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    Studies in protein structure : the structure and properties of the iron superoxide dismutase from Methanobacterium thermoautotrophicum : the structures of [beta]-lactoglobulin in two new crystal forms : a dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy in the Institute of Fundamental Sciences at Massey University
    (Massey University, 2002) Adams, Julian James
    The crystal structure of Methanobacterium thermoautotrophicum iron superoxide dismutase (Mt-FeSOD) has been determined by X-ray diffraction to a resolution of 2.6 Å. The crystals were grown from PEG 6000 at a pH of 5.5, and the structure was solved by molecular replacement. The structure, in concert with structural and functional data from other Fe and Mn SODs, provides insights into aspects of metal specificity, reactivity of superoxide dismutase towards toward the inhibitor azide and deactivator hydrogen peroxide, and how the primary structure is involved in subtle tuning of these properties. The structure reveals how the protein is designed for thermal and chemical stability, yet retains moderate superoxide dismutase activity at ambient temperature. Bovine β-lactoglobulin (BLG) has been studied for many decades; numerous X-ray and NMR structures are available. Here we present two new X-ray structures, one from a crystal grown at very low ionic strength, and at the lowest pH (~5.2) of any X-ray structure. This structure provides validation of the other, high ionic strength X-ray structures. The core elements of BLG, an eight-stranded β-barrel, a three-turn α-helix external to the barrel, and an external β-strand (that forms the dimeric interface), are almost invariant across all structures. Four flexible loops have a variety of positions in the known structures and this represents a set of snapshots of the in vivo states of BLG. These flexible loops play an important role in the entropic stabilization of the β-barrel.
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    β-Lactoglobulin nanofibrils: Effect of temperature on fibril formation kinetics, fibril morphology and the rheological properties of fibril dispersions
    (Elsevier Ltd, 2012-05) Loveday SM; Wang XL; Rao MA; Anema SG; Singh H
    Almost all published studies of heat-induced b-lactoglobulin self-assembly into amyloid-like fibrils at low pH and low ionic strength have involved heating at 80 C, and the effect of heating temperature on self-assembly has received little attention. Here we heated b-lactoglobulin at pH 2 and 75 C, 80 C, 90 C, 100 C, 110 C or 120 C and investigated the kinetics of self-assembly (using Thioflavin T fluorescence), the morphology of fibrils, and the rheological properties of fibril dispersions. Self-assembly occurred at all temperatures tested. Thioflavin T fluorescence increased sigmoidally at all temperatures, however it decreased sharply with >3.3 h heating at 110 C and with >5 h heating at 120 C. The sharp decreases were attributed partly to local gelation, but destruction of fibrils may have occurred at 120 C. Thioflavin T fluorescence results indicated that maximal rates of fibril formation increased with increasing temperature, especially above 100 C, but fibril yield (maximum Thioflavin T fluorescence) was not affected by temperature. At 100 C and 110 C, fibrils were slightly shorter than at 80 C, but otherwise they looked very similar. Fibrils made by heating at 120 C for 1 h were also similar, but heating at 120 C for 8 h gave predominantly short fibrils, apparently the products of larger fibrils fragmenting. Heating at 100 C gave consistently higher viscosity than at 80 C, and heating for >2 h at 120 C decreased viscosity, which may have been linked with fibril fragmentation seen in micrographs.
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    Interactions of whey protein isolate and human saliva, as related to the astringency of whey protein beverages : a thesis in partial fulfilment of the requirement of the degree of Master of Technology in Food Technology at Riddet Institute, Massey University, New Zealand
    (Massey University, 2010) Streicher, Christina
    Interactions between 3 different proteins (lactoferrin, beta-lactoglobulin and Whey Protein Isolate) and human saliva were determined. Lactoferrin and whey proteins are known to be astringent at low pH. Astringency is defined as the tactile sensation, mainly on the tongue, caused by astringent compounds when in contact with human saliva. Proline-rich proteins are already known to be directly involved in the astringency of polyphenols. Whey proteins do not contain polyphenols. However, because whey proteins at low pH develop an astringent sensation when consumed, it was expected to detect proline-rich proteins in the interaction between Whey Protein Isolate (WPI) and saliva as well. The protein solutions were adjusted to different pH-levels, ranging from neutral to high acidic, where a part of each protein solution was heat-treated. All solutions were mixed with human saliva in the same ratio (w/w). One part of all mixtures was pH-readjusted. Additionally, WPI model solutions were prepared, adjusted to different pH-levels, heat-treated and then consumed by voluntary participants, who swirled each solution in their mouth for at least 10 seconds. These mixtures of WPI and saliva were collected for further analysis. After consuming the WPI model solutions, followed by rinsing the mouth with water, tongue swabs were taken to determine the particle sizes and zeta-potentials of the remaining material on the tongue. Control tongue swabs of the clean tongue were taken by the participants before any consumption of the WPI model solutions. All mixtures as well as lactoferrin, beta-lactoglobulin (beta-lg), WPI and saliva on their own, were analysed for particle size, zeta-potential and turbidity, which may give an indication for possible aggregation/precipitation of the proteins as well as the analysis of the SDS-PAGE profile of the sediments of the sample mixtures. Saliva is negatively charged between neutral pH and 3.0, whereas lactoferrin has a positive charge below pH 8.0. WPI has a positive charge below pH 5.1; the same applies to beta-lg. None of the proteins themselves showed aggregation/precipitation at pH-levels 6.8, 3.6, 3.4, 3.0, 2.5 or 2.0. However, after the proteins were mixed with saliva, the pH of mixtures shifted towards neutral pH. The mixtures of lactoferrin (unheated/heat-treated) and saliva neither showed any significant increases in particle size nor the presence of turbidity. Salivary proteins were not detected in any mixtures at any observed pH either, despite the known fact that lactoferrin causes astringency. The mixtures of beta-lg (unheated/heated) and saliva displayed high particle sizes below final pH 3.6, whereas the high turbidities of both mixtures were measured between final pH 3.6 and 3.4. Furthermore, only at final pH 2.8 were salivary proteins (mainly glycosylated proline-rich proteins and alpha-amylase) detected. However, higher concentrations of salivary proteins were measured when heat-treated beta-lg was mixed with saliva. The mixtures of WPI and saliva presented the strongest interaction compared to lactoferrin and beta-lg. High aggregation/precipitation occurred in the mixtures between pH 4.3 and 3.0, where significantly high particle sizes and turbidities were detected. The pH-readjusted mixtures of lactoferrin/beta-lactoglobulin/WPI and saliva showed similar values in particle size and turbidity as the mixtures of the proteins and saliva without pH-readjustment at similar pH-values. Furthermore, the pH-readjusted mixtures of the proteins and saliva showed in their sediments the presence of alpha-amylase and glycosylated proline-rich proteins.The mixtures of heat-treated WPI and saliva, collected from the mouth after taking a sip (ratio unknown), revealed that the strongest interactions occurred when WPI-solutions were adjusted to pH 3.6 and 3.4. Similar observations were made for heat-treated WPI-solutions, which were adjusted to pH 3.6 and 3.4, when mixed with saliva 1:1 (w/w). However, additionally to the glycosylated proline-rich proteins and alpha-amylase, faint bands of mucin as well as basic proline-rich proteins were detected in the mixtures collected from the mouth. The proteins of the material remaining on the tongue followed the consumption of WPI-solutions and rinsing with water showed that the particle size measurementswere not reliable. However, pH-levels between 6.8 and 5.7 occurred and negative charges were measured on the tongue after rinsing the mouth twice with water. The strongest interactions between the proteins and human saliva occurred when the proteins, in particular beta-lg and WPI, were positively charged and then mixed with saliva (negative charge). Concluding from that it is suggested that electrostatic interactions may cause the astringent sensations. However, since no evidence could be found that salivary proteins were involved in the interaction between lactoferrin and saliva (without pH-readjustment), it is suggested that other interactions than electrostatic interactions cause the astringent sensation of lactoferrin.
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    Effects of environmental factors on heat-induced [beta]-lactoglobulin fibril formation : a thesis presented in partial fulfilment of the requirements for the degree of Master of Technology in Food Technology, Riddet Institute, Massey University, Palmerston North, New Zealand
    (Massey University, 2010) Wang, Xiangli
    The heat-induced fibrillar aggregation of β-lactoglobulin was studied under various environmental conditions. The formation of β-lactoglobulin fibrils was monitored by Thioflavin T (ThT) fluorescence and their morphology was studied using transmission electron microscopy (TEM). Amyloid-like fibrils were formed under standard conditions (pH 2.0, 80°C and low ionic strength). The β-lactoglobulin fibrillation kinetics exhibited sigmoidal behaviour, and the two-step autocatalytic reaction model fitted ThT fluorescence data well. The studies of the individual effect of pH, temperature, NaCl, CaCl2 on β-lactoglobulin fibril formation showed that decreasing pH (2.4 - 1.6), increasing temperature (75 - 120°C) and increasing salt concentration (NaCl 0-100 mM; CaCl2 0-100 mM) accelerated the fibril formation process and altered the morphology of fibrils. The two-step autocatalytic reaction model did not fit the ThT fluorescence data well at higher temperature (>100°C) or at low pH (1.6). The effects of the four factors (pH, temperature, NaCl and CaCl2) on β-lactoglobulin fibril formation were studied by using a central composition design (CCD) experiment. Results showed that the four main and some of the non-linear effects were significant (95%) on fibril formation, including fibrillation time and the fibril yield. Taking all results together, it can be implied that β-lactoglobulin fibril formation can be promoted by choosing the external incubation conditions. This study is the first step towards the application of protein fibrils as texture-modifying ingredients in food systems.
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    Characterization of heat-induced aggregates of beta-lactoglobulin, alpha-lactalbumin and bovine serum albumin in a whey protein concentrate environment
    (Cambridge University Press, 2001) Havea P; Singh H; Creamer LK
    Bovine b-lactoglobulin (b-lg), a-lactalbumin (a-la) and bovine serum albumin (BSA), dispersed in ultra®ltration permeate, that had been prepared from whey protein concentrate solution (100 g}kg, pH 6±8), were heated at 75 °C. The consequent protein aggregation was studied by one-dimensional (1D) and twodimensional (2D) polyacrylamide gel electrophoresis (PAGE). When 100 g b-lg}kg permeate solution was heated at 75 °C, cooled and examined, large aggregates were observed. These aggregates were partially dissociated in SDS solution to give monomers, disulphide-bonded dimers, trimers and larger aggregates. When mixtures of b-lg and a-la or BSA were heated, homopolymers of each protein as well as heteropolymers of these proteins were observed. These polymer species were also observed in a heated mixture of the three proteins. Two-dimensional PAGE of mixtures demonstrated that these polymers species contained disulphide-bonded dimers of b-lg, a-la and BSA, and 1:1 disulphide-bonded adducts of a-la and b-lg, or BSA. These results are consistent with a mechanism in which the free thiols of heattreated b-lg or BSA catalyse the formation of a range of monomers, dimers and higher polymers of a-la. It is likely that when whey protein concentrate is heated under the present conditions, BSA forms disulphide-bonded strands ahead of b-lg and that a-la aggregation with b-lg and with itself is catalysed by the heat-induced unfolded BSA and b-lg.