Thomas F. Kumosinski

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 Wymans 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.

[14] Measurements of protein hydration by various techniques

Publisher Summary This chapter deals with hydration measurements made on globular proteins in solution z by three techniques which have been found in recent years to afford certain useful insights: nuclear magnetic resonance (NMR) relaxation, small-angle X-ray scattering (SAXS), and hydrodynamics (velocity sedimentation). The latter two is treated together, because sedimentation, which cannot by itself give definitive values of hydration, is very useful in corroborating SAXS data. Conversely, sedimentation coefficients can be calculated independently from SAXS. NMR methodology should be treated in particular detail, both because of its potential utility for the present purpose of the chapter. Moreover, specific discussions of these methods are then followed by some general remarks, including an evaluation of the different approaches. It may be noted that correlations between SAXS and hydrodynamic methods other than sedimentation (viscosity, diffusion), and between various combinations of hydrodynamic data with each other, have been less successful.

Quantitation of the global secondary structure of globular proteins by FTIR spectroscopy: Comparison with X-ray crystallographic structure

Fourier transform infrared spectroscopy (FTIR) is potentially a powerful tool for determining the global secondary structure of proteins in solution, providing the spectra are analyzed using a statistically and theoretically justified methodology. We have performed FTIR experiments on 14 globular proteins and two synthetic polypeptides whose X-ray crystal structures are known to exhibit varying types and amounts of secondary structures. Calculation of the component structural elements of the vibrational bands was accomplished using nonlinear regression analysis, by fitting both the amide I and amide II bands of the Fourier self-deconvoluted spectra, the second-derivative spectra, and the original spectra. The methodology was theoretically justified by comparing (via nonlinear regression analysis) the global secondary structure determined after deconvolving into component bands the vibrational amide I envelopes with the calculated structure determined by first principles from Ramachandran analysis of the X-ray crystallographic structure of 14 proteins from the Brookhaven protein data bank. Justification of the nonlinear regression analysis model with respect to experimental and instrumental considerations was achieved by the decomposition of all the bands of benzene and an aqueous solution of ammonium acetate into component bands while floating the Gaussian/Lorentzian character of the line shapes. The results for benzene yield all pure Lorentzian line shapes with no Gaussian character while the ammonium acetate spectra yielded all Gaussian line shapes with no Lorentzian character. In addition, all-protein spectra yielded pure Gaussian line shapes with no Lorentzian character. Finally, the model was statistically justified by recognizing random deviation patterns in the regression analysis from all fits and by the extra sum of squares F-test which uses the degrees of freedom and the root mean square values as a tool to determine the optimum number of component bands required for the nonlinear regression analysis. Results from this study demonstrate that the globular secondary structure calculated from the amide I envelope for these 14 proteins from FTIR is in excellent agreement with the values calculated from the X-ray crystallographic data using three-dimensional Ramachandran analysis, providing that the proper contribution from GLN and ASN side chains to the 1667 and 1650 cm(-1) component bands has been taken into account. The standard deviation of the regression analysis for the per cent helix, extended, turn and irregular conformations was found to be 3.49%, 2.07%, 3.59% and 3.20%, respectively.

Determination of the global secondary structure of proteins by Fourier transform infrared (FTIR) spectroscopy

In the development of new foods or in the control of traditional process, protein functionality plays a paramount role. It has long been theorized that changes in protein structure can alter functionality, but there has been a lack of reliable methodologies for observing protein structural changes in ‘real-world’ food samples. Here, a technique for determination of the global secondary structure of proteins using Fourier transform infrared (FTIR) spectroscopy in H2O instead of D2O is assessed and contrasted with other methodologies for structural determinations. A quantitative procedure is presented for preparing and analysing FTIR spectra of proteins for the determination of their global secondary structural components. As an example, an analysis of the FTIR spectra of egg-white lysozyme is presented and correlated with global secondary structure values calculated from its X-ray crystallographic structure. Other examples of FTIR analyses of food proteins are presented with consideration given to qualitative procedures for more rapid structural analysis of processed samples.

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.

Estimation of sedimentation coefficients of globular proteins: An application of small-angle X-ray scattering

A new semiempirical procedure is presented which relates solution small-angle X-ray scattering parameters to sedimentation coefficients. With this method, sedimentation coefficients were calculated for a set of 20 globular macromolecules with molecular weights ranging from 1.3 × 104 to 7.0 × 106; all were in excellent agreement with experimental values. Best results were arrived at by obtaining: (1) the Stokes radius in Svedbergs equation by way of the scattering volume V of the macromolecule instead of the commonly used partial specific volume v; and (2) the structural frictional ratio (ff0)s from an axial ratio derived from RGSV instead of the usual 3V(4πRG3 relationship, where RG is the radius of gyration and S is the external surface area of the molecule. This indicates that the frictional ratio is a function of the surface roughness of the macromolecule, in agreement with similar conclusions in the literature. In addition, structural parameters from the X-ray crystallographic structure are compared with those from small-angle X-ray scattering for a better insight into the contribution of hydration to the frictional coefficient.

An energy-minimized casein submicelle working model

To develop a molecular basis for structure-function relationships of the complex milk protein system, an energy-minimized, three-dimensional model of a casein submicelle was constructed consisting ofκ-casein, fourαs1-casein, and fourβ-casein molecules. The models for the individual caseins were from previously reported energy-minimized, three-dimensional structures. Docking of oneκ-casein and fourαs1-casein molecules produced a framework structure through the interaction of two hydrophobic antiparallel sheets ofκ-casein with two small hydrophobic antiparallel sheets (residue 163–174) of two preformedαs1-casein dimers. The resulting structure is approximately spherically symmetric, with a loose packing density; its external portion is composed of the hydrophilic domains of the fourαs1-caseins, while the central portion contains two hydrophbic cavities on either side of theκ-casein central structure. Symmetric and asymmetric preformed dimers ofβ-casein formed from the interactions of C-terminalβ-spiral regions as a hinge point could easily be docked into each of the two central cavities of theα-κ framework. This yielded two plausible energy-minimized, three-dimensional structures for submicellar casein, one with two symmetricβ-casein dimers and one with two asymmetric dimers. These refined submicellar structures are in good agreement with biochemical, chemical, and solution structural information available for submicellar casein.

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 A) with slightly decreased anisotropy and with substantial increases in molecular volume (55,600 ± 530 vs 66,500 ± 240 A3), surface (11,840 ± 120 vs 13,470 ± 140 A), 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.

A deuteron and proton magnetic resonance relaxation study of β-lactoglobulin A association: Some approaches to the scatchard hydration of globular proteins☆

Abstract A general expression for the concentration-dependent relaxation increment, ( dR dc ) μ , (where R may be R1, the spin-lattice, or R2, the spin-spin relaxation rate of water), has been derived from multicomponent theory for a protein in salt solution. Emphasis is placed on the addition of salt to the aqueous protein to minimize potentially high virial effects due to charge repulsion or to the charge fluctuations predicted by the Kirkwood-Shumaker theory; under conditions where the protein has a high net charge-to-mass ratio the calculation of relaxation increments must employ protein activities in place of concentrations. This treatment was applied to the molecular states of β-lactoglobulin A under associating and nonassociating conditions. In contrast to data in the literature obtained in the absence of salt, where correlation times τc were excessively high and hydration values too low, here values of τc from 2H NMR were in quantitative agreement with those expected from parameters of the known structural states of this protein. With these values, hydrations were obtained by three different ways of calculating the relaxation rate of the bound water from 1H and 2H NMR data. Preferential hydrations, derived from linked functions, for the association of the protein at pH 4.65 were obtained from sedimentation velocity measurements. Combination of the results from the temperature dependence of the deuteron NMR and the linked functions, on the basis of a three-state model, yields slow-tumbling hydration values and correlation times comparable to those obtained from the two-state model. Based on either an isotropic bound-water mechanism or an anisotropic orientational distribution of the water molecules, enthalpies of hydration determined from the three-state model are in accord with those calculated from the two-state model.

[11] Structural interpretation of hydrodynamic measurements of proteins in solution through correlations with X-ray data

Publisher Summary This chapter deals with the structural interpretation of hydrodynamic measurements of proteins in solution through correlations with X-Ray data. Analysis of protein structure by X-ray crystallography has revealed irregular surfaces consisting of many clefts, grooves, and protuberances. Hydrodynamic parameters, which are sensitive to surface characteristics, have been calculated from X-ray crystallographic coordinates and compared with solution values in attempts to answer such questions, but with only marginal success. There appears to be a need for structural information from other nonhydrodynamic sources for use in conjunction with hydrodynamic parameters, so that the combined results may be compared with those from X-ray crystallography. The three hydrodynamic parameters under consideration here are obtained experimentally by the observation of flow under the influence of an applied force. This chapter also presents an alternative procedure for estimating the contribution of hydration to the total frictional coefficient to obtain the contribution of the axial ratio by itself, and thus estimates of geometric parameters from sedimentation coefficients.