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PubMed1998

Kinetic refolding of beta-lactoglobulin. Studies by synchrotron X-ray scattering, and circular dichroism, absorption and fluorescence spectroscopy.

beta-Lactoglobulin (beta LG) is a predominantly beta-sheet protein with a markedly high helical propensity and forms non-native alpha-helical intermediate in the refolding process. We measured the refolding reaction of beta LG with various techniques and characterized the folding kinetics and the structure of the intermediate formed within the burst phase of measurements, i.e. the burst-phase intermediate. Time-resolved stopped-flow X-ray scattering measurements using the integral intensity of scattering show that beta LG forms a compact, globular structure within 30 ms of refolding. The averaged radius of gyration within 100 ms is only 1.1 times larger than that in the native state, ensuring that the burst-phase intermediate is compact. The presence of a maximum peak in a Kratky plot shows a globular shape attained within 100 ms of refolding. Stopped-flow circular dichroism, tryptophan absorption and fluorescence spectroscopy show that pronounced secondary structure regains rapidly in the burst phase with concurrent non-native alpha-helix formation, and that the subsequent compaction process is accompanied by annealing of non-native secondary structure and slow acquisition of tertiary structure. These findings strongly suggest that both compaction and secondary structure formation in protein folding are quite rapid processes, taking place within a millisecond time-scale. The structure of the burst-phase intermediate in beta LG refolding was characterized as having a compact size, a globular shape, a hydrophobic core, substantial beta-sheets and remarkable non-native alpha-helical structure, but little tertiary structure. These results suggest that both local interactions and non-local hydrophobic interactions are dominant forces early in protein folding. The interplay of local and non-local interactions throughout folding processes is important in understanding the mechanisms of protein folding.

Animals↗
PubMed1999

Unfolding and refolding of bovine beta-lactoglobulin monitored by hydrogen exchange measurements.

Bovine beta-lactoglobulin (beta-LG) is a widely studied protein belonging to the lipocalin family, whose structural characterisation has been reported by X-ray crystallography and NMR studies at physiological and acidic pH, respectively. Bovine beta-LG consists of nine antiparallel beta-sheets and a terminal alpha-helix segment. The beta-sheets form a calyx structure with a hydrophobic buried cluster conferring stability to the protein while a hydrophobic surface patch provides stabilising interactions between the barrel and the flanking terminal helix. Here, the stability and the folding properties of bovine beta-LG in the presence of a chemical denaturant is probed. The analysis of the NMR spectra recorded in aqueous solution with increasing amounts of urea revealed that the intensities of the backbone cross-peaks decrease upon increasing urea concentration, while their secondary shifts do not change significantly on going from 0 to 5 M urea, thus suggesting the presence of slow exchange between native and unfolded protein. Hydrogen exchange measurements at different urea concentrations were performed in order to evaluate the exchange rates of individual backbone amide protons. The opening reactions that determine protein exchange can be computed for the most slowly exchanging hydrogen atoms, and the measured exchange rates and the corresponding free energies can be expressed in terms of the equilibrium energetic for the global transition and the local fluctuations. Most of the residues converge to define a common isotherm identifying a unique cooperative folding unit, encompassing all the strands, except strand betaI, and the terminal region of the helix. The amides that do not join the same global unfolding isotherm are characterised by low DeltaGH20op and especially by low m values, indicating that they are already substantially exposed in the native state. A two-state unfolding model N <==> U is therefore proposed for this rather big protein, in agreement with CD data. Renaturation studies show that the unfolding mechanism is reversible up to 6 M urea and suggest a similar unfolding and refolding pathway. Present results are discussed in light of the hypothesis of an alpha-->beta transition proposed for bovine beta-LG refolding.

Amino Acid Motifs↗
PubMed1998

Isotopically labeled bovine beta-lactoglobulin for NMR studies expressed in Pichia pastoris.

beta-Lactoglobulin (beta-Lg) is the major whey protein in ruminant milk and has been implicated in the irreversible denaturation of milk proteins and its associated poor processing behavior during heat treatment. In order to help understand this behavior, as well as to facilitate other studies into the relationship between the molecular structure and its behavior in solution, we have prepared and purified 15N-labeled and 13C/15N-double-labelled beta-Lg in sufficient quantities to permit a full determination of the structure and dynamics using heteronuclear NMR spectroscopy. The overexpression of the labeled protein using the Pichia pastoris system proceeds with good yield but requires the removal of significant quantities of copurifying carbohydrate which otherwise interfere with the NMR experiments. At pH 2, the resulting material gives triple resonance NMR spectra of good quality that are consistent with a monomeric, globular protein rich in beta-sheet.

Amino Acid Sequence↗
PubMed1999

Expression of a lipocalin in prokaryote and eukaryote cells: quantification and structural characterization of recombinant bovine beta-lactoglobulin.

In this paper we quantify and characterize the expression of recombinant beta-lactoglobulin (rBLG) in prokaryote and eukaryote cells. In Escherichia coli we used the pET26 vector, which permits the secretion of rBLG in periplasm. We studied the expression of rBLG in COS-7 cells and in vivo in mouse tibialis muscle. The expression of rBLG was measured by two immunoassays specific, respectively, for BLG in its native and denatured conformation. We observed that rBLG was essentially expressed in a denatured form in E. coli even in the periplasm, whereas rBLG in eukaryote cells was found in its native conformation.

Animals↗
PubMed2000

Nucleosome organisation of the beta-lactoglobulin gene. Transcription complex formation.

In most mammals, the major whey protein beta-lactoglobulin (beta lg) represents a marker for tissue-specific, temporally regulated gene expression in the mammary gland. Prolactin, the major lactogenic stimulus which activates beta lg expression acts through a cytoplasmic signalling cascade ending in the activation of the transcription factor STAT5. Although much is known about the complexities of this signalling cascade, little is known about how this transcription factor functions within the context of chromatin. Using DNaseI as a probe of chromatin structure we have identified temporally regulated elements within the ovine beta lg gene domain. The appearance of these hypersensitive sites accompanies changes in expression state of the beta lg gene. Changes in DNaseI hypersensitivity at the proximal promoter region, while reflecting STAT5 activation, is not dependent upon STAT5 interaction at this site. We have mapped the nucleosome positions over the entire beta lg gene, both in vitro using the monomer extension assay and in vivo using cuprous phenanthroline to probe for nucleosome-linker positions. The specific positioning pattern detected, which reflects strong sequence-directed positioning over the proximal promoter, complement the STAT5 consensus sites within this region. The comparison of both the functional and chromatin data enables a model for beta lg gene transcription to be developed.

Animals↗
PubMed1993

Mapping of the beta-lactoglobulin gene and of an immunoglobulin M heavy chain-like sequence to homoeologous cattle, sheep, and goat chromosomes.

With a combination of non-isotopic in situ hybridization and simultaneous fluorescent R-banding, this study presents the first map of the short arm of sheep Chromosome (Chr) 3 and of the homoeologous cattle and goat Chrs 11 with two DNA sequences: the beta lactoglobulin gene (LGB) and an immunoglobulin M heavy chain-like sequence (IGHML). The results are in agreement with the high degree of banding pattern similarity, previously reported, among cattle, sheep, and goat karyotypes.

Animals↗
PubMed1984

Labelling of colloidal gold with protein. A quantitative study using beta-lactoglobulin.

Markers prepared by labelling colloidal gold with macromolecules such as lectins, antibodies and protein A are gaining wide acceptance both in transmission and scanning electron microscopy. However detailed information on the process and extent of adsorption of macromolecules onto gold particles are still lacking. The adsorption isotherm of protein onto gold particles was studied quantitatively using goat beta-lactoglobulin (beta L) tritiated in vivo. When this protein was modified chemically by iodination with 125I, the adsorption isotherm was not significantly different (Langmuir type for monolayer). In the presence of saturating amount of beta L, a maximum of 13-14 molecules was adsorbed per particle of 12 nm in diameter for a theoretical maximum of 20 (compact monolayer). Ellipsometric measurements on nickel-coated slides indicated that betas L was adsorbed onto metallic surfaces as a compact monolayer. The molecules were irreversibly adsorbed on gold particles, kept to a large extent their capacity to bind anti-beta L antibodies and could not be displaced by polyethylene glycol, a stabilizer commonly used in the preparation of gold markers. Only markers labelled with more than 5 beta L molecules per particle could be completely bound by immobilized anti-beta L antibodies. Preliminary data indicated that the energetics of adsorption of beta L onto colloidal gold was in agreement with that expected from the mutual interaction of surface and adsorbate.

Adsorption↗
PubMed1988

Labelling of colloidal gold with IgE. A quantitative study using monoclonal IgE anti-beta-lactoglobulin and evaluation of the biological activity of the gold complex with RBL-1 cells.

Immunocytochemical markers prepared by labelling colloidal gold with antibodies are gaining wide acceptance both in transmission and scanning electron microscopy. However, detailed information on the process and extent of adsorption of IgG and IgE in particular are still lacking. The adsorption isotherm of mouse monoclonal 125I-IgE antibovine milk beta-lactoglobulin was studied quantitatively with colloidal gold buffered at pH 6.1-8.8 (28 nm in particle diameter). At low coverage of the particles (less that or equal to 5 molecules per particle), the isotherm was independent of pH. In the presence of a large excess of IgE, the highest coverage was obtained at pH 6.1 near the pI of IgE (5.2-5.8). The binding constants were higher at low coverage (side-on adsorption) than at high coverage where desorption was observed. IgE-Au markers were unreactive towards the immobilized antigen and did not bind to receptors for IgE of rat basophilic leukemia cells (RBL-1). The reactivity of immobilized anti-IgE antibodies with IgE-Au markers increased as a function of particle coverage. Mapping of RBL-1 cell membrane IgE receptors was achieved by incubating successively IgE-sensitized RBL-1 cells with anti-IgE antibodies and a protein A-gold marker at 4 degrees C. Surface clusters developed when the cells were incubated at 37 degrees C.

Animals↗
PubMed1977

[Formation of dehydroalanine, lanthionine and lysinoalanine during heat treatment of beta-lactoglobuline A (author's transl)].

Solutions of beta-lactoglobulin A were heated at 97.5 degrees C at pH 6.9 and 9.8 80 min. The formation of dehydroalanine, lanthionine and lysinoalanine as well as changes in the levels of cysteine, cystine and the basic amino acids were recorded. Lanthionine (equal amounts of the 1- and the mesoform) was formed only in small amounts, while the lysinoalanine levels were rather high. Beside this, lysine was converted to unknown compounds. The main sources for the formation of dehydroalanine are the cystine moieties of the protein. Cysteine residues are first oxidized to cystine before beta-elimination occurs.

Alanine↗
PubMed1994

Ectopic expression of beta-lactoglobulin/human serum albumin fusion genes in transgenic mice: hormonal regulation and in situ localization.

We produced transgenic mice carrying the native sheep beta-lactoglobulin (BLG) or fusion genes composed of the BLG promoter and human serum albumin (HSA) minigenes. BLG was expressed exclusively in the mammary glands of the virgin and lactating transgenic mice evaluated. In contrast, transgenic females carrying the BLG/HSA fusion constructs also expressed the HSA RNA ectopically in skeletal muscle, kidney, brain, spleen, salivary gland and skin. Ectopic expression of HSA RNA was detected only in strains that express the transgene in the mammary gland. There was no obvious correlation between the level of the HSA RNA expressed in the mammary gland and that found ectopically. In three transgenic strains analysed, the expression of HSA RNA in kidney and skeletal muscle increased during pregnancy and lactation, whereas in the brain HSA expression decreased during lactation in one of the strains. HSA protein was synthesized in skeletal muscle and skin of strain #23 and its level was higher in lactating mice compared with virgin mice. Expression of HSA was also analysed in males and was found to be more stringently controlled than in females of the same strains. In situ hybridization analyses localized the expressed transgene in the skin, kidney, brain and salivary glands of various transgenic strains. Distinct strain-specific and cell-type specific HSA expression patterns were observed in the skin. This is in contrast to the exclusive expression of the HSA transgene in epithelial cells surrounding the alveoli of the mammary gland. Taken together, these results suggest that the absence of sufficient mammary-specific regulatory elements in the BLG promoter sequences and/or the juxtaposition of the BLG promoter with the HSA coding sequences leads to novel tissue- and cell-specific expression in ectopic tissues of transgenic mice.

Animals↗
PubMed1996

Comparable processing of beta-lactoglobulin pre-mRNA in cell culture and transgenic mouse models.

Eukaryotic pre-mRNAs undergo a variety of post-transcriptional modifications, including the removal of intronic sequences by splicing, leading to creation of a functional mRNA. We have compared the processing of transcripts generated from ovine beta-lactoglobulin gene constructs in stably transfected cells and in transgenic mice. In both the in vitro and in vivo model systems the removal of the middle two introns resulted in the inefficient splicing of the downstream, terminal intron. This intron-containing transcript was detected in the cytoplasmic RNA fraction. Thus, the initial in vitro analysis in cell lines of minigene constructs destined for expression in transgenic animals may provide a rapid and reliable indicator of the processing efficiency of the pre-mRNA produced by the construct in vivo. This is in contrast to the apparent limitations of in vitro systems in the analysis of transcription regulatory elements required for transgene expression.

Animals↗
PubMed2002

Probing protein aggregation by time-resolved fluorescence during beta-lactoglobulin crystal growth.

We have used the fluorescence anisotropy (FA) decay of retinol bound to bovine beta-lactoglobulin to monitor the time evolution of protein aggregation during the early stages of crystal growth. With this approach we have followed the formation of aggregates at different concentrations of ammonium sulfate, the precipitant used for crystallization. The average aggregation number is found to depend on precipitant concentration, and to be restricted to small numbers ranging from 2 to 5, also in the presence of visible growing crystals. The effect of particle distribution and of low probe-to-protein saturation on the FA response is also discussed in detail.

Ammonium Sulfate↗
PubMed1999

Isolation, sequencing and relative quantitation by fluorescent-ratio PCR of feline beta-lactoglobulin I, II, and III cDNAs.

Beta-lactoglobulin (betaLG) is a whey protein found in the milk of most mammals, except those of humans and rodents. In ruminants, only one type of betaLG is expressed in milk, although the presence of pseudogenes has been reported. The milk of other mammals (dog, horse, dolphins) contains two types of betaLG: type I is related to ruminant betaLG, while type II has a strong similarity to the ruminant pseudogenes. The presence of three types of betaLG has been described only in cat milk. For the first time, we have cloned, sequenced, and characterized the three types of feline betaLG cDNAs from a mammary gland sample of a lactating cat. Since no specific probe could be easily used to differentiate them by Northern Blotting, we developed a new technique named Fluorescent-Ratio PCR (FR-PCR) in order to assess their level of expression during lactation. The relative amounts of each feline betaLG cDNA was quantified in two cats by capillary electrophoresis of the restricted RT-PCR product, labeled with fluorescent primers. We observed the same percentage of expression of the three betaLG genes in two cats. The differences of expression could be due to changes in the promoter and 3' non-coding region affecting the level of transcription and the mRNA stability. The FR-PCR technique was shown to be reproducible and accurate even for small percentage differences, being very useful when a small amount of sample is available.

Animals↗
PubMed2006

Conformational and structural analysis of bovine beta lactoglobulin-A upon interaction with Cr+3.

Thermodynamic studies have been made on the effect of Cr(+3) on the conformation and structure of bovine beta lactoglobulin-A, (Blg-A) in 50 mM sodium chloride solution at 27 degrees C using isothermal titration calorimetry (ITC), circular dichroism (CD) and fluorescence spectroscopy. There is a set of six identical and independent binding sites for Cr(+3) by a dissociation binding constant of 124 microM and the molar enthalpy of binding -17.8 kJ/mol. Circular dichroism studies do not show any significant change in the secondary structure of the protein after the binding of chromium ion on the Blg-A. Fluorescence spectroscopy studies do not show any considerable change in the tertiary structure of Blg-A due to the increasing of Cr(+3) in low concentration. However, occupation of fourth and fifth binding sites for chromium ions induce partially unfolding in the tertiary structure of the protein resulted from solvent - exposed hydrophobic patches on BLG-A.

Animals↗
PubMed2005

Development of transgenic mice expressing calcitonin as a beta-lactoglobulin fusion protein in mammary gland.

Expression of foreign proteins in mammalian milk is becoming a widespread strategy for high-level production of recombinant pharmaceuticals, especially those with the most complex post-translational modifications. A milk-specific ovine beta-lactoglobulin (oBLG) promoter was used to drive expression of recombinant calcitonin in mouse milk. A gene construct was generated, consisting of 10.7 kbp of the oBLG gene including its promoter and 3' flanking region with the calcitonin coding sequences inserted in-frame into the oBLG fifth exon. After microinjection, six founder mice transmitted the transgene to their progeny. RT-PCR confirmed mammary-gland specific expression of recombinant mRNA in most transgenic mice and Western blot analysis confirmed expression of chimeric protein. Calcitonin can thus be expressed under the oBLG promoter and regulatory elements in a mammary-gland specific manner.

Animals↗
PubMed1983

Secondary structure of bovine beta-lactoglobulin B.

Secondary-structure-prediction algorithms have been used to find the segments of beta-lactoglobulin sequence most likely to fit the circular dichroism assignment of 15% alpha-helix, 50% beta-sheet, and 15-20% reverse turn. A number of segments may have an alpha-helical conformation but the most prominent region of alpha-helix is from residue 129 to 143. A further probable alpha-helix segment is residues 65-76. The number of residues predicted to occur in segments of beta-sheet structure is less than expected. However, the most likely segments are for residues 1-6, 11-16, 39-45, 80-85, 92-96, 101-107, 117-123, and 145-151. Predicted reverse-turn tetrapeptides are residues 7-10, 49-52, 61-64, 88-91, and 112-115. These predicted secondary structures are consistent with the low-resolution structure of the molecule determined by X-ray diffraction studies.

Amino Acid Sequence↗
PubMed1976

Effect of temperature on tryptophan fluorescence of beta-lactoglobulin B.

The effect of heat on the conformation of bovine beta-lactoglobulin has been studied using intrinsic fluorescence spectroscopy. Changes in the intensity, wave-length of maximum emission and emission peak width at half height of tryptophan fluorescence over the range 15-90 degrees C at pH 6.4-6.5 has allowed the environments of the two tryptophans in the molecule to be discriminated. At 20 degrees C both tryptophans are in hydrophobic environments. As the temperature is raised the conformation changes such that at about 50 degrees C one of the tryptophans is transferred to a more polar environment accessible to solvent. Conformational changes appear to be reversible if the protein is cooled to 20 degrees C after heat treatments up to 70 degrees C. Above 70 degrees C the second tryptophan residue becomes exposed to solvent. Complete exposure of one residue occurs at 80 degrees C while the other is still partially buried even at 90 degrees C. When the protein is then cooled to 20 degrees C the conformational changes appear to be irreversible with only one tryptophan residue returning to the hydrophobic interior of the molecule.

Animals↗
PubMed1979

The reaction of cow beta-lactoglobulin with tetracyanoaurate(III).

The reaction of cow beta-lactoglobulin with Au(CN)-4 is shown to label the free sulphydryl group on the protein by a reductive mechanism yielding a stable complex. High salt concentrations are shown to affect the pH-dependent conformational transition (N in equilibrium R yields S). It is hoped that Au(CN)-4 may be of more widespread use as a specific heavy-atom label for the isomorphous replacement method.

Animals↗