Reconstituted micelle formation using reduced, carboxymethylated bovine kappa-casein and human beta-casein.

In milk, kappa-casein, a mixture of disulfide-bonded polymers, stabilizes and regulates the size of the unique colloidal complex of protein, Ca2+ and inorganic phosphate (Pi) termed the casein (CN) micelle. However, reduced, carboxymethylated bovine kappa-CN (RCM-kappa) forms fibrils at 37 degrees C and its micelle-forming ability is in question. Here, the doubly- and quadruply-phosphorylated human beta-CN forms and 1:1 (wt:wt) mixtures were combined with RCM-kappa at different beta/kappa weight ratios. Turbidity (OD(400 nm)) and a lack of precipitation up to 37 degrees C were used as an index of micelle formation. Studies were with 0, 5 and 10 mM Ca2+ and 4 and 8 mM Pi. The RCM-kappa does form concentration-dependent micelles. Also, beta-CN phosphorylation level influences micelle formation. Complexes were low-temperature reversible and RCM-kappa fibrils were seen. There appears to be equilibrium between fibrillar and soluble forms since the solution still stabilized after fibril removal. The RCM-kappa stabilized better than native bovine kappa-CN.

A folding pattern that is stable to thermal cycling is achieved by long term storage of recombinant human beta-casein with four extra N-terminals amino-acid residues at -20 degrees C.

Studies have followed the turbidity (OD400 nm) of beta-casein (CN) as temperature (T) increased from 4 to 37 degrees C. Native non-phosphorylated beta-CN showed a turbidity increase above 25 degrees C and precipitated at about 22 degrees C in 5mM Ca+2. These patterns were reproducible upon T-cycling while those of recombinant beta-CN proteins are not. Here, a wild-type recombinant that was thermally stable after being frozen in solution and stored at -20 degrees C for a prolonged period of time was denatured with guanidine HCl and refolded by dialysis against buffer. This protein was again not stable to T-cycling. A recombinant mutant with four extra N-terminal amino acids was very stable to T-cycling, both with and without 5mM Ca+2. However, it was still much different than the native protein. These results indicate that there are probably many energy minima for this protein and emphasize the possibility of "chaperon-like" conditions for proper folding of human beta-CN.

Colloidal calcium phosphate in the reconstituted milk micelle may direct wild-type recombinant human beta-casein to fold like the native protein.

Native human beta-casein (CN) at all phosphorylation levels exhibits reproducible behavior and appears to have a unique, stable folding pattern. In contrast, the recombinant non-phosphorylated form of human beta-CN (beta-CN-0P) with the exact amino acid sequence (wild-type), expressed and purified from Escherichia coli, differs greatly in its behavior from the native protein and the complexes formed are unstable to thermal cycling. However, when it was incorporated into reconstituted milk micelles, using bovine kappa-CN at a kappa/beta molar ratio of 1/3 with added Ca2+ ions and inorganic phosphate (P(i)) at levels that would ordinarily precipitate, its association behavior vs. temperature as monitored by turbidity (OD(400 nm)) approximated that of native beta-CN-0P. This suggests that the milk micelle system, and particularly the colloidal calcium phosphate, may act as a 'molecular chaperon' to direct the folding of the molecule into the highly stable conformation found in the purified native human beta-CN molecule.

The formation of casein micelles reconstituted with Ca+2 and added inorganic phosphate is influenced by the non-phosphorylated form of human beta-casein.

The beta-casein (CN) human milk fraction is comprised of a single protein phosphorylated at levels from 0 to 5. Component interactions are dependent on the phosphorylation level. Here, 3 mg/ml of beta-CN-0P, beta-CN-2P, beta-CN-4P, a 2P/4P 1:1 (wt:wt) mixture, or a mixture of all six forms in the ratio in human milk, were mixed with bovine kappa-CN at a kappa/beta molar ratio of 0.33. Measurements were with 0, 5 and 10 mM Ca+2 and 4 and 8 mM added inorganic phosphate (Pi). The turbidity (OD400 nm) and a lack of precipitation as T increased from 4 to 37 degrees C was an index of micelle formation. The results indicate: (1) while micelles will form with Ca+2 alone, added Pi has a significant enhancing effect on micelle formation; (2) the patterns of micelle formation as a function of T are influenced by the beta-CN-0P and beta-CN-1P forms of beta-CN to an unexpected extent.

The effect of C-terminal deletion on the folding and self-association of recombinant non-phosphorylated human beta-casein.

Recombinant human beta-casein (CN) mutants were prepared having 11, 22 and 31 amino acids (aa) deleted from the C-terminus. The temperature-dependent self-association of these and the wild-type recombinant was studied by turbidity (OD400) while possible folding differences were examined by intrinsic and extrinsic fluorescence intensity and fluorescence resonance energy transfer. There were major self-association and some conformational differences. Hydrophobicity profile and hydrophobic cluster analysis for bovine and human beta-CN suggested that the ability of the 31 aa deletion mutant in human beta-CN to self-associate when a comparable bovine deletion peptide would not may be due to the presence of additional hydrophobic regions in the middle, indicating that the human protein may contain more than a single hydrophobic binding locus and suggesting that the process for the formation and structure of the micelles of human milk may be quite different from that for bovine milk. A new model may be needed.

The effect of conserved residue charge reversal on the folding of recombinant non-phosphorylated human beta-casein.

A short stretch of 13 amino acids in the central portion of human beta-casein contains four positively charged conserved residues, three Lys and one Arg. We changed these individually to Glu, reversing their charge, and compared the resulting recombinant proteins to the wild-type recombinant, monitoring thermal aggregation with turbidity as well as using the fluorescence of the intrinsic Trp, of hydrophobically bound ANS and fluorescence resonance energy transfer from Trp to ANS to detect differences in structure. The results demonstrate the need to maintain the actual or functional identity of these conserved charged amino acid residues in order to attain the protein folding and functional properties of the wild-type human beta-casein molecule. They emphasize the probability that native human beta-casein has a unique folding pattern that is important for its function of suspending minerals and delivering the protein and minerals to the neonate in a readily ingestible form.

Comparison of native and recombinant non-phosphorylated human beta-casein: further evidence for a unique beta-casein folding pattern.

Recombinant wild-type non-phosphorylated human beta-casein was obtained from Escherichia coli. Turbidity vs. temperature (T) without Ca(2+) showed wild-type self-association like native except for irreversibility upon T-cycling with the original pattern re-established after concentrated urea/dialysis. With Ca(2+), wild-type was more native-like. Intrinsic Trp fluorescence spectra were similar but with lowered intensity for the wild-type protein. Changes in extrinsic ANS fluorescence from 4 to 37 degrees C showed less exposure of hydrophobic surface for wild-type than native. Trp to ANS fluorescence resonance energy transfer was higher for wild-type than native at 4 degrees C but 2- to 3-fold lower at 37 degrees C. The native protein must be directed by the environment and/or a chaperone to fold into a unique, somewhat flexible, conformation, unaltered by urea during purification. Wild-type protein, with many native properties, does not spontaneously fold to the native conformation, even after solubilization with urea. T-cycling gives a stable conformation that is different from the native.