A multinuclear, high-resolution NMR study of bovine casein micelles and submicelles

High-resolution, natural abundance 13C[1H] (100.5 MHz), 31P[1H] (161.8 MHz) and 1H (400.0 MHz) NMR spectroscopy was used to identify the calcium-binding sites of bovine casein and to ascertain the dynamic state of amino acid residues within the casein submicelles (in 125 mM KCl, pD = 7.4) and micelles (in 15 mM CaCl2/80 mM KCl, pD = 7.2). The presence of numerous, well-resolved peaks in the tentatively assigned 13C-NMR spectra of submicelles (90 A radius) and micelles (500 A radius) suggests considerable segmental motion of both side chain and backbone carbons. The partly resolved 31P-NMR spectra concur with this. Upon Ca2+ addition, the phosphoserine beta CH2 resonance (65.8 ppm vs DSS) shifts upfield by 0.2 ppm and is broadened almost beyond detection; a general upfield shift (up to 0.3 ppm) is also observed for the 31P-NMR peaks. The T1 values of the alpha CH envelope for submicelles and micelles are essentially identical corresponding to a correlation time of 8 ns for isotropic rotation of the caseins. Significant changes in the 31P T1 values accompany micelle formation. Data are consistent with a loose and mobile casein structure, with phosphoserines being the predominant calcium-binding sites.     

Codon usage in the vertebrate hemoglobins and its implications

A study of codon usage in vertebrate hemoglobins revealed an evolutionary trend toward elevated numbers of CpG codon boundary pairs in mammalian hemoglobin alpha genes. Selection for CpG codon boundaries countering the generally observed CpG suppression is strongly suggested by these data. These observations parallel recently published experimental results that indicate that constitutive expression of the human alpha-globin gene appears to be determined by regulatory information encoded within the structural gene. The possibility is raised that, in the absence of selection, CpG decay can be used to date the evolutionary origin of a mammalian alpha pseudogene from its active alpha gene.      

Structure and mechanism of action of riboflavin-binding protein: Small-angle X-ray scattering,sedimentation, and circular dichroism studies on the holo- and apoproteins

Riboflavin-binding protein, a transport protein occurring in egg whites, binds riboflavin tightly at pH values above 4.5 but releases it readily at pH values below 4.0. Structural aspects of this biologically important binding were studied by several methods. Analysis of sedimentation equilibrium data gave an average molecular weight of 32,500 ± 1000 for all forms of the protein and showed the absence of changes in quaternary structure when riboflavin was bound at neutral pH or released at pH 3.7. Sedimentation velocity showed no change in tertiary structure on binding at pH 7.0 but revealed a significant change in sedimentation constant at pH 3.7. While circular dichroism showed no appreciable change in secondary structure, it gave evidence of a marked change in the aromatic region at the lower pH. Small-angle X-ray scattering, going from the holoprotein at neutral pH to the apoprotein at low pH, showed a small but significant increase in radius of gyration (19.8 ± 0.2 vs 20.6 ± 0.1 Å) with slightly decreased anisotropy and with substantial increases in molecular volume (55,600 ± 530 vs 66,500 ± 240 ų), surface (11,840 ± 120 vs 13,470 ± 140 Å), and hydration (0.27 ± 0.01 vs 0.38 ± 0.01 g H₂O/g dry protein). Hydration values were obtained from small-angle X-ray scattering in two different ways for comparison with those calculated from sedimentation coefficients by way of frictional coefficients (derived from two different dimensionless ratios based independently on the structural small-angle X-ray scattering data). For either form of the protein, the surface calculated from an ellipsoidal model could account for only about 62% of the surface found experimentally. The excess surface was ascribed to topographic features of the molecule. Relative changes in this new parameter, together with the circular dichroism data and the known association of riboflavin binding with aromatic residues, suggested the opening of an aromatic-rich cleft concomitant with the release of riboflavin as a consequence of lowered pH.     

Investigation of differences in the tertiary structures of food proteins by small-angle X-ray scattering

Summary With current emphasis in bioengineering on developing new and better structure-function relationships for proteins (e.g., the need for predictability of expected properties prior to cloning), practical and reliable methodology for providing characterization of appropriate features has become of increasing importance. The most potent and detailed technique, X-ray crystallography, has severe limitations: it is so demanding and time-consuming that X-ray coordinates are frequently unavailable for materials of interest; its data relate to static and essentially unhydrated structures, whereas proteins exhibit a variety of dynamic features and function in an aqueous environment; and many proteins of technological importance may never be crystallized. Small-angle X-ray scattering, however, is particularly suitable as a methodology that can provide a substantial number of significant geometric parameters consistent with crystallographic results, that can readily show tertiary structural changes occurring under varying conditions, and that can deal with solutions and gels. Results are presented here from small-angle X-ray scattering investigations of the apo and holo forms of chicken egg-white riboflavin-binding protein, chicken egg-white lysozyme, bovine milk-whey -lactalbumin and -lactoglobulin, and bovine ribonuclease. We utilize these observations to compare tertiary structures of these proteins as well as conformational changes in these structures, and to provide a basis for discussion of their physical and biological significance.Agricultural Research Service, U.S. Department of Agriculture. Reference to brand or firm name does not constitute endorsement by the U.S. Department of Agriculture over others of a similar nature not mentioned.     

Calcium-induced associations of the caseins: a thermodynamic linkage approach to precipitation and resolubilization

Calcium-induced changes in protein solubility play a role in a variety of important biological processes including the deposition of bone and dentin and the secretion of milk. The phenomena of salt-induced (calcium) precipitation of proteins (salting-out), and the resolubilization of these proteins at higher salt concentrations (salting-in) have been studied and quantitated using an approach based on the concepts of Wyman's thermodynamic linkage. Salting-out has been described by a salt-binding constant, k1, the number of moles of salt bound per mole of protein, n, and S1, the fraction soluble at saturation of n; salting-in has been described by corresponding constants k2, m, and S2. Analysis of salt-induced solubility profiles was performed using nonlinear regression analysis. Results of calcium-induced solubility profiles of two genetic variants of alpha s1-casein (alpha s1-A), (alpha s1-B), and beta-casein C (beta-C) at 37 degrees C, where hydrophobic interactions are maximized, showed no salting-in behavior and for salting-out, yielded k1 values of 157, 186, and 156 liters.mol-1 and n values of 8, 8, and 4, respectively. The values of k1 can be correlated with the apparent association constant for calcium binding to casein, while the values of n can be correlated with the number of calcium binding sites of the respective caseins. At 1 degree C, where hydrophobic interactions are minimized, nominally only hydrophilic and electrostatic interactions can be linked to the salt-induced solubility profiles; here beta-C is totally soluble at all calcium concentrations and alpha s1-B and alpha s1-A were now found to have salting-in parameters, k2 and m, of 2.5 liters.mol-1 and 4, and 11 liters.mol-1 and 8, respectively. alpha s1-A is more readily salted-in and studies on the variation of S1 with added KCl for this protein at 1 degree C indicated that salting-in is also mainly electrostatic in nature and may result from competition between K+ and Ca2+ for binding sites rather than from solute-solvent interactions as previously proposed. Comparison of k1 and k2 values between the two genetic variants, coupled with the known sequence differences (the A variant is a linear deletion of 13 amino acids) suggest the existence of a hydrophobically stabilized ion pair in alpha s1-B which is deleted in alpha s1-A; it is speculated that such bonds may play a role in other calcium-induced changes in protein solubility.     

Effects of multivalent cations on cell wall-associated Acid phosphatase activity

Primary cell walls, free from cytoplasmic contamination were prepared from corn (Zea mays L.) roots and potato (Solanum tuberosum) tubers. After EDTA treatment, the bound acid phosphatase activities were measured in the presence of various multivalent cations. Under the conditions of minimized Donnan effect and at pH 4.2, the bound enzyme activity of potato tuber cell walls (PCW) was stimulated by Cu²⁺, Mg²⁺, Zn²⁺, and Mn²⁺; unaffected by Ba²⁺, Cd²⁺, and Pb²⁺; and inhibited by Al³⁺. The bound acid phosphatase of PCW was stimulated by a low concentration but inhibited by a higher concentration of Hg²⁺. On the other hand, in the case of corn root cell walls (CCW), only inhibition of the bound acid phosphatase by Al³⁺ and Hg²⁺ was observed. Kinetic analyses revealed that PCW acid phosphatase exhibited a negative cooperativity under all employed experimental conditions except in the presence of Mg²⁺. In contrast, CCW acid phosphatase showed no cooperative behavior. The presence of Ca²⁺ significantly reduced the effects of Hg²⁺ or Al³⁺, but not Mg²⁺, to the bound cell wall acid phosphatases. The salt solubilized (free) acid phosphatases from both PCW and CCW were not affected by the presence of tested cations except for Hg²⁺ or Al³⁺ which caused a Ca²⁺-insensitive inhibition of the enzymes. The induced stimulation or inhibition of bound acid phosphatases was quantitatively related to cation binding in the cell wall 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 Wyman's Thermodynamic Linkage Theory. Ca²⁺-induced colloid stability profiles were obtained experimentally for model micelles consisting of only _s1- (a calcium insoluble casein) and the stabilizing protein -casein, eliminating the complications arising from - and minor casein forms. Two distinct genetic variants _s1-A andB were used. Analysis of _s1-A colloid stability profiles yielded a precipitation (salting-out) constantk ₁, as well as colloid stability (salting-in) parameterk ₂. No variations ofk ₁ ork ₂ were found with increasing amounts of -casein. From the variation of the amount of colloidal casein capable of being stabilized vs. amount of added -casein an association constant of 4 L/g could be calculated for the complexation of _s1-A and -casein. For the _s1-B and -casein micelles, an additional Ca²⁺-dependent colloidal destabilization parameter,k ₃, was added to the existingk ₁ andk ₂ parameters in order to fully describe this more complex system. Furthermore, the value ofk ₃ decreased with increasing concentration of -casein. These results were analyzed with respect to the specific deletion which occurs in _s1-caseinA in order to determine the sites responsible for these Ca²⁺-induced quaternary structural effects.     

Water interactions with varying molecular states of bovine casein: 2H NMR relaxation studies

The caseins occur in milk as spherical colloidal complexes of protein and salts with an average diameter of 1200 A, the casein micelles. Removal of Ca2+ is thought to result in their dissociation into smaller protein complexes stabilized by hydrophobic interactions and called submicelles. Whether these submicelles actually occur within the micelles as discrete particles interconnected by calcium phosphate salt bridges has been the subject of much controversy. A variety of physical measurements have shown that casein micelles contain an inordinately high amount of trapped water (2 to 7 g H2O/g protein). With this in mind it was of interest to determine if NMR relaxation measurements could detect the presence of this trapped water within the micelles, and to evaluate whether it is a continuum with picosecond correlation times or is associated in part with discrete submicellar structures with nanosecond motions. For this purpose the variations in 2H NMR longitudinal and transverse relaxation rates of water with protein concentration were determined for bovine casein at various temperatures, under both submicellar and micellar conditions. D2O was used instead of H2O to eliminate cross-relaxation effects. From the protein concentration dependence of the relaxation rates, the second virial coefficient of the protein was obtained by nonlinear regression analysis. Using either an isotropic tumbling or an intermediate asymmetry model, degrees of hydration, v, and correlation times, tau c, were calculated for the caseins; from the latter parameter the Stokes radius, r, was obtained. Next, estimates of molecular weights were obtained from r and the partial specific volume. Values were in the range of those published from other methodologies for the submicelles. Temperature dependences of the hydration and Stokes radius of the casein submicelles were consistent with the hypothesis that hydrophobic interactions represent the predominant forces responsible for the aggregation leading to a submicellar structure. The same temperature dependence of r and v was found for casein under micellar conditions; here, the absolute values of both the Stokes radii and hydrations were significantly greater than those obtained under submicellar conditions, even though tau c values corresponding to the great size of the entire micelle would result in relaxation rates too fast to be observed by these NMR measurements. The existence of a substantial amount of trapped water within the casein micelle is, therefore, corroborated, and the concept that this water is in part associated with submicelles of nanosecond motion is supported by the results of this study.     

A benefit-finding intervention for family caregivers of persons with Alzheimer disease: study protocol of a randomized controlled trial

Background

Patients with ST-elevation myocardial infarction (STEMI) not treated with primary or rescue percutaneous coronary intervention (PCI) are at risk for recurrent ischemia, especially when viability in the infarct-area is present. Therefore, an invasive strategy with PCI of the infarct-related coronary artery in patients with viability would reduce the occurrence of a composite end point of death, reinfarction, or unstable angina (UA).

Methods

Patients admitted with an (sub)acute myocardial infarction, who were not treated by primary or rescue PCI, and who were stable during the first 48 hours after the acute event, were screened for the study. Eventually, we randomly assigned 216 patients with viability (demonstrated with low-dose dobutamine echocardiography) to an invasive or a conservative strategy. In the invasive strategy stenting of the infarct-related coronary artery was intended with abciximab as adjunct treatment. Seventy-five (75) patients without viability served as registry group. The primary endpoint was the composite of death from any cause, recurrent myocardial infarction (MI) and unstable angina at one year. As secondary endpoint the need for (repeat) revascularization procedures and anginal status were recorded.

Results

The primary combined endpoint of death, recurrent MI and unstable angina was 7.5% (8/106) in the invasive group and 17.3% (19/110) in the conservative group (Hazard ratio 0.42; 95% confidence interval [CI] 0.18-0.96; p = 0.032). During follow up revascularization-procedures were performed in 6.6% (7/106) in the invasive group and 31.8% (35/110) in the conservative group (Hazard ratio 0.18; 95% CI 0.13-0.43; p < 0.0001). A low rate of recurrent ischemia was found in the non-viable group (5.4%) in comparison to the viable-conservative group (14.5%). (Hazard-ratio 0.35; 95% CI 0.17-1.00; p = 0.051).

Conclusion

We demonstrated that after acute MI (treated with thrombolysis or without reperfusion therapy) patients with viability in the infarct-area benefit from a strategy of early in-hospital stenting of the infarct-related coronary artery. This treatment results in a long-term uneventful clinical course. The study confirmed the low risk of recurrent ischemia in patients without viability.

Trial registration

ClinicalTrials.gov: NCT00149591.