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T F Kumosinski

Publications and source records attributed to T F Kumosinski.

At least 19 recordsLinked to original sources
PubMed1998

Changes in the secondary structure of bovine casein by Fourier transform infrared spectroscopy: effects of calcium and temperature.

Bovine casein submicelles and reformed micelles, produced by addition of Ca2+, were examined by Fourier transform infrared spectroscopy at 15 and 37 degrees C in aqueous salt solutions of K+ and Na+. Previous measurements of caseins, made in D2O and in the solid form, can now be made in a more realistic environment of H2O. When analyzed in detail, data obtained by Fourier transform infrared spectroscopy have the potential to show subtle changes in secondary structural elements that are associated with changes in protein environment. Electrostatic binding of Ca2+ to casein resulted in a redistribution of the components of the infrared spectra. Addition of Ca2+ in salt solutions of K+ and Na+ led to apparent decreases in large loop or helical structures at 37 degrees C with concomitant increases in the percentage of structures having greater bond energy, such as turns and extended helical structures. At 15 degrees C, Na+ and K+ have differential effects on the Ca(2+)-casein complexes. All of these observations are in accordance with the important role of serine phosphate side chains as sites for Ca2+ binding in caseins and the swelling of the casein structure upon incorporation into reformed micelles at 37 degrees C. This new open, hydrated structure is buttressed by a change in backbone as evidenced by a shift in absorbance to higher wave numbers (greater bond energies) as colloidal micelles are reformed.

Animals↗
PubMed1997

Salt and pH effects on electrochemistry of myoglobin in thick films of a bilayer-forming surfactant.

Salt concentration and pH of external solutions were shown to control the electrochemistry of the heme protein myoglobin (MbFe(III)-H2O) in stable, ordered films of didodecyldimethylammonium bromide (DDAB). Protonation of aquometmyoglobin (MbFe(III)-H2O) in these films precedes electron transfer from electrodes, causing formal potentials to shift negative as pH increases from 5 to 8. At pH > 8, MbFe(III)-H2O dissociates to MbFe(III)-OH, which is reduced directly at the electrode at higher rates than MbFe(III)-H2O. Correlations of voltammetric data with FT-IR spectra suggested that at pH < 4.6, an unfolded form of Mb resides in the films and is reduced directly. The concentration of salt in solution influences electrochemical properties of Mb-DDAB films by its influence on Mb conformation and by effects on interfacial Donnan potentials. NMR indicated strong binding of anions to Mb within DDAB films. Bound anions may neutralize positive charge on Mb's surface so that it can reside in a partly hydrophobic environment, as postulated on the basis of previous ESR and linear dichroism studies.

Animals↗
PubMed1996

Particle sizes of purified kappa-casein: metal effect and correspondence with predicted three-dimensional molecular models.

kappa-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 the kappa-casein particles and leads to a broder 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 for kappa-casein that actually predicted participation of divalent cations in the structure.

Amino Acids↗
PubMed1995

alpha-Crystallin quaternary structure: molecular basis for its chaperone activity.

alpha-Crystallin, the major protein in all vertebrate lenses, functions as a chaperone. In the present analysis, an 'open' micellar structure composed of alpha A subunits is used to simulate chaperoning of partially heat denatured soluble gamma-crystallin. The interaction is both electrostatic and hydrophobic and satisfies experimental evidence for a 1:1 alpha/gamma molar ratio, a doubling of molecular mass and a minimal increase in the dimensions of the complex [J. Biol. Chem. (1994) 269, 13601-13608; Invest. Opthalmol. Vis. Sci. (1995) 36, 311-21]. These data are also in accord with Farahbaksh et al. [Biochemistry (1995) 34, 509-16]; i.e. the bound gamma-crystallin monomers are not in a central cavity, but are separated by alpha A subunits.

Amino Acid Sequence↗
PubMed1995

Identification of the active-site residues of the L proteinase of foot-and-mouth disease virus.

The foot-and-mouth disease virus (FMDV) leader (L) protein is involved in autocatalytic cleavage at the L/P1 junction and in the cleavage of translation initiation factor p220, a subunit of the cap-binding protein complex. It has been suggested that this proteinase has homology to the papain-like family of cysteine proteinases, and from this information, we have investigated the active-site residues by introducing specific mutations into the L gene. Mutations of Cys-23 to Ala or His-120 to Leu resulted in enzymes that lacked cis activity at the L/VP4 cleavage site, trans activity on a truncated L-P1 substrate, and p220 cleavage activity. Mutations of Cys-23 to ser or His-110 to Leu resulted in enzymes that retained some or all cis activity and had reduced p220 cleavage. These mutations were introduced separately into a full-length FMDV cDNA, and RNA transcripts derived from these cDNAs were translated in a cell-free system and transfected into cells. The C23S mutant inefficiently cleaved at the L/P1 junction and within P1, and virus obtained from transfected cells reverted to wild type. The H110L mutant cleaved the L/P1 junction almost as well as the wild-type enzyme, and virus recovered from transfected cells retained the mutation and displayed wild-type viral protein synthesis and host shut-off kinetics.

Animals↗
PubMed1994

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 of kappa-casein, four alpha s1-casein, and four beta-casein molecules. The models for the individual caseins were from previously reported energy-minimized, three-dimensional structures. Docking of one kappa-casein and four alpha s1-casein molecules produced a framework structure through the interaction of two hydrophobic antiparallel sheets of kappa-casein with two small hydrophobic antiparallel sheets (residue 163-174) of two preformed alpha s1-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 four alpha s1-caseins, while the central portion contains two hydrophbic cavities on either side of the kappa-casein central structure. Symmetric and asymmetric preformed dimers of beta-casein formed from the interactions of C-terminal beta-spiral regions as a hinge point could easily be docked into each of the two central cavities of the alpha-kappa framework. This yielded two plausible energy-minimized, three-dimensional structures for submicellar casein, one with two symmetric beta-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.

Animals↗
PubMed1994

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 the kappa-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.

Animals↗
PubMed1993

Three-dimensional molecular modeling of bovine caseins: an energy-minimized beta-casein structure.

To obtain a molecular basis for the similarities and dissimilarities in the functional, chemical, and biochemical properties between beta-casein and the other caseins, a predicted three-dimensional model is presented. The predicted structure was assembled using molecular modeling techniques, as well as secondary structural prediction algorithms, in conjunction with global secondary structural information from Raman spectroscopy. To add validity to this model, the structure was refined using energy minimization techniques to diminish the likelihood of structural overlaps and energetically unfavorable van der Waals contacts arising from the large number of proline residues present in the beta-casein sequence. The refined model overall showed a loosely packed, asymmetrical structure with an axial ratio of 2:1. Hydrophobic side chains were uniformly dispersed over one end (C terminal) and the center surface of the structure; the other end (N terminal) was hydrophilic. The hydrophobic section also possessed a large loop through which water could easily travel. Such a suprasurfactant structure could account for the micellar type of hydrophobically driven self-association exhibited by beta-casein. Other chemical and biochemical data are in good agreement with the refined structure.

Amino Acid Sequence↗
PubMed1993

Three-dimensional molecular modeling of bovine caseins: a refined, energy-minimized kappa-casein structure.

A refined three-dimensional molecular model of kappa-casein has been produced using energy minimization techniques and a Kollman force field on a previously reported predicted three-dimensional structure. This initial model was constructed via molecular modeling techniques from sequence-based secondary structural prediction algorithms. Both the initial and refined structures agreed with global secondary structure analysis from vibration spectroscopy. The refined structure contained many of the features of the initial model, including two sets of antiparallel beta-sheet structures containing predominantly hydrophobic side chains, which could form interaction sites with alpha s1-casein. Two types of energy-minimized dimer and tetramer models are presented: 1) using Cys as potential intermolecular disulfide binding sites and 2) using the two sheets as possible hydrophobic self-association sites, without Cys interactions. All structures yielded good stabilization energies and are in agreement with chemical, biochemical, and physical chemical results obtained for kappa-casein.

Amino Acid Sequence↗
PubMed1993

Regulation of the soluble form of nicotinamide adenine dinucleotide phosphate-specific isocitrate dehydrogenase from lactating bovine mammary gland: effects of metabolites on activity and structure.

The cytosolic form of NADP+: isocitrate dehydrogenase, a primary source of the NADPH required for de novo fatty acid synthesis in lactating bovine mammary gland, was studied to determine possible mechanisms of regulation by metabolites. The enzymatic reduction of NADP+ exhibits lag-burst (hysteretic) kinetics that are eliminated by the noncatalytic binding of the substrate, a complex (1:1) of a metal ion (Mn2+ or Mg2+) and isocitrate. Preincubation of the enzyme with metal-citrate complex also nearly abolished the lag or activation time. In steady-state experiments, analyses of velocity versus metal-citrate complex as a binding isotherm, following the assumptions of Wyman's theory of thermodynamic linkage, showed that binding of metal-citrate complex could both stimulate and inhibit the enzyme. This analysis suggested hyperactivation by binding to sites with an average dissociation constant of .25 mM, inhibition by binding to sites with an average dissociation constant of 3.83 mM, and modulation (reactivation) by binding to sites with an average dissociation constant of 1.54 mM. Conformational changes induced by the binding of ligands were assessed using circular dichroism. The results suggest that binding of metal-isocitrate induces a conformational transition involving tyrosyl residues that is related to the altered kinetic processes. Reexamination of Michaelis-Menten kinetics using non-linear regression analysis also demonstrated hyperactivation of enzyme activity by metal-isocitrate with a dissociation constant equal to 21 microM (which is nearly seven times greater than the Michaelis constant). Concentration ranges observed for these transitions are compatible with physiological conditions, suggesting that complexes of metal-citrate and metal-isocitrate serve to modulate the activity of NADP+: isocitrate dehydrogenase.

Animals↗
PubMed1991

Molecular dynamics of water in foods and related model systems: multinuclear spin relaxation studies and comparison with theoretical calculations.

A review of recent studies of molecular dynamics of water in foods and model systems is presented, and the theoretical results are compared with experimental data obtained by several techniques. Both theoretical and experimental approaches are discussed for electrolytes, carbohydrates, and food proteins in solution. Theoretical results from Monte Carlo simulations are compared with experimental NMR relaxation data for quadrupolar nuclei such as those of deuterium and oxygen-17. Hydration studies of wheat, soybean, corn, and myofibrillar proteins by multinuclear spin relaxation techniques are discussed, and several new approaches to the analysis of the experimental data are considered. Correlation times of water motions in hydrated food systems are determined from NMR and dielectric relaxation data. The values of the correlation times for dilute solutions of electrolytes and carbohydrates estimated by NMR are in good agreement with those calculated from dielectric relaxation data, but seem to differ significantly from those proposed from Monte Carlo simulations. Several new and important results concerning the hydration of potato and cereal starches are presented, showing the very different hydration behaviors of these two major groups of starches. The combination of molecular dynamics computations with NMR relaxation techniques will hopefully stimulate novel technological developments in food engineering based on such fundamental studies.

Carbohydrates↗
PubMed1991

Protein-water interactions from 2H NMR relaxation studies: influence of hydrophilic, hydrophobic, and electrostatic interactions.

The importance of water interactions with proteins in food systems is well documented. A controversy exists, however, as to the nature of these interactions and the effect of protein structural changes on them. To clarify these questions, a method has been developed for determining hydration from the protein concentration-dependence of deuteron resonance relaxation rates. Measurements were made in D2O on beta-lactoglobulin A to study effects of hydrophilic interactions, and on both casein micelles and submicelles to study hydrophobic and electrostatic effects. From the protein concentration-dependent relaxation rates, the second viral coefficients of the proteins were obtained by nonlinear regression analysis. Using either an isotropic tumbling or an intermediate asymmetry model, hydrations, upsilon, and correlation times, tau c, were calculated for the protein-associated water; from tau c, the Stokes radius, R, was obtained. Variations in upsilon and R were in accord with known structural changes in molecular states of the proteins. The NMR results are compared with hydrations and structural information derived independently from small-angle X-ray scattering.

Chemical Phenomena↗
PubMed1991

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 A 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 1500 g. 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. Ca(2+)-induced colloid stability profiles were obtained experimentally for model micelles consisting of only alpha s1- (a calcium insoluble casein) and the stabilizing protein kappa-casein, eliminating the complications arising from beta- and minor casein forms. Two distinct genetic variants alpha s1-A and B were used. Analysis of alpha s1-A colloid stability profiles yielded a precipitation (salting-out) constant k1, as well as colloid stability (salting-in) parameter k2. No variations of k1 or k2 were found with increasing amounts of kappa-casein. From the variation of the amount of colloidal casein capable of being stabilized vs. amount of added kappa-casein an association constant of 4 L/g could be calculated for the complexation of alpha s1-A and kappa-casein. For the alpha s1-B and kappa-casein micelles, an additional Ca(2+)-dependent colloidal destabilization parameter, k3, was added to the existing k1 and k2 parameters in order to fully describe this more complex system. Furthermore, the value of k3 decreased with increasing concentration of kappa-casein. These results were analyzed with respect to the specific deletion which occurs in alpha s1-casein A in order to determine the sites responsible for these Ca(2+)-induced quaternary structural effects.

Amino Acid Sequence↗
PubMed1991

Tertiary and quaternary structural differences between two genetic variants of bovine casein by small-angle X-ray scattering.

The casein complexes of bovine milk consist of four major protein fractions, alpha s1, alpha s2, beta, and kappa. Colloidal particles of casein (termed micelles) contain inorganic calcium and phosphate; they are very roughly spherical with an average radius of 650 A. Removal of Ca2+ leads to the formation of smaller protein aggregates (submicelles) with an average radius of 94 A. Two genetic variants, A and B, of the predominant fraction, alpha s1-casein, result in milks with markedly different physical properties, such as solubility and heat stability. To investigate the molecular basis for these differences, small-angle X-ray scattering was performed on the respective colloidal micelles and submicelles. Scattering curves for submicelles of both variants showed multiple Gaussian character; data for the B variant were previously interpreted in terms of two concentric regions of different electron density, i.e., a "compact" core and a relatively "loose" shell. For the submicelle of A, there was a third Gaussian, reflecting a negative contribution due to interparticle interference. Molecular parameters for submicelles of both A and B are in agreement with hydrodynamic data in the literature. Data for the micelles, for which scattering yields cross-sectional information, were fitted by a sum of three Gaussians for both variants; for these, the corresponding two lower radii of gyration represent the two concentric regions of the submicelles, while the third reflects the average packing of submicelles within the micellar cross section. Most of the molecular parameters obtained showed small but consistent differences between A and B, but for submicelles within the micelle several differences were particularly notable: A has a greater molecular weight for the "compact" region of the constituent submicelle (82,000 vs 60,000) and a much greater submicellar packing number (6:1 vs 3:1). Reasons for these and other differences are to be sought in sequence differences and in differences in calcium-binding sites and charge distribution.

Animals↗
PubMed1991

Three-dimensional molecular modeling of bovine caseins: kappa-casein.

Three-dimensional structures derived from X-ray crystallography are extremely important in elucidating relationships between structure and function for many proteins. However, not all proteins can be crystallized. The caseins of bovine milk are one class of noncrystallizable proteins. The complete primary and partial secondary structures of these proteins are known, but homologous proteins with known crystallographic structure are not available. In this report, sequence-based predictions of secondary structure were made and adjusted to conform with global secondary structures derived from Fourier transform infrared spectroscopy. With this information, a three-dimensional structure for kappa-casein was constructed using molecular modeling computer programs. The constructed model contains two unstranded beta-sheets; both are predominantly hydrophobic and capable of forming quaternary structural interaction sites with alpha s1-casein. This unrefined structure is in good agreement with much of the biochemical information available for kappa-casein.

Amino Acid Sequence↗
PubMed1991

Three-dimensional molecular modeling of bovine caseins: alpha s1-casein.

Structures derived from X-ray crystallography are extremely important in elucidating functional relationships for many proteins. However, the caseins of bovine milk are one class of noncrystallizable proteins. The complete primary and partial secondary structures of these proteins are known, but homologous proteins of known crystallographic structure cannot be found. Therefore, sequence-based predictions of secondary structure were made and adjusted to conform with global secondary structures determined by Raman spectroscopy. With this information, a three-dimensional structure for alpha s1-casein was constructed using molecular modeling programs. The predicted structure of alpha s1-casein contains a hydrophobic and a hydrophilic domain, which are connected by a segment of alpha-helix. This unrefined structure shows good agreement with global biochemical and chemical information concerning alpha s1-caseins A, B, and C.

Amino Acid Sequence↗