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    Alternative proteins vs animal proteins: The influence of structure and processing on their gastro-small intestinal digestion
    (Elsevier Ltd, 2022-04) Kaur L; Mao B; Beniwal AS; Abhilasha; Kaur R; Chian FM; Singh J
    Background: Digestibility, an indicator of protein bioavailability, is essentially a measure of the susceptibility of a protein towards proteolysis. Proteins with higher digestibility have been linked with better health outcomes. Animal proteins are generally considered to be of better nutritional value than plant proteins not only because they are a good source of essential amino acids but also due to their higher digestibility in the human gastro-intestinal tract. With the recent emergence of alternative food protein sources, which are now processed in a completely new way to design new foods or new versions of the conventional foods, it has become extremely important to understand their digestion characteristics. Scope and approach: This review discusses the factors that affect protein digestibility, including protein source, structure, type of processing, and modification, with a particular focus on the effects of non-protein components present in food matrix. Key findings and conclusions: To obtain the desired functionality, particularly for alternate proteins, numerous physical, chemical, and enzymatic methods for modification have been reported. These modifications may alter structural characteristics of proteins by inducing structural modifications such as protein unfolding, crosslinking, and aggregation. Depending upon the protein reactivity during processing, the susceptibility of proteins towards hydrolysis by digestive enzymes might change, affecting not only the overall protein digestibility but also the rates of release of polypeptides and amino acids. The faster rates of protein digestion have been linked with muscle anabolism, suggesting the need and importance of classifying the new, emerging and alternative protein sources according to their rates of digestion into rapidly (RDP), slowly digestible (SDP) and resistant (RP) proteins. More research needs to be focussed on converting, through processing, the undigestible or RP into RDP or SDP to achieve better health outcomes.
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    An investigation of Rheo-NMR techniques to improve the capture of residual dipolar couplings : a thesis submitted in partial fulfillment of the requirements for the award of the degree of Master of Science in Chemistry, Institute of Fundamental Sciences, Massey University, Palmerston North, New Zealand
    (Massey University, 2015) Munro, Ben
    Residual Dipolar Couplings (RDCs) are an increasingly important structural restraint that can be used to help generate high quality structural models of proteins by Nuclear Magnetic Resonance (NMR) methods. They are captured with the aid of an alignment medium that imposes some anisotropy to the protein’s tumbling. Current methods for the capture of multiple sets of these couplings are tedious, expensive, and do not always result in unique sets being captured. This thesis set out to investigate whether multiple RDC sets could be captured from a single sample by controllably shearing the liquid crystal alignment medium used. Initial experiments focused on the ability to controllably realign a number of different nematic phase liquid crystals. These experiments found that controlling the director angle of the liquid crystal is possible, and that a number of stable alignments can be achieved through the application of different shear stresses. The application of RDCs to small molecules is a very young field that is still developing and finding potential uses. In this thesis a small molecule system of (+)-isopinocampheol ((+)-IPC) was investigated with RDCs being collected from this molecule within a liquid crystal phase with the director at a number of different orientations relative to the external magnetic field. The fitting of these captured RDCs to a structural model of the (+)-IPC was not able to generate a high quality fit for any of the RDC sets collected, leading to some puzzling results. It is hypothesized that inhomogeneity of the alignment phase was responsible for these difficulties. As the application of RDCs is so heavily dominated by protein structure studies, a small protein was investigated. The protein of choice, ubiquitin, has been heavily investigated in the past, and is often used as a demonstrator protein for new NMR techniques. This work presents several RDC data sets measured from ubiquitin which were successfully captured at a variety of different director orientations of the alignment media. These RDC sets were all successfully fitted to a previously known X-Ray crystallographic structure of ubiquitin, and unique alignment tensors for each RDC data set were extracted. Finally, structure calculations were carried out incorporating these captured ubiquitin RDC data sets with the goal of investigating how the variation in the ensembles of structures generated was modified. The results from these calculations showed that the addition of RDC data (over and above NOE constraints) to the simulated annealing process results in ensembles of higher quality structures being obtained. However, the addition of multiple sets of RDC data (collected with different director alignments) did not appear to cause any further improvement.
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    In search of novel folds : protein evolution via non-homologous recombination : a dissertation presented in partial fulfillment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University, Albany, New Zealand
    (Massey University, 2014) Saraswat, Mayank
    The emergence of proteins from short peptides or subdomains, facilitated by the duplication and fusion of the minigenes encoding them, is believed to have played a role in the origin of life. In this study it was hypothesised that new domains or basic elements of protein structure, may result from nonhomologous recombination of the genes coding for smaller subdomains. The hypothesis was tested by randomly recombining two distantly related (βα)8-barrel proteins: Escherichia coli phosphoribosylanthranilate isomerase (PRAI), and β subunit of voltage dependent K+ channels (Kvβ2) from Rattus norvegicus. The aim was to identify new, folded structures, which may or may not be (βα)8-barrels. Incremental truncation (ITCHY), a method for fragmenting and randomly recombining genes, was used to mimic in vivo non-homologous recombination and to create a library of chimeric variants. Clones from the library were selected for right reading frame and solubility (foldability) of the recombined chimeras, using the pSALect selection system. Out of the six clones identified as soluble by pSALect, only one (P25K86) was found to be actually soluble. The protein, P25K86, was found to form oligomers and on treatment with a reducing agent, β-mercaptoethanol the multimeric state disappeared. The protein has three cysteines and one of the cysteines (Cys56) was found to mediate in the bond formation, thus giving a dimeric state. An engineered version of P25K86 that has the Cys56 replaced by serine was expressed as a monomer and additionally it was found to be ! iv! more stable. As the pSALect folding selection system reported false positives, i.e. only one of the six chimeras was actually soluble, it was concluded that the in vivo solubility selection system was leaky. A series of experiments were conducted so as to improve pSALect that led to the creation of pFoldM – a more stringent selection system, discussed in chapter 4. Comparing the newer improved version with the old, two more interesting chimeras were discovered. A total of 240,000 non-homologous recombination events were created in vitro and three soluble chimeras (evolutionary solutions) were found. Data from circular dichroism spectroscopy (CD) combined with heteronuclear single quantum coherence (HSQC) spectra suggest that the proteins, P24K89 and P25K86, are present in a molten globule state. ITCHY, as a means of mimicking the subdomain assembly model, was applied in vitro. The discovery of two interesting chimeras (P25K86 and P24K89) using highthroughput engineering experiments widens the possibilities of exploring the protein structure space, and perhaps offers close encounters with these never born proteins that may be trapped in an ensemble of fluctuating (structured and unstructured) states.
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    The nutritional value of proteins with special reference to A) The availability of amino acids in meat meals to chicks, and B) The chemical changes with heat-damage of pure proteins : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Biochemistry at Massey University
    (Massey University, 1974) Wilson, Margaret Nellie
    Meat meals are highly variable in protein quality and this may be due in part to heat-damage. This possibility was investigated by estimating the available lysine content of meat meals by chick growth assay. In addition, the combined urinary and faecal excretion of dietary amino acids by chicks, fed a meat meal as the sole source of protein, was determined, and by subtraction from the amount consumed, values for the apparent retention of dietary amino acids were obtained. In a second part of the study, the mechanism of heat-damage to pure proteins was investigated. Since cross-linkages may form during heat-damage of proteins, enzymatic digests were examined for the presence of peptides with enzyme-resistant linkages. Samples of unheated and heat-damaged haemoglobin or globin were digested with either trypsin, or exhaustively, using several proteolytic enzymes. Chick growth assay for lysine. A chick growth assay for lysine was developed using wheat gluten as the protein source in a semi-purified diet. The meat meals were added to the basal diet either at the expense of starch or by isonitrogenous substitution of wheat gluten. Estimated potencies based on weight gain varied with the method of meat meal addition to the basal diet. This variation was probably due to an effect on the appetite of the chicks as estimates based on food conversion efficiency did not differ significantly with the method of meat meal inclusion. The percentage of lysine biologically available in eight meat meals ranged from 61 to 105%, suggesting that some meals had been heat-damaged. Apparent retention of dietary amino acids. Estimates of the apparent retention of essential amino acids in six meat meals ranged from 79 to 100%. The apparent retention of lysine was generally much higher than the estimated potencies by chick growth assay. The difference in the two biological estimates indicated that other factors, apart from digestibility, and absorption and urinary excretion of peptides and amino acids, must be responsible for the reduced availability of lysine in heat-damaged proteins. Tryptic digests of unheated and heated haemoglobin and globin. Several large fragments were isolated from digests of heated globin which were not present in digests of unhealed globin. The fragments had more than one amino-terminal but individual peptides could not be separated. It was not possible to determine if cross-linkages were present. Exhaustive enzyme digests of unheated and heated globin. A peptide was isolated from digests of heated globin which was not present in unheated globin digests. Results obtained indicated that the peptide was a cyclic tetrapeptide composed of equal quantities of lysine and aspartic acid. It was suggested that the peptide was the result of cross-linkages formed during heat-damage of globin, between the β-carboxyl groups of aspartic acid and the E-amino groups of lysine. It is considered that the formation of covalent bonds with the E-amino groups would account for an appreciable proportion of the decreased availability of lysine in heat-damaged proteins.
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    X-ray crystallographic investigations of the structures of enzymes of medical and biotechnological importance : a dissertation submitted in partial satisfaction of the requirements for the degree of Doctor of Philosophy in the Department of Biochemistry at Massey University, New Zealand
    (Massey University, 1996) Kingston, Richard Lawrence
    This thesis is broadly in three parts. In the first, the problem of identifying conditions under which a protein will crystallize is considered. Then structural studies on two enzymes are reported, glucose-fructose oxidoreductase from the bacterium Zymomonas mobilis, and the human bile salt dependent lipase (carboxyl ester hydrolase). The ability of protein crystals to diffract X-rays provides the experimental data required to determine their three dimensional structures at atomic resolution. However the crystallization of proteins is not always straightforward. A systematic procedure to search for protein crystallization conditions has been developed. This procedure is based on the use of orthogonal arrays (matrices whose columns possess certain balancing properties). The theoretical and practical background to the problem is discussed, and the relationship of the presented procedure to other published search methods is considered. The anaerobic Gram-negative bacterium Zymomonas mobilis occurs naturally in sugar-rich growth media, and has attracted much interest because of its potential for industrial ethanol production. In this organism the periplasmic enzyme glucose-fructose oxidoreductase (GFOR) is involved in a protective mechanism to counter osmotic stress. The enzyme is unusual in that it contains tightly associated NADP which is not released during its catalytic cycle. The crystal structure of Z mobilis GFOR has been determined by the method of multiple isomorphous replacement, and refined by restrained least squares methods using data extending to an effective resolution of 2.7 Å. The structure determination reveals that each subunit of the tetrameric protein is folded into two domains, one of which is the classical dinucleotide binding domain, or Rossmann fold. The C-terminal domain is a nine-stranded predominantly antiparallel β-sheet around which the tetramer is constructed. Preceding the Rossmann fold there is a 30 amino acid proline rich 'arm' which wraps around an adjacent subunit in the tetramer. The N-terminal arm buries the adenine ring of the NADP, and may also be involved in stabilization of the quaternary structure of the enzyme. The tight association of NADP is accounted for by the structure. An unsuspected structural relationship has been discovered between GFOR and the cytoplasmic enzyme glucose-6-phosphate dehydrogenase (G6PD). It is proposed that GFOR and G6PD derive from an common ancestral gene, and GFOR has evolved to allow it to function in the bacterial periplasm where it is required. The human bile salt dependent lipase (BSDL) is secreted by the pancreas into the digestive tract, and by the lactating mammary gland into human milk, and is integral to the effective absorption of dietary lipids. It is markedly non-specific, and as its name implies is only active against water-insoluble substrates in the presence of primary bile salts. This differentiates the enzyme from conventional lipases. Diffraction data has been collected from crystals of native BSDL (isolated from human milk), and from crystals of recombinant BSDL (including a truncated variant which lacks a C-terminal heavily glycosylated tandem repeat region found in the native enzyme). The structure of the truncated variant has been partially determined at 3.5 Å resolution, by the method of molecular replacement. The recent collection of a higher resolution (2.8 Å) data set should allow the completion of the structure. The current status of the crystallographic investigations of the human bile salt dependent lipase are reported.
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    PAW - the Protein Analysis Workshop for 2D nuclear magnetic resonance spectroscopy : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Physics at Massey University, New Zealand
    (Massey University, 1999) Lie, Wilford; Lie, Wilford
    An X Window-based software package for SGI workstations has been developed to process and assign NMR spectra. Special consideration has been given to the assignment of two-dimensional 1H NMR spectra of proteins. The program combines features from the packages PROSPA [Eccles 1995], EASY [Eccles 1991] and FELIX [Biosym 1995] as well as having its own capabilities. It allows simultaneous display of multiple toolboxes and spectra, which can be flexibly manipulated by mouse operations, command entries, and user-editable macros. NMR spectra can be processed either interactively or with macros containing commands with parameters. A unique filter that combines the exponential and sine-bell functions has been frequently used. A water suppression technique based on fitting averaged time-domain data, as well as an efficient algorithm for calculating fast Fourier transform and Hilbert transform [Eccles 1995] are discussed and implemented. NMR spectral assignment is done interactively in three steps: peak picking, spin-system identification, and sequence-specific assignment. The process utilises three peak lists: a raw-peak list that contains records of all possible peaks in a NOESY spectrum, a diagonal peak list that contains records of peaks that define a curve about which the spectrum is symmetric, and a cross-peak list that contains records of peaks that are assigned. Details of the peak-picking methods are discussed. By reference to a list of diagonal peaks, a common calibration problem caused by Bloch-Siegert shifts [Bloch and Siegert 1940, Ernst 1987] has been minimised. Automatically produced NOE summaries allow a quick identification of peaks that are unassigned or incorrectly assigned. The peak position and integration parameters can be calculated through non-linear curve fitting with Gaussians. NMR data processing and spectral assignment using the package has been completed for Caerin 4.1, a 23-residue protein. Linear-prediction has been applied to increase the spectral resolution. Detailed results for this protein are presented. The NOE summary of the sequential assignments indicates a well-defined secondary structure that is different from Caerin 1.1 [Wong 1996, 1997].
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    Structural studies on the nuclear lamins and other intermediate filament proteins : a thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in biophysics at Massey University
    (Massey University, 1989) Conway, James Frederick; Conway, James Frederick
    A number of aspects of IF chain and molecular structure, as well as molecular aggregation, have been examined. These include the delineation of periodicities in the sequences of structural domains of IF proteins, the distribution of amino acid residues within the heptad substructure, the flexibility of the peptide backbone, the extent of homology among the IF proteins, the packing of chains in the dimeric molecule, and the axial packing of molecules in the IF. Particular focus has been placed on the newly sequenced type V IF proteins (the nuclear lamins and the Helix pomotia B protein) and on a type III IF protein (peripherin). A parallel in-register arrangement of chains in the molecule is predicted for peripherin and the type V chains from a consideration of interchain ionic interactions. Also, periodicities in the linear distribution of charged residues in the rod domains of these proteins are shown to be comparable with periods in other IF chains. Ionic interactions between lamin molecules have been used to assess the likely modes of molecular aggregation in an in vitro assembly and a model is presented which also satisfies the constraints imposed by electron microscope data. In this model, antiparallel arrays of molecules are half-staggered and an extended conformation for the carboxy-terminal domains is predicted. Simple explanations are given for the transition between paracrystalline and lattice structures and for the disassembly of the lamin meshwork concommitant with hyperphosphorylation. The method of calculating intermolecular ionic interaction profiles is enhanced and a new, three-dimensional method is developed. The inhomogeneous distribution of residues in the heptad substructure can be correlated to the coiled-coil structure and chain packing in the molecule. In particular, the ~75% occupancy rate of apolar residues in the internal a and d heptad positions is shown to be a general feature of α-fibrous proteins. Variability of residues in the outer b,c and f positions indicates that structural or functional specificity in the rod domain may be determined by these parts of the sequence. The predicted flexibilities of IF chains have been compared to the underlying structure for the chains. Evidence from sequence homology studies suggests that several new subtypes are appropriate in the classification scheme. For the hard keratins the terms types Ia and IIa are proposed and for the soft keratins, types Ib and IIb; the need to separate the neurofilaments into the type IV class separate from the type III IF chains is confirmed; and division of the type V chains into cytoskeletal and karyoskeletal groups is indicated. A more detailed delineation is made of regions within the amino- and carboxy-terminal domains than has been possible previously. Periodic features of the homology profiles for the rod domain are examined and found to be similar to those in the linear distribution of residues in the amino acid sequences. Comparison between amphibian and mammalian keratins, and also between hard and soft keratins reveals that type II chains are maintained at a higher level of fidelity than type I chains. Consensus rod domain sequences are derived for the various IF subtypes: absolutely conserved regions of primary structure identify types or subtypes.