H.M. Farrell

Secondary Structural Studies of Bovine Caseins: Structure and Temperature Dependence of β-Casein Phosphopeptide (1-25) as Analyzed by Circular Dichroism, FTIR Spectroscopy, and Analytical Ultracentrifugation

The defining structural feature of all of the caseins is their common phosphorylation sequence. In milk, these phosphoserine residues combine with inorganic calcium and phosphate to form colloidal complexes. In addition, nutritional benefits have been ascribed to the phosphopeptides from casein. To obtain a molecular basis for the functional, chemical, and biochemical properties of these casein peptides, the secondary structure of the phosphopeptide of bovine β-casein (1–25) was reexamined using Fourier transform infrared (FTIR) and circular dichroism (CD) spectroscopies. Both methods predict secondary structures for the peptide which include polyproline II elements as well as β-extended sheet and turn-like elements. These structural elements were highly stable from 5° to 70°C. Reexamination of previously published 1H NMR data using chemical shift indices suggests structures in accord with the CD and FTIR data. Dephosphorylation showed little or no secondary structural changes, as monitored by CD and FTIR, but the modified peptide demonstrated pronounced self-association. The polymers formed were not highly temperature sensitive, but were pressure sensitive as judged by analytical ultracentrifugation at selected rotor speeds. Molecular dynamics (MD) simulations demonstrated relatively large volume changes for the dephosphorylated peptide, in accord with the pressure dependent aggregation observed in the analytical ultracentrifuge data. In contrast the native peptide in MD remained relatively rigid. The physical properties of the peptide suggest how phosphorylation can alter its biochemical and physiological properties.

Review of the chemistry of αS2-casein and the generation of a homologous molecular model to explain its properties

alpha(S2)-Casein (alpha(S2)-CN) comprises up to 10% of the casein fraction in bovine milk. The role of alpha(S2)-CN in casein micelles has not been studied in detail in part because of a lack of structural information on the molecule. Interest in the utilization of this molecule in dairy products and nutrition has been renewed by work in 3 areas: biological activity via potentially biologically active peptides, functionality in cheeses and products, and nutrition in terms of calcium uptake. To help clarify the behavior of alpha(S2)-CN in its structure-function relationships in milk and its possible applications in dairy products, this paper reviews the chemistry of the protein and presents a working 3-dimensional molecular model for this casein. The model was produced by threading the backbone sequence of the protein onto a homologous protein: chloride intracellular channel protein-4. Overall, the model is in good agreement with experimental data for the protein, although the amount of helix may be over-predicted. The model, however, offers a unique view of the highly positive C-terminal portion of the molecule as a surface-accessible area. This region may be the site for interactions with kappa-carrageenan, phosphate, and other anions. In addition, most of the physiologically active peptides isolated from alpha(S2)-CN occur in this region. This structure should be viewed as a working model that can be changed as more precise experimental data are obtained.

Comparison of the three-dimensional molecular models of bovine submicellar caseins with small-angle X-ray scattering. Influence of protein hydration.

To test the applicability of two energy-minimized, three-dimensional structures of the bovine casein submicelle, theoretical small-angle X-ray scattering curves in the presence and absence of water were compared to experimental data. The published method simulates molecular dynamics of proteins in solution by employing adjustable Debye-Waller temperature factors (B factors) for the protein, for the solvent, and for protein-bound water. The programs were first tested upon bovine pancreatic trypsin inhibitor beginning with its known X-ray crystal structure. To approximate the degree of protein hydration previously determined by NMR relaxation experiments (0.01 g water/g protein), 120 water molecules were docked into the large void of theκ-casein portion of the structure for both the symmetric and asymmetric casein submicelle models. To approximate hydrodynamic hydration (0.244 g water/g protein), 2703 water molecules were added to each of the above structures using the “droplet” algorithm in the Sybyl molecular modeling package. All structures were then energy-minimized and their solvation energies calculated. Theoretical small-angle X-ray scattering curves were calculated for all unhydrated and hydrated structures and compared with experimentally determined scattering profiles for submicellar casein. Best results were achieved with the 120-bound-water structure for both the symmetric and asymmetric submicelle models. Comparison of results for the protein submicelle models with those for the theoretical and literature values of bovine pancreatic trypsin inhibitor demonstrates the applicability of the methodology.

Particle sizes of purified κ-casein: Metal effect and correspondence with predicted three-dimensional molecular models

Abstractκ-Casein as purified from bovine milk exhibits a rather unique disulfide bonding pattern as revealed by SDS-PAGE. The disulfide-bonded caseins present range from dimer to octamer and above and preparations contain about 10% monomer. All of these heterogenous polymers, however, self-associated into nearly spherical uniform particles with an average radius of 8.9 nm as revealed by negatively stained transmission electron micrographs. Evidence is presented that multivalent cations play a role in the stabilization of these spherical particles. Treatment with EDTA causes disruption of theκ-casein particles and leads to a broader size distribution as judged by electron microscopy and dynamic light scattering. The size and shape of the particles are in accord with earlier proposed 3D models forκ-casein that actually predicted participation of divalent cations in the structure.

Calcium-induced associations of the caseins: Thermodynamic linkage of calcium binding to colloidal stability of casein micelles

The caseins occur in milk as colloidal complexes of protein aggregates, calcium, and inorganic phosphate. As determined by electron microscopy, these particles are spherical and have approximately a 650 Å radius (casein micelles). In the absence of calcium, the protein aggregates themselves (submicelles) have been shown to result from mainly hydrophobic interactions. The fractional concentration of stable colloidal casein micelles can be obtained in a calcium caseinate solution by centrifugation at 1500g. Thus, the amount of stable colloid present with varying Ca2+ concentrations can be determined and then analyzed by application of equations derived from Wymans Thermodynamic Linkage Theory. Ca2+-induced colloid stability profiles were obtained experimentally for model micelles consisting of only αs1- (a calcium insoluble casein) and the stabilizing protein κ-casein, eliminating the complications arising from β- and minor casein forms. Two distinct genetic variants αs1-A andB were used. Analysis of αs1-A colloid stability profiles yielded a precipitation (salting-out) constantk1, as well as colloid stability (salting-in) parameterk2. No variations ofk1 ork2 were found with increasing amounts of κ-casein. From the variation of the amount of colloidal casein capable of being stabilized vs. amount of added κ-casein an association constant of 4 L/g could be calculated for the complexation of αs1-A and κ-casein. For the αs1-B and κ-casein micelles, an additional Ca2+-dependent colloidal destabilization parameter,k3, was added to the existingk1 andk2 parameters in order to fully describe this more complex system. Furthermore, the value ofk3 decreased with increasing concentration of κ-casein. These results were analyzed with respect to the specific deletion which occurs in αs1-caseinA in order to determine the sites responsible for these Ca2+-induced quaternary structural effects.

Pyrolysis Gas Chromatography of Wool Part II: Detection and Quantitation of Tryptophan in Wool and Simple Proteins

Although determination of tyrosine and most other amino acids is straightforward by conventional methods of amino acid analysis, determination of tryptophan is dif ficult and time-consuming due to its sensitivity to acid-catalyzed protein hydrolysis. Pyrolysis gas chromatography (Py-GC) therefore was investigated as an alternative method for tryptophan analysis. Py-GC revealed that wool gives rise to indole and skatole from degradation of its tryptophanyl residues, along with phenol and para- cresol from its tyrosyl residues. To test Py-GC for determining tryptophan content relative to tyrosine content, mixtures of poly-tryptophan and poly-tyrosine were used to establish a linear relationship between the peak area ratio indole/(phenol + para- cresol) and the tryptophan / tyrosine molar ratio (Trp/Tyr). The polypeptide data were supplemented with data from pyrolysis of several proteins of known Trp/Tyr ratio. The supplemental data also were used to confirm the utility of the linear rela tionship in deriving a Trp/Tyr ratio for an unknown protein. The accuracy of the derived Trp/Tyr ratio diminished somewhat when the tryptophan content was low; using Py-GC, wool was predicted to have one tryptophan for every eight to nine tyrosines, as opposed to one for every seven tyrosines using conventional amino acid analysis. The method permitted facile monitoring of tryptophan degradation in wool during treatment with dimethyl sulfoxide in hydrochloric and acetic acid. There also was a strong indication of tryptophan degradation during wool carbonizing.

Isolation and properties of goat β2-microglobulin

Abstract 1. 1. Goat β 2 - microglobulin was isolated and purified from colostrum. 2. 2. Comparisons of the amino acid composition and amino-terminal sequence the goat protein with the bovine and human homologues, indicates a high degree of similarity. Both goat and bovine β 2 - microglobulins differ slightly in composition from the human molecule, most notably in threonine and proline values. 3. 3. For the first 32 residues, bovine and goat differ only at two positions, one of which is a valyl/isoleucyl substitution consistent with the amino acid compositions. The equivalent goat/human sequence comparison shows seven differences. 4. 4. Immunological studies, using the ELISA method, also confirm the close relatedness of goat and bovine β 2 - microglobulin and their more distant relatedness to the human homologue.