative contents of - and -_L-crystallins in the complex suggest that several -crystallin molecules are involved in complex formation.     

The Human α-Lactalbumin Molten Globule: Comparison of Structural Preferences at pH 2 and pH 7

Structural investigations of molten globules provide an important contribution towards understanding protein folding pathways. A close similarity between equilibrium molten globule states and kinetic species observed during refolding has been reported for several proteins. However, the experimental conditions, and in particular the pH, under which the equilibrium and kinetic species are studied often differ significantly. For human α-lactalbumin (α-LA), the equilibrium molten globule is most often studied at pH 2, the so-called A-state, while kinetic refolding experiments are performed at neutral pH. α-LA contains a large number of acidic amino acid residues that may influence the properties of the molten globule differently at low and neutral pH. In this study, we investigate the structural preferences of the α-LA molten globule at pH 7 at the level of individual residues using nuclear magnetic resonance spectroscopy and compare these data with previous results obtained at pH 2. We show that differences exist in the conformational ensemble that describes the α-LA molten globule at these two pH values. The molten globule at pH 7 is generally less stable than that at the low pH A-state. Most notable are differences in the stability of structure for the C-helix and the calcium-binding loop that precedes it and differences in the contribution of long-range hydrophobic contacts between the N-terminal and C-terminal regions of the α-domain to the stability of the molten globule. Our results are discussed in the context of previous studies of the α-LA molten globule and can be used to reconcile apparent discrepancies in published data relating to the C-helix. In the light of our results, the low pH A-state may not be the best model for the kinetic molten globule observed during refolding of α-LA. The pH-dependent effects reported here for α-LA may be of relevance in comparisons of equilibrium and kinetic molten globules of other proteins.     

Structure and stability analysis of cytotoxic complex of camel α-lactalbumin and unsaturated fatty acids produced at high temperature

α-Lactalbumin α-La), together with oleic acid can be converted to a complex, which kills tumor cells selectively. Cytotoxic α-La -oleic acid and α-La -linoleic acid complexes were generated by adding fatty acid to camel holo α-La at 60 ° C (referred to as La-OA-60 and La-LA-60 state, respectively). Structural properties of these complexes were studied and compared to the camel α-La. The experimental results show that linoleic acid induces α-La partial unfolding but oleic acid does not change the protein structure significantly. Also the stability of La-OA-60 and La-LA-60 toward thermal denaturation was measured. The order of temperature at the transition midpoint is as follows: La-LA-60 < La-OA-60 < α-La. La-OA-60 complex inhibited tubulin polymerization in vitro. Although the structures of La-OA-60 and La-LA-60 were different, these two complexes had similar cytotoxic effect to DU145 human prostate cancer cells. Samples of La-OA-60 that have been renatured after denaturation lost the specific biological activity toward tumor cells.     

Chemical Structure of Mating Pheromone Peptides, αsk1 and αsk3 Pheromones,Produced by Mating Type α Cells of Saccharomyces kluyveri

Three peptides, α^sk1, α^sk2 and α^sk3 pheromones, have been isolated as α-mating pheromones of Saccharomyces kluyveri, the primary structure of the main active component, α^sk2 pheromone, having already been determined. The unknown N-terminus of α^sk1 pheromone was elucidated to be 1,2,3,4-tetrahydro-β-carboline-3-carboxylic acid (β-CAR) by mass and NMR spectrometric analyses. Synthetic β-CAR-His-Trp-OH was identical with N-terminal tripeptide fragment obtained from α^sk1 pheromone, and the primary structure of α^sk1 pheromone was determined as β-CAR-His-Trp-Leu-Ser-Phe-Ser-Lys-Gly-Glu-Pro-Met(O)-Tyr-OH. The amino acid sequence of α^sk3 pheromone was determined as H-Trp-His-Trp-Leu-Ser-Phe-Ser-Lys-Gly-Glu-Pro-Met-OH by comparing the enzymatic fragments with those of α^sk2 pheromone.     

Stability of α-chymotrypsin conjugated with poly (ethylene glycols) and proxanols at high temperature and in watercosolvent mixtures

Summary -Chymotrypsin has been modified with poly(ethylene glycols) and proxanols, block-copolymers of poly(propylene oxide) and poly(ethylene oxide). These conjugates were several-fold more thermostable and showed high catalytic activity at elevated concentrations of water-miscible organic cosolvents (alcohols and dimethyl sulfoxide) which caused inactivation of free (non-modified) -chymotrypsin.     

The First N-terminal Amino Acids of α-Synuclein Are Essential for α-Helical Structure Formation In Vitro and Membrane Binding in Yeast

α-Synuclein (α-syn), a protein implicated in Parkinson's disease, is structurally diverse. In addition to its random-coil state, α-syn can adopt an α-helical structure upon lipid membrane binding or a β-sheet structure upon aggregation. We used yeast biology and in vitro biochemistry to detect how sequence changes alter the structural propensity of α-syn. The N-terminus of the protein, which adopts an α-helical conformation upon lipid binding, is essential for membrane binding in yeast, and variants that are more prone to forming an α-helical structure in vitro are generally more toxic to yeast. β-Sheet structure and inclusion formation, on the other hand, appear to be protective, possibly by sequestering the protein from the membrane. Surprisingly, sequential deletion of residues 2 through 11 caused a dramatic drop in α-helical propensity, vesicle binding in vitro, and membrane binding and toxicity in yeast, part of which could be mimicked by mutating aspartic acid at position 2 to alanine. Variants with distinct structural preferences, identified here by a reductionist approach, provide valuable tools for elucidating the nature of toxic forms of α-syn in neurons.     

The Structure of the T190M Mutant of Murine α-Dystroglycan at High Resolution: Insight into the Molecular Basis of a Primary Dystroglycanopathy

The severe dystroglycanopathy known as a form of limb-girdle muscular dystrophy (LGMD2P) is an autosomal recessive disease caused by the point mutation T192M in α-dystroglycan. Functional expression analysis in vitro and in vivo indicated that the mutation was responsible for a decrease in posttranslational glycosylation of dystroglycan, eventually interfering with its extracellular-matrix receptor function and laminin binding in skeletal muscle and brain. The X-ray crystal structure of the missense variant T190M of the murine N-terminal domain of α-dystroglycan (50-313) has been determined, and showed an overall topology (Ig-like domain followed by a basket-shaped domain reminiscent of the small subunit ribosomal protein S6) very similar to that of the wild-type structure. The crystallographic analysis revealed a change of the conformation assumed by the highly flexible loop encompassing residues 159–180. Moreover, a solvent shell reorganization around Met190 affects the interaction between the B1–B5 anti-parallel strands forming part of the floor of the basket-shaped domain, with likely repercussions on the folding stability of the protein domain(s) and on the overall molecular flexibility. Chemical denaturation and limited proteolysis experiments point to a decreased stability of the T190M variant with respect to its wild-type counterpart. This mutation may render the entire L-shaped protein architecture less flexible. The overall reduced flexibility and stability may affect the functional properties of α-dystroglycan via negatively influencing its binding behavior to factors needed for dystroglycan maturation, and may lay the molecular basis of the T190M-driven primary dystroglycanopathy.     

High-cholesterol Diets Induce Changes in Lipid Composition of Rat Erythrocyte Membrane Including Decrease in Cholesterol,Increase in α-Tocopherol and Changes in Fatty Acids of Phospholipids

Effects of high dietary cholesterol on erythrocyte membrane lipids were studied. Feeding rats with a diet containing 0.5% cholesterol and 0.15% sodium cholate for two weeks induced changes in erythrocyte membrane lipids including a decrease in cholesterol, an increase in α-tocopherol (α-Toc) and changes in the fatty acid composition of phospholipids. Oleic acid and linoleic acid increased, while arachidonic acid decreased in phosphatidylcholine. Saturated fatty acids decreased and unsaturated fatty acids increased in phosphatidylethanolamine. Almost the same changes in membrane lipids were also noted after six weeks of feeding rats with the diet. A diet containing 0.5% cholesterol but without sodium cholate caused a decrease in erythrocyte cholesterol and an increase in erythrocyte α-Toc after two weeks of feeding, as compared to the basal diet, indicating that high dietary cholesterol, but not sodium cholate, was responsible for these changes in the erythrocyte membrane.     

Crystal Structure of the Complex Formed Between Bovine β-Trypsin and MCTI-A, a Trypsin Inhibitor of Squash Family, at 1.8-? Resolution

The stoichiometric complex formed between bovine -trypsin and Momordica charantia, Linn. Cucurbitaceae trypsin inhibitor A (MCTI-A) was crystallized and its X-ray crystal structure was refined to a final R value of 0.179 using data of 7.0- to 1.8-Å resolution. Combination with results on the complex of MCTI-A with porcine trypsin gives the sequence of MCTI-A definitely, of which 13 residues are conserved compared with other squash family trypsin inhibitors. Its spatial structure and the conformation of its primary binding segment from Cys3I (P3) to Glu7I (P3), which contains a reactive scissile bond Arg5I C–Ile6I N, were found to be very similar to the other squash family proteinase inhibitors.