Biomedical subjects

L K Creamer

Publications and source records attributed to L K Creamer.

At least 19 recordsLinked to original sources

PubMed2001

Characterization of heat-induced aggregates of beta-lactoglobulin, alpha-lactalbumin and bovine serum albumin in a whey protein concentrate environment.

Bovine beta-lactoglobulin (beta-lg), alpha-lactalbumin (alpha-la) and bovine serum albumin (BSA), dispersed in ultrafiltration permeate, that had been prepared from whey protein concentrate solution (100 g/kg, pH 6.8), were heated at 75 degrees C. The sequent protein aggregation was studied by one-dimensional (1D) and two-dimensional (2D) polyacrylamide gel electrophoresis (PAGE). When 100 g beta-lg/kg permeate solution was heated at 75 degrees C, cooled and examined, large aggregates were observed. These aggregates were partially dissociated in SDS solution to give monomers, disullphide-bonded dimers, trimers and larger aggregates. When mixtures of beta-lg and alpha-la or BSA were heated, homopolymers of each protein as well as heteropolymers of these proteins were observed. These polymer species were also served in a heated mixture of the three proteins. Two-dimensional PAGE of mixtures demonstrated that these polymers species contained disulphide-bonded dimers of beta-lg. alpha-la and BSA, and 1:1 disulphide-bonded adducts of alpha-la and beta-lg, or BSA. These results are consistent with a mechanism in which the free thiols of heat-treated beta-lg or BSA catalyse the formation of a range of monomers, dimers and higher polymers of alpha-la. It is likely that when whey protein concentrate is heated under the present eonditions. BSA forms disulphide-bonded strands ahead of beta-lg and that alpha-la aggregation with beta-lg and with itself is catalysed by the heat-induced unfolded BSA and beta-lg.

Animals↗

PubMed2000

RNA folding argues against a hot-start origin of life.

Opinion is strongly divided on whether life arose on earth under hot or cold conditions, the hot-start and cold-start scenarios, respectively. The origin of life close to deep thermal vents appears as the majority opinion among biologists, but there is considerable biochemical evidence that high temperatures are incompatible with an RNA world. To be functional, RNA has to fold into a three-dimensional structure. We report both theoretical and experimental results on RNA folding and show that (as expected) hot conditions strongly reduce RNA folding. The theoretical results come from energy-minimization calculations of the average extent of folding of RNA, mainly from 0-90 degrees C, for both random sequences and tRNA sequences. The experimental results are from circular-dichroism measurements of tRNA over a similar range of temperatures. The quantitative agreement between calculations and experiment is remarkable, even to the shape of the curves indicating the cooperative nature of RNA folding and unfolding. These results provide additional evidence for a lower temperature stage being necessary in the origin of life.

Hot Temperature↗

PubMed2000

Formation of new protein structures in heated mixtures of BSA and alpha-lactalbumin.

The heat-induced protein-protein interactions of alpha-lactalbumin (alpha-La) and bovine serum albumin (BSA), dispersed in a pH 6.8, 10% whey protein concentrates (WPC) permeate, were followed using alkaline and sodium dodecyl sulfate (SDS) 1D and 2D polyacrylamide gel electrophoresis (PAGE) and size-exclusion high-performance liquid chromatography (SE-HPLC). Heated (75 degrees C) 5% BSA solution contained large disulfide-bonded BSA aggregates, although some monomer BSA (SDS-monomeric BSA) could be dissociated from the aggregates by SDS. In contrast, similarly heated alpha-La solutions contained small quantities of several monomeric forms of alpha-La and dimeric alpha-La but no large aggregates. When 10% solutions of 1:1 (w/w) mixtures of alpha-La and BSA were heated, large disulfide-bonded aggregates and SDS-monomeric BSA and alpha-La were present. However, heated 2% mixtures contained more modified alpha-La monomers, alpha-La-dimers, and alpha-La-trimers, fewer large disulfide-bonded aggregates, and less SDS-monomeric alpha-La or BSA. These results suggest that BSA forms disulfide-bonded aggregates that contain available thiol groups that can catalyze the formation of differently structured alpha-La monomers, dimers, higher polymers, and adducts of alpha-La with BSA.

Animals↗

PubMed1999

Structural features of a peptide corresponding to human kappa-casein residues 84-101 by 1H-nuclear magnetic resonance spectroscopy.

The peptide Val-Arg-Arg-Pro-Asn-Leu-His-Pro-Ser-Phe-Ile-Ala-Ile-Pro-Pro- Lys-Lys-Ile, which corresponds to residues 84-101 of human kappa-casein, has been synthesized and its conformation preferences determined by 1H-nuclear magnetic resonance spectroscopy in dimethyl sulphoxide. The peptide adopted a largely extended chain conformation in solution and there was evidence for the presence of a beta-turn involving residues Pro87-His90 of human kappa-casein. The presence of a turn in this position would make the physiologically significant Arg85 residue of human kappa-casein (which is equivalent to Arg97 in bovine kappa-casein) unavailable for interaction with Asp249 of bovine chymosin, and may partly explain why human kappa-casein is hydrolysed more slowly than its bovine counterpart by bovine chymosin.

Amino Acid Sequence↗

PubMed1999

Effect of heat treatment on the circular dichroism spectra of bovine beta-lactoglobulin A, B, and C.

Dilute solutions of beta-lactoglobulin (beta-Lg) A, B, and C were heated in phosphate buffer at temperatures between 40 and 94 degrees C for 10 min, cooled, and analyzed using near-UV and far-UV circular dichroism (CD). The decrease in near-UV CD intensity at 293 nm (Deltaepsilon(293)) could be analyzed in terms of a two-state model, and the stability was beta-Lg C > beta-Lg A > beta-Lg B on the basis of the midpoint temperatures for samples heated at pH 6.7 and 7.4. However, the slopes of the curves at the midpoint temperature for variant A were generally less than those for beta-Lg B and beta-Lg C, indicating that the substitution of Val (beta-Lg A) for Ala (beta-Lg B or beta-Lg C) at position 118 had altered the entropic contribution to unfolding of the protein. The changes in CD at 270 nm (Deltaepsilon(270)), an index of significant alteration to disulfide bond dihedral angles, occurred at higher temperatures than those for the Deltaepsilon(293) results. The far-UV CD showed some small changes as a consequence of heat treatment, and the shifts at 205 nm ([theta](205)) fitted a two-state model. Plotting the changes in both Deltaepsilon(293) and [theta](205) against the loss of nativelike and sodium dodecyl sulfate-monomeric protein (assessed by polyacrylamide gel electrophoresis) showed a strong 1:1 relationship between Deltaepsilon(293) or [theta](205) and the loss of nativelike beta-Lg. These results indicated that the initial irreversible stage in the heat-induced aggregation of beta-Lg (nativelike monomer to unfolded monomer) altered the chirality of the environment of Trp(19) and modified the secondary structure of beta-Lg slightly. The differences in the behavior of variants A-C were explicable on the basis of generalized electrostatic and hydrophobicity effects as well as specific amino acid effects.

Animals↗

PubMed1999

Effect of heat treatment on bovine beta-lactoglobulin A, B, and C explored using thiol availability and fluorescence.

Dilute solutions of beta-lactoglobulin (beta-Lg) A, B, and C were heated at temperatures between about 40 and 94 degrees C for 10 min, cooled, and analyzed using Trp fluorescence and extrinsic fluorescence spectra of the probe 1,8-anilinonaphthalene sulfonate (ANS). Thiol availabilities using 5,5'-dithiobis(2-nitrobenzoic acid) (DTNB) were determined using a separate set of samples. The normalized ANS fluorescence emission intensity and the thiol availability results showed a 1:1 relationship with the loss of nativelike but not SDS-monomeric protein, as determined by PAGE analysis. The normalized Trp emission intensity results did not show a comparable 1:1 relationship with the loss of nativelike protein, indicating that the Trp intensity arose from consequential disulfide bond reorganization and not the initial unfolding reaction. The results were also analyzed in terms of two-state models, and the midpoint temperatures (T(mid)) for the proteins were generally beta-Lg C > beta-Lg A > beta-Lg B, and the slopes at the midpoint temperatures for the A variant were generally less than those for the B and C variants indicating that beta-Lg A may denature by a different mechanism from that of beta-Lg B or beta-Lg C. The T(mid) parameters derived from the ANS fluorescence intensity results were similar to those for thiol availability and both were lower than the T(mid) values for Trp emission intensity showing that creation of an ANS binding site on a beta-Lg molecule was linked to the irreversible exposure of a thiol group and the loss of native beta-Lg but preceded the decrease in Trp(61) fluorescence quenching. These results for the differences between the behavior of the A and B or the C variants involved the creation of a destabilizing cavity by the Val(118)Ala (A --> B) substitution and the changed charge distribution within the CD loop caused by the Asp(64)Gly (A --> B) substitution.

Animals↗

PubMed1999

Functional implications of structural differences between variants A and B of bovine beta-lactoglobulin.

The structure of the trigonal crystal form of bovine beta-lactoglobulin variant B at pH 7.1 has been determined by X-ray diffraction methods at a resolution of 2.22 A and refined to values for R and Rfree of 0.239 and 0.286, respectively. By comparison with the structure of the trigonal crystal form of bovine beta-lactoglobulin variant A at pH 7.1, which was determined previously [Qin BY et al., 1998, Biochemistry 37:14014-14023], the structural consequences of the sequence differences D64G and V118A of variants A and B, respectively, have been investigated. Only minor differences in the core calyx structure occur. In the vicinity of the mutation site D64G on loop CD (residues 61-67), there are small changes in main-chain conformation, whereas the substitution V118A on beta-strand H is unaccompanied by changes in the surrounding structure, thereby creating a void volume and weakened hydrophobic interactions with a consequent loss of thermal stability relative to variant A. A conformational difference is found for the loop EF, implicated in the pH-dependent conformational change known as the Tanford transition, but it is not clear whether this reflects differences intrinsic to the variants in solution or differences in crystallization.

Amino Acid Substitution↗

PubMed1998

12-Bromododecanoic acid binds inside the calyx of bovine beta-lactoglobulin.

The X-ray structure of bovine beta-lactoglobulin with the ligand 12-bromododecanoic acid as a model for fatty acids has been determined at a resolution of 2.23 A in the trigonal lattice Z form. The ligand binds inside the calyx, resolving a long-standing controversy as to where fatty-acid like ligands bind. The carboxylate head group lies at the surface of the molecule, and the lid to the calyx is open at the pH of crystallization (pH 7.3), consistent with the conformation observed in ligand-free bovine beta-lactoglobulin in lattice Z at pH 7.1 and pH 8.2.

Animals↗

PubMed1998

Structural basis of the Tanford transition of bovine beta-lactoglobulin.

The structures of the trigonal crystal form of bovine beta-lactoglobulin variant A at pH 6.2, 7.1, and 8.2 have been determined by X-ray diffraction methods at a resolution of 2.56, 2. 24, and 2.49 A, respectively. The corresponding values for R (Rfree) are 0.192 (0.240), 0.234 (0.279), and 0.232 (0.277). The C and N termini as well as two disulfide bonds are clearly defined in these models. The glutamate side chain of residue 89 is buried at pH 6.2 and becomes exposed at pH 7.1 and 8.2. This conformational change, involving the loop 85-90, provides a structural basis for a variety of pH-dependent chemical, physical, and spectroscopic phenomena, collectively known as the Tanford transition.

Animals↗

PubMed1998

Micelle stability: kappa-casein structure and function.

The stability of the casein micelle is dependent on the presence of kappa-casein (CN) on the surface of the micelle where it functions as an interface between the hydrophobic caseins of the micelle interior and the aqueous environment. kappa-Casein is also involved in thiol-catalyzed disulfide interchange reactions with the whey proteins during heat treatments and, after rennet cleavage, in the facilitation of micelle coagulation. These functions of kappa-CN are regulated by the three-dimensional structure of the protein on the micelle surface. The usual means of determining structure are not available for kappa-CN because this protein is strongly self-associating and has never been crystallized. Instead, algorithms were used to predict selected secondary structures and circular dichroism spectroscopy on kappa-CN and the macropeptide released by chymosin. Three peptides were synthesized to cover the chymosin-sensitive site (His98-Lys111), the region in the macropeptide that could be helical (Pro130-Ile153), and the region between. Nuclear magnetic resonance spectroscopy showed that the peptide His98-Lys111 was probably a beta-strand with tight turns at each end. This hypothesis was confirmed by a study of the molecular dynamics showing that the C variant of kappa-CN interacted less strongly with chymosin; consequently, the slow renneting time of milk that contains this protein was explainable. Both circular dichroism and nuclear magnetic resonance indicated that the peptide Pro130-Ile153 was probably helical under normal physiological conditions. A preliminary study using nuclear magnetic resonance showed that the intervening peptide had no discernible secondary structure. Consequently, most of the beta-sheet structure of kappa-CN is likely in the para-kappa-CN region.

Amino Acid Sequence↗

PubMed1997

Solution conformation of a peptide corresponding to bovine kappa-casein B residues 130-153 by circular dichroism spectroscopy and 1H-nuclear magnetic resonance spectroscopy.

The peptide Pro130-Thr-Ser-Thr-Pro-Thr-Ile-Glu-Ala-Val-Glu140- Ser-Thr-Val-Ala-Thr-Leu-GLu-Ala-Ser-Pro150-Glu-Val-Ile, which corresponds to residues 130-150 of kappa-casein B, was synthesized and the conformation of the peptide in solution investigated by circular dichroism (CD) spectroscopy, structure prediction algorithms and 1H-nuclear magnetic resonance spectroscopy. In a solution containing the structure-enhancing solvent trifluoroethanol the CD spectrum was typical of a peptide in the alpha-helical conformation and nuclear magnetic resonance showed that the amino acids between Ile136 and Ser149 (kappa-casein numbering) were predominantly in the alpha-helical conformation but that Pro130 to Thr135 and Pro150 to Ile153 were not. In addition, Thr133-Pro134 and Ser-149-Pro150 were primarily in the trans conformation, the residues from Thr131 to Thr135 were in unordered structures and the residues from Glu151 to Ile153 were in an extended conformation. Residues Glu137 to Glu140 and Thr145 to Ala148 also displayed some 3(10)-helix character. When the peptide was dissolved in 10 mM-cetyltrimethylammonium chloride solution at pH 6, the CD spectra indicated that the proportion of helical structure was comparable to that of the peptide in trifluoroethanol solution (400 ml/l), whereas when the peptide was dissolved in buffer alone in 10 mM-SDS solution, the CD spectra were consistent with a low helical content. Acidification of these solutions to pH 2.85 resulted in a slight increase in the helical content of the peptide in buffer and more markedly in buffer containing SDS. When the peptide was in 5 mM-CaCl2 solution at neutral pH, the CD spectrum indicated that some ordered structure was present. Taken together these results indicate that the ionizable residues Glu137, Glu140, Glu147 and Glu151 could be important in determining the stability of the putative helix. The structure predictions found that the sequence from Glu137 to Pro150 would be more likely to be in a helical than any other conformation in the intact bovine protein, but that pig, sheep and goat kappa-caseins did not give a prediction of a strongly helical region in this part of the molecule.

Amino Acid Sequence↗

PubMed1995

Effect of sodium dodecyl sulfate and palmitic acid on the equilibrium unfolding of bovine beta-lactoglobulin.

The unfolding of bovine beta-lactoglobulin, a small globular protein that unfolds reversibly at low pH in the presence of urea or guanidine hydrochloride, has been studied at pH 6.72 in phosphate buffer at 21 degrees C. The midpoint urea concentration for the loss of CD intensity at 220 nm, loss of CD intensity at 293 nm, quenching of intrinsic fluorescence, shift in the wavelength of the maximum of the intrinsic fluorescent emission, and loss of fluorescence intensity from 1-anilino-8-naphthalenesulfonate (ANS) (and probably the hydrophobic binding site) was close to 4.4 M. Addition of sodium dodecyl sulfate (SDS) at concentrations less than 100 microM to the beta-lactoglobulin solutions increased the midpoint urea concentration for the CD and intrinsic fluorescence parameters to about 5.8 M. Palmitic acid had a similar effect to that shown by SDS in altering the CD intensity at 293 nm, and both SDS and palmitic acid attained a maximum effect in altering the CD at 293 nm at a 1:1 molar ratio to beta-lactoglobulin. It seems likely that the beta-sheet structure of beta-lactoglobulin breaks down simultaneously with the loss of the hydrophobic binding site and exposure of tryptophan-19 to the external environment, supporting the view that the major hydrophobic binding site of beta-lactoglobulin is closely involved with the beta-sheet core of the protein.(ABSTRACT TRUNCATED AT 250 WORDS)

Anilino Naphthalenesulfonates↗

PubMed1995

Restrained molecular dynamics study of the interaction between bovine kappa-casein peptide 98-111 and bovine chymosin and porcine pepsin.

The cleavage of bovine kappa-casein at the Phe105-Met106 bond by chymosin or pepsin is the first stage in casein micelle coagulation and casein digestion. The nature of the interaction of the peptide His98-Pro-His-Pro-His-Leu-Ser-Phe105-Met-Ala-Ile-Pro-Pro- Lys111 with chymosin and porcine pepsin was investigated using molecular modelling and energy minimization techniques. This study verified and extended a proposed model that electrostatic binding (involving His98, His100, His102 and Lys111 or Lys112) at either end of the active site cleft of chymosin is important for the positioning of residues 103-108 in the cleft. The peptide conformation remained unchanged in going from solution to binding into the active site cleft, with the exception that optimum binding of substrate to chymosin required the isomerization of the His98-Pro99 peptide bond from the trans to the cis conformation. The study also identified an acidic region in porcine pepsin that is in a position to form strong electrostatic interactions with the histidines at the N-terminus of the peptide.

Amino Acid Sequence↗

PubMed1993

Effect of the A and B variants of both alpha s1- and kappa-casein on bovine casein micelle solvation and kappa-casein content.

Casein micelle solvation, a micelle characteristic that is sensitive to many factors, has been measured by a centrifugation technique at 30 degrees C for a series of uncooled fresh skim milks at pH 6.3, 6.6, 6.9 and 7.1. The relative alpha s-(alpha s1-plus alpha s2-), beta- and kappa-casein contents of all centrifuge pellets and supernatants were determined by a standardized electrophoretic method. The calcium and phosphate contents of a number of the pellets and milk samples were also determined. Solvation of micelles from milks with various genetic variants of beta-lactoglobulin (A and B), alpha s1-casein (A and B) and kappa-casein (A and B) was often found to be lower for milks containing either the B variant of alpha s1-casein or the A variant of kappa-casein. It was also found that these two variant caseins were associated with a lower kappa-casein. It was also found that these two variant caseins were associated with a lower kappa-casein content of the milks and the micelles, which is consistent with the lower solvation as kappa-casein is associated with smaller micelle size and greater solvation. The solvations also seemed to increase during the lactation period. It is possible that some of the other features of milk and its products that have been ascribed to the differences in functional character between the A and B variants of alpha s1-casein may be partly caused by the increased level of kappa-casein. The reason for the association of the A variant of alpha s1-casein with higher concentrations of kappa-casein (and micelle solvation) is not obvious but possibly the haplotype alpha s1-casein A, beta-casein A1, kappa-casein A contains a controlling sequence in the chromosomal DNA that enhances expression of the kappa-casein gene.

Amino Acid Sequence↗

PubMed1992

Genetic manipulation of milk proteins and its consequences for the dairy industry.

Genetic selection of cattle by selective breeding patterns dates back to prehistoric times and has resulted in the diversity of breeds we see today. Selection in New Zealand has been for fat production earlier in the century, and more recently for protein production as well as fat. There is a lot of interest today in the naturally occurring variants of the milk proteins, as these can confer interesting differences in the molecular behaviour of the proteins as well as being correlated with compositional differences in the milk. Genetic modification holds great promise for the future in the dairy industry, but present constraints due to cost, lack of basic knowledge, and difficulty in producing genetically-modified calves, mean that only the biopharmaceutical area is likely to be affected in the near future. Coupled to this is an apparent lack of acceptance of food from genetically-modified animals by consumers. It will therefore need a change in public attitude as well as some development in science and technology before dairy products from genetically modified cattle become a commercial reality.

Adult↗

PubMed1989

Cloning and sequencing of a complementary deoxyribonucleic acid coding for a bovine alpha s1-casein A from mammary tissue of a homozygous B variant cow.

A cDNA clone for bovine alpha s1-casein variant A was isolated from a mammary gland cDNA library using a synthetic degenerate oligonucleotide probe. The largest Pst I insert containing an EcoR I site was sequenced. It contained 1090 base pairs, 47 in the 5' noncoding region, 603 in the coding region and 440 in the 3' noncoding region. The nucleotide sequence was compared with three published cDNA sequences for alpha s1-casein variant B. The most obvious difference was the absence of the 39 bases encoding the 13 amino acids that are present in the B variant but absent from the A variant. In addition, five other single base positions differed within individual codons among the four sequences at the third base for each codon, but this did not change the amino acids encoded. There were, however, a number of differences found in the 3' noncoding region. The isolated cDNA was subjected to site-directed mutagenesis to replace a Val-Ile dipeptide with Phe-Phe to increase the chymosin sensitivity of the protein. When the milk proteins from mammary gland tissue extracts were typed, the alpha s1-casein A gene product was not detected.

Amino Acid Sequence↗

PubMed1984

Anomalous behavior of bovine alpha s1- and beta-caseins on gel electrophoresis in sodium dodecyl sulfate buffers.

Electrophoresis in the presence of sodium dodecyl sulfate (SDS) provides a relatively simple means of determining molecular weights of proteins. This technique relies on the validity of a correlation between some function of Mr and the mobility of the protein through the gel matrix. However, bovine caseins (especially alpha s1-casein) have lower mobilities than expected on the basis of their known Mr. The binding of SDS to both alpha s1-casein (Mr 23,600) and beta-casein (Mr 24,000) reached a maximum at the slightly low value of 1.3 g SDS/g protein. Gel-filtration chromatography showed, however, that the alpha s1-casein:SDS complex was larger than the beta-casein:SDS complex at pH 6.8 or 7.0, but that they were similar in size at pH 2.9 or 3.0. Circular dichroism spectra indicated that the low helical structure content of both alpha s1- and beta-casein increased with the addition of SDS and/or decreasing the pH to 1.5. 13C NMR results showed that SDS bound to alpha s1- and beta-casein in the same way as it did to bovine serum albumin. Either esterification or dephosphorylation followed by amidation of alpha s1-casein increased its mobility in SDS-gel electrophoresis, but neither modification affected beta-casein mobility. These and other results indicate that the low electrophoretic velocity of alpha s1-casein in SDS-gel electrophoresis results from its unexpectedly large hydrodynamic size. This is caused by localized high negative charges on certain segments of alpha s1-casein, which would induce a considerable amount of inter- and intrasegmental electrostatic repulsion, leading to an expanded or extended structure for portions of the alpha s1-casein molecule in the presence of SDS. It is clear that the conformation, and hence the equivalent radius, of an SDS:protein complex is determined by the sequence of amino acids in the protein and that, a priori, it cannot be anticipated that the electrophoretic mobility of such a complex will bear more than a casual relationship to the Mr of the protein.

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↗