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PubMed1978

Binding of iodomercurates to sulfhydryl-blocked beta-lactoglobulin-A, -B, and -C.

Sulfhydryl-blocked beta-lactoglobulins (beta-LG-S-SCH2CH2OH)-A, -B, and -C bind only one iodomercurate species, HgI3-, at only one site, with a dissociation constant of 4.0 X 10(-5) M at 25 degrees, pH 5.0, 0.10 ionic strength. (Binding to native beta-LG-SH-A, -B, and -C is more complex, involving the sulfhydryl and two other sites and several iodomercurates.) The red shift of the HgI3- spectrum on binding would ordinarily suggest a hydrophobic site, but the HgI3- site is distinct from, and independent of, the alkane-binding site of native and blocked beta-LG; HgI3- may bind a group that shifts its trigonal planar structure toward the tetrahedron of HgI4(2-). Binding of HgI3- to blocked beta-LG interferes with the well-known association of beta-LG-A to octamers at pH 4.6 and low temperature. The relation of the HgI3- site to the crystallographic iodomercurate-binding sites of beta-LG-SH is examined. To facilitate these and future studies of iodomercurate binding, the 200-400 nm spectra of HgI2, HgI3-, and HgI4(2-) in aqueous solutions and the thermodynamic formation constants at 25 degrees for the equilibria HgI2 + I- = HgI3- (4.9 X 10(3) M-1) and HgI3- + I- = HgI4(2-) (0.118 X 10(3) M-1) were obtained.

Animals↗
PubMed1996

Partially folded structure of monomeric bovine beta-lactoglobulin.

Bovine beta-LG (beta-lactoglobulin) has been studied under a variety of solution conditions by one- and two-dimensional NMR spectroscopy. At highly acidic pH (pH=2) and low ionic strength the protein is present in a monomeric form, exhibiting a highly structured beta-sheet core and less ordered regions as evidenced by both CD data and the NOESY spectra. Marginal protection was observed for most of the amide protons as a result of high conformational mobility. This structural state of beta-LG may be considered as an attractive model for a partially folded structure occurring late in the folding process of the protein.

Amino Acid Sequence↗
PubMed1993

Temperature-induced folding changes of beta-lactoglobulin in hydro-methanolic solutions.

It has been demonstrated using circular dichroism that methanol induces important structural changes of beta-lactoglobulin (BLG). The secondary structure of BLG dissolved in 45% methanol (v/v) at 30 degrees C (dielectric constant epsilon approximately 50) is predominantly alpha-helical and unable to complex retinol any more. However, when the dielectric constant of such a medium is raised back to epsilon approximately 70 by decreasing the temperature, both the refolding of BLG into a beta-structure and the formation of the retinol/BLG complex are observed. On the other hand, in the case of BLG solution in 30% methanol, the decrease of the dielectric constant from epsilon approximately 69 to epsilon approximately 53 by heating from 20 degrees C to 60 degrees C leads to the transition from a predominantly beta-structure into a predominantly random one and the dissociation of the retinol/BLG complex. The reversibility of the conformation changes by cooling was demonstrated by circular dichroism curves and tryptophan fluorescence. The retinol fluorescence intensity could not be completely recovered.

Animals↗
PubMed1996

Effect of pressure on the deuterium exchange reaction of alpha-lactalbumin and beta-lactoglobulin.

The effect of pressure on the deuterium exchange reaction of alpha-lactalbumin (LA) and beta-lactoglobulin (LG) was investigated to determine the structural change in these proteins induced by elevated pressure. LG, one of the main components of milk whey, has been degraded selectively from other milk proteins including LA by protease treatment under high pressure (Hayashi, R., Kawamura, Y. and Kunugi, S. J. Food Sci. 1987; 52: 1107-1108). This was considered to occur because LG lost its native structure under high pressure more remarkably than LA. In the present study, the H/D exchange reaction was carried out under high pressure and the resulting structures were analysed by Fourier-transform infra-red (FTIR) and nuclear magnetic resonance (NMR) spectroscopy, after the release of elevated pressure. The wavenumber of amide I bands in the FTIR spectrum assigned to alpha-helix and beta-sheet structures of the proteins, shifted to lower regions as the H/D exchange of protons proceeded. The integral band area of the amide proton signal in low-field regions of the NMR spectrum is related to the H/D exchange of less stable protons in the protein. H/D exchanges for LA at 200 MPa and LG at 50 MPa were detectable by NMR as a decrease in the amide proton signals, but they were detected less unambiguously by FTIR. This apparent difference may be explained by reference to an intermediary unfolding stage of the protein that is generated under moderately high pressure.

Deuterium↗
PubMedDate not supplied

Bovine beta-lactoglobulin H: isolation by preparative isoelectric focusing in immobilized pH gradients and preliminary characterization.

In spite of the fact that beta-lactoglobulin (beta-lg) was first discovered in bovine milk more than fifty years ago, and that it represents the main whey protein component in all the milks in which it has been found, its biological role and genetic evolution still remain rather uncertain. From comparative studies of the primary and tertiary structures of beta-lg and of other proteins of a similar size, the existence of a new superfamily of proteins with the function of transporter of hydrophobic molecules has been conjectured. The elucidation of the structure of beta-lg either from different species or from different genetic variants of the same species should give useful information on the evolution and function of this protein family. With this aim in mind we have now undertaken the isolation and characterization of a recently discovered, new genetic variant of bovine beta-lg. A two-step purification procedure involving preparative HPLC gel filtration and preparative IEF-IPG has been successfully carried out; it affords a good recovery of the new beta-lg in highly purified form.

Amino Acids↗
PubMedDate not supplied

A rapid microbore HPLC method for determination of primary structure of beta-lactoglobulin genetic variants.

A rapid method for determination of the primary structures for beta-lactoglobulin (beta-LG) genetic variants is described. This included rapid microbore HPLC, amino acid analyses, and wherever necessary, direct peptide sequencing. Two novel variants of beta-LG have been identified, bovine beta-LG W and ovine beta-LG C. The proteins were oxidized, digested with trypsin and separated using RP-HPLC. All peptides were recovered in a single run. Peptides with amino acid exchanges were identified by retention time and subjected to amino acid and sequence analyses. Ovine beta-LG C differs from the ovine beta-LG A variant by a single amino acid exchange at position 148 where Arg is replaced by Gln. Bovine beta-LG W differs from bovine beta-LG B by having Leu at position 56 instead of Ile. The method described here is reliable and can be used for mapping of 20-1000 pmol of material.

Amino Acid Sequence↗
PubMed1993

Localization of T-cell determinants on bovine beta-lactoglobulin.

T-cell determinants of bovine beta-lactoglobulin (beta-LG) in BALB/c(H-2d), C57BL/6(H-2b) and C3H/He(H-2k) mice were identified using a set of overlapping synthetic peptides encompassing the entire primary structure of the protein. Lymph node cells from mice immunized with beta-LG were subjected to cell proliferation assay in the presence of these peptides and uptake of 3H-labeled thymidine was measured. Determinant regions were indicated to lie in residues 42-56, 62-76 and 139-153 in BALB/c mice, residues 11-26, 72-86, 100-113 and 119-133 in C57BL/6 mice, residues 72-86, 91-104, 129-143 and 139-153 in C3H/He mice. Some of these fragments included the antigenic motifs predicted by hypotheses according to amphipathicity and sequential patterns of peptides. We reported elsewhere that residues 42-56 and 72-86 represent one of the B-cell antigenic determinants in BALB/c and C3H/He, respectively. These peptides serve as good models of colinear T- and B-cell determinants as they contain both of T- and B-cell determinants.

Amino Acid Sequence↗
PubMed1989

Monoclonal antibodies as probes for monitoring the denaturation process of bovine beta-lactoglobulin.

Five monoclonal antibodies (MAbs) of different idiotypes were produced against bovine beta-lactoglobulin (beta-LG). Among them, MAbs 61B4 and 62A6 reacted preferentially to native beta-LG, while MAbs 21B3 and 31A4 reacted more strongly to the reduced carboxymethylated (denatured) beta-LG than to the native material. These two types of MAb were used to analyze the denaturation process of a beta-LG molecule during heating. The binding affinity of MAbs 21B3 and 31A4 with beta-LG was increased by increasing the heating temperature, the transition temperature being 67-68 degrees C, while that of MAbs 61B4 and 62A6 was reduced by increasing the temperature, this transition temperature being about 80 degrees C. Epitopes recognized by MAbs 31A4 and 61B4 were shown to be included in the segments, Lys8-Trp19 (mostly in the random-coil region) and Thr125-Lys135 (helical region), respectively. The heat-induced conformational change of the beta-LG molecule is, therefore, likely to start in random-coil region as Lys8-Trp19, and to be followed by a structural change in a helical region as Thr125-Lys135. This study demonstrates that MAb is a useful probe to monitor local conformational changes of a protein molecule during denaturation.

Animals↗
PubMed1994

Tryptophan-19 of beta-lactoglobulin, the only residue completely conserved in the lipocalin superfamily, is not essential for binding retinol, but relevant to stabilizing bound retinol and maintaining its structure.

Residue 19 of tryptophan in bovine beta-lactoglobulin (beta-LG) is the only invariant residue throughout the lipocalin superfamily having two characteristic features: binding ability for small hydrophobic molecules and the unique beta-barrel three-dimensional structure. In this study, we investigated whether this strictly conserved Trp-19 of beta-LG would be indispensable for its structure and function such as maintaining the molecular structure and biological activity of beta-LG. Spectroscopic and enzymatic oxidation experiments on retinol bound to W19Y, in which Tyr was substituted for Trp-19, showed that Trp-19 was not critical for this binding, but was important for stably maintaining the environment surrounding retinol and the bound retinol. An using four anti-beta-LG monoclonal antibodies as probes, revealed a structural change in region 20-29, but not in the reverse region of Trp-19. A guanidine hydrochloride-induced unfolding study showed that the conformational stability of W19Y was greatly reduced by 6.9 kcal/mol compared to that of wild-type beta-LG. These facts indicated that Trp-19 is one of the important residues in correctly maintaining the local structure of beta-LG and stably retaining its overall structure, thereby conserving the bound retinol molecule.

Base Sequence↗
PubMed1994

beta-Lactoglobulin binding properties during its folding changes studied by fluorescence spectroscopy.

The milk protein, beta-lactoglobulin (BLG) exhibits structural and binding properties which vary widely, depending on the medium. These properties of BLG are reflected in fluorescence intensities, steady-state anisotropies and phase lifetimes of BLG tryptophan residues and of retinol and diphenyl hexatriene (DPH) bound to BLG, as functions of pH, ethanol concentration and protein modifications (22% ethylated, 90% methylated and 85% acetylated BLGs). Tryptophan quenching experiments show that retinol and DPH bind to BLG in 1:1 molar ratios with apparent dissociation constants around 10(-7) - 10(-8) M. The strength of retinol binding is pH-dependent in the range 3-8, whereas that of DPH binding is not. Two different binding sites for these two ligands coexist on the protein. Modified BLGs exhibit higher affinities for DPH than the unmodified protein. At all pH values investigated, the fluorescence emission at 480 nm of retinol/BLG mixtures and retinol, DPH and tryptophan anisotropies and lifetimes change dramatically with midpoint at 27% ethanol for the first parameter and 35% for the others, suggesting simultaneous beta-strand to alpha-helix transition and the dissociation of BLG complexes at 35% ethanol. An intermediate state, possibly 'molten globular', occurs around 20% ethanol, as deduced from anisotropy and lifetime measurements.

Diphenylhexatriene↗
PubMed1989

Calorimetric and circular dichroic studies of the thermal denaturation of beta-lactoglobulin.

The thermal denaturation of beta-lactoglobulin in aqueous solutions at pH 5.5 and 2.0 was investigated by differential scanning calorimetry (DSC) and circular dichroic (CD) measurements. By calorimetry, the denaturation temperatures (Td), denaturation enthalpies, and specific heat capacity changes for thermal denaturation in the temperature range scanned, i.e., 20-100 degrees C. The unfolding process was found to be only partially reversible. Analysis of the far-ultraviolet CD spectra reveals that with increasing temperature the mean residue ellipticity [( theta]) becomes less negative, which reflects unfolding of the native protein. At the highest temperature of CD measurements, i.e., 80 degrees C, conformational changes are to a large extent reversible.

Calorimetry, Differential Scanning↗
PubMed1995

The proximal milk protein binding factor binding site is required for the prolactin responsiveness of the sheep beta-lactoglobulin promoter in Chinese hamster ovary cells.

To identify cis-acting prolactin (PRL) response elements within the sheep beta-lactoglobulin (BLG) promoter, CHO cells were co-transfected with a rabbit PRL-receptor (PRL-R) expression plasmid and a number of BLG-CAT constructs. Resection through the 4200 bp BLG promoter diminished the PRL response. Mutation of the proximal binding site for milk protein binding factor (MPBF), a previously described mammary gland transcription factor, abolished the PRL inducibility of full length and shorter forms of the promoter. MPBF was shown to be similar to the Stat protein mammary gland factor (MGF) which has been shown to mediate PRL responsiveness of the rat beta-casein gene in mammary cells. MPBF binding activity was detected in the nucleus of CHO cells and was increased 2-6-fold in cells stably transfected with the PRL-R. The lactating mammary gland has high levels of MPBF binding activity and it is likely that this has an important role in the PRL induction of a variety of milk protein genes.

Animals↗
PubMed1990

Characterisation of the alleles encoding ovine beta-lactoglobulins A and B.

beta-Lactoglobulin (BLG) is the major whey protein in the milk of ruminants and is produced in the mammary gland during pregnancy and lactation. Here, we compare the nucleotide sequences of two BLG-encoding clones isolated from a sheep genomic library. The two clones are very similar differing by only 1 bp in their coding regions, giving rise to a Tyr/His difference in the gene product, and suggesting that the two clones correspond to A and B allelic variants of BLG. The isoelectric points (pI) of BLGs A and B were estimated as 5.7 and 6.0, respectively. Transgenic mice carrying a particular clone secrete BLG of the expected pI into their milk. Restriction fragment length polymorphism analysis of a small sheep population demonstrated the existence of at least four BLG haplotypes.

Alleles↗
PubMed1996

Characterization of a caprine beta-lactoglobulin pseudogene, identification and chromosomal localization by in situ hybridization in goat, sheep and cow.

A beta-lactoglobulin (beta-LG) pseudogene has been isolated and sequenced (7634 bp) in goat. Its structure is remarkably similar to that of the beta-LG gene with all seven exons placed in the same relative position. The pseudogene seems to have originated by gene duplication, but a non uniform distribution of similarities along the sequence suggests that events of gene conversion have also occurred during its evolution. The comparison of the predicted ancestral protein encoded by the pseudogene shows its evolutionary relationship to the monomeric beta-LG II forms of the beta-LG reported in some non-ruminant species. Southern-blot analysis shows that similar pseudogenes are also found in the genome of sheep and cow. The pseudogene has been mapped by fluorescent in situ hybridization (FISH) to sheep chromosome 3p28 and cattle and goat chromosomes 11q28, in the same mapped position as that found for the beta-LG gene in all these species.

Animals↗
PubMed2005

The pH-dependent conformational transition of beta-lactoglobulin modulates the binding of protoporphyrin IX.

We have investigated the interaction between PPIX and beta-lactoglobulin (beta-lg) as a function of the pH of the solution. beta-lg is a small globular protein (MW approximately 18 kDa) with a very well characterized structure that reveals several possible binding sites for ligands. The interaction with beta-lg affects the photophysical properties of PPIX. The shift of PPIX emission maximum, excitation maximum and the increase of the fluorescence intensity is an indicator that binding between the porphyrin and beta-lg occurs. The binding constant appears to be modulated by the pH of the solution. Spectroscopic measurements do not reveal any significant energy transfer between the Trp residues of beta-lg and PPIX, however, fluorescence anisotropy decay measurements confirm the binding and the modulation introduced by the pH of the solution. Since beta-lg has been shown to be stable within the range of pH adopted in our experiments (5.0-9.0), the results suggest that PPIX binds a site affected by the pH of the solution. Because of the crystallographic evidence an obvious site is near the aperture of the interior beta-barrel however an alternative (or concurrent) binding site may still be present.

Animals↗
PubMed2006

Effects of heating at neutral and acid pH on the structure of beta-lactoglobulin A revealed by differential scanning calorimetry and circular dichroism spectroscopy.

The structural change of beta-lactoglobulin A (betaLG A) on heating was measured at pH 3.0 and 7.5 with UV absorption difference spectra, differential scanning calorimetry (DSC), and circular dichroism (CD). At pH 3.0, betaLG A showed a reversible structural change by heating at 80 degrees C, while an irreversible change was observed and molecular aggregates of betaLG were formed by heating at 95 degrees C. DSC analysis of betaLG A gave endothermic peaks at 75 degrees C and 90 degrees C at pH 7.5, and 90 degrees C at pH 3.0. At pH 7.5, betaLG A modified with N-ethylmaleimide (NEM-betaLG A) gave two endothermic peaks: at 72 degrees C and 90 degrees C. CD spectra of betaLG A heated at various temperatures and pHs were measured and the spectra at pH 3.0 and 7.5 were not changed by heating to 95 degrees C and 80 degrees C, respectively. Unheated NEM-betaLG A gave a spectrum similar to that of heated betaLG A, suggesting that the secondary structure was changed by NEM treatment.

Calorimetry, Differential Scanning↗
PubMed2005

Complete refolding of bovine beta-lactoglobulin requires disulfide bond formation under strict conditions.

beta-Lactoglobulin (beta-LG) denatured with 6 M guanidine hydrochloride (GdnHCl) containing a reducing agent and subsequently dialysed against phosphate-buffered saline (PBS) resulted in incomplete refolding of this protein despite the fact that the biological activity for retinol-binding was recovered to almost the same degree as that of the native molecule [Hattori, M., Ametani, A., Katakura, Y., Shimizu, M., Kaminogawa, S. J., Biol. Chem. 268 (1993) 22414-22419]. The enzyme probe method, evaluation of hydrophilicity values, in-gel mobility on SDS-PAGE, and evaluation of disulfide bonds with the Ellman method showed exposure of the hydrophobic region(s) and incorrect disulfide bond formation in such dialyzed beta-LG molecules. We reveal in this present work that complete refolding could be attained by diluting denatured beta-LG with PBS containing a reducing agent, before slow reoxidation of the sulfhydryl groups upon dialysis for gradient removal of the reducing agent in 6 steps. Complete renaturation was confirmed by analyzing the retinol-binding activity, CD spectra, intrinsic fluorescence, binding ability of monoclonal antibodies (mAbs), and SDS-PAGE. Step-by-step disulfide bond formation was considered to be critical for the complete refolding of denatured beta-LG. Our method can contribute to establish a procedure for complete refolding of useful recombinant proteins in vitro without such biological aids as chaperones.

Animals↗
PubMed2005

beta-lactoglobulin under high pressure studied by small-angle neutron scattering.

We used small-angle neutron scattering to study the effects of the high hydrostatic pressure on the structure of beta-lactoglobulin. Experiments were carried out at pH 7 on the dimeric form of the protein in a pressure range going from 50 MPa to 300 MPa. These measurements allow the protein size and the interactions between macromolecules to be studied during the application of pressure. Increasing pressure up to 150 MPa leads to a swollen state of the protein that gives rise to an increase of the radius of gyration by about 7%. Within this pressure range, we also show that the interaction between macromolecules weakens although it remains repulsive. The measurements show an aggregation process occurring above 150 MPa. From the spectra analysis, it appears that the aggregation occurs mainly by association of the dimeric units.

Animals↗