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Doctor of Philosophy / Grain Science and Industry / Scott R. Bean / Hulya Dogan / The role of sorghum protein cross-linking into high M[subscript w] polymeric groups in grain hardness was investigated using a number of protein analytical techniques to study the protein composition (reduced and non-reduced) of isolated vitreous and floury endosperm. The relative molecular weight distributions of polymeric proteins within two of three differentially extracted fractions were determined by size exclusion chromatography (SEC). The proteins in vitreous endosperm showed more protein cross-linking and a larger M[subscript w] distribution than found in the floury endosperm. An improved method for fractionating sorghum proteins designed to obtain intact disulfide linked protein polymers was developed. Three protein fractions obtained by application of the method represented proportionally different protein polymer contents as evidenced by comparative SEC and provides an improved tool for polymeric protein content comparison and measurement. The improved method was applied to a highly diverse non-tannin wild-type sorghum sample set spanning a range of in-vitro protein digestibility (IVPD) values to determine polymers involved with and influencing IVPD. Grain traits other than cross-linked proteins were also investigated for significant relationships to IVPD. Three protein fractions (F1, F2, F3) containing intact protein polymers were obtained for analysis by SEC and RP-HPLC. Proteins represented by four of five individual SEC peaks from F3 were significantly negatively correlated to IVPD, with three of the correlated peaks being polymeric. A 2-dimensional (2-D) technique involving peak collection after size exclusion chromatography followed by reverse phase high performance liquid chromatography (SEC x RP-HPLC) of the collected peaks was applied to protein polymers previously determined to be correlated with IVPD. RP-HPLC chromatogram patterns unique to each collected SEC peak from three selectively extracted protein fractions allowed qualitative and quantitative comparisons of protein polymer components. A pair of early eluting peaks appearing in the [gamma]-kafirin region of 2nd-dimension RP-HPLC chromatograms from a protein fraction with the largest M[subscript w] distribution were significantly correlated to IVPD. The correlated peak of interest was collected and characterized using SDS-PAGE and was preliminarily identified as 27kDa [gamma]-kafirin. By combining techniques using differing selectivity’s (solvent based, molecular size based, hydrophobicity based), it was possible to disassemble and compare components of protein polymers significantly correlated to IVPD.

<p>Mikrokapsule, kao nosači aktivnih supstanci, imaju sve veću primenu u različitim granama industrije, naročito prehrambene i farmaceutske. Inkorporiranje biolo&scaron;ki aktivnih supstanci unutar mikrokapsula omogućava maskiranje neprijatnih mirisa i ukusa, za&scaron;titu osetljivih i lako isparljivih komponenata.<br />Cilj ove doktorske disertacije je dobijanje mikrokapsula za istovremeno inkorporiranje hidrosolubilnih i liposolubilnih aktivnih materija, radi njihove za&scaron;tite i kontrolisanog otpu&scaron;tanja. Mikrokapsule su formirane iz duplih emulzija tipa voda-ulje-voda (V/U/V) metodom koacervacije, odnosno deponovanjem koacervata, koji nastaje u sistemu dva suprotno naelektrisana proteina, želatina i natrijum kazeinata (NaKN), na granicu faza ulje/voda. Kao model supstance za hidrosolubilne i liposolubilne biolo&scaron;ki aktivne materije, kori&scaron;ćeni su vitamini C i E.<br />Najpre su detaljno ispitane interakcije u sistemu želatin/NaKN primenom različitih metoda (merenje zeta potencijala, tenziometrija, viskozimetrija, reolo&scaron;ka ispitivanja). Na osnovu ovih rezultata definisane su promene, kako na granici faza, tako i unutar rastvora, kao i mehanizama formiranja koacervata između ova dva suprotno naelektrisana proteina. Utvrđeno je da se pri masenom odnosu želatin:NaKN od 2:1 dolazi do formiranja nerastvornog koacervata. Ispitan je uticaj interakcija u ovom sistemu na osobine duplih, V/U/V emulzija dobijenih emulgovanjem primarnih voda/ulje (V/U) emulzija u sme&scaron;i želatin/NaKN, pri njihovim odabranim masenim odnosima i zaključeno je da interakcija između proteina u kontinualnoj fazi utiče na<br />osobine emulzija.<br />S obzirom da je prvi korak ka dobijanju stabilne V/U/V emulzije, dobijanje stabilne primarne V/U emulzije, ispitana je mogućnost primene lipofilnih emulgatora, poliglicerol poliricinoleata (PGPR) i poliglicerol estra jestivih masnih kiselina i njihovih sme&scaron;a, za dobijanje 20% V/U emulzija. Ispitivanjem uticaja sastava sme&scaron;e emulgatora i njegove koncentracije na formiranje adsorpcionog sloja na graničnoj povr&scaron;ini ulje/voda i osobine formiranih V/U emulzija odabran je najpogodniji sistem za stabilizaciju primarnih emulzija.<br />Nakon formulisanja stabilnih duplih V/U/V emulzija sa inkorporiranim vitaminima C i E, optimizovani su uslovi za dobijanje mikrokapsula umrežavanjem kompleksa proteina na kapima ulja pomoću genipina, a njihovo izdvajanje iz rastvora ostvareno je primenom Spray drying postupka. Karakterizacijom dobijenih mikrokapsula (ispitivanjem morfologije povr&scaron;ine, efikasnosti inkapsulacije vitamina C i E, kinetike otpu&scaron;tanja vitamina C u in vitro uslovima) zaključeno je da na osobine mikrokapsula utiče koncentracija umreživača, kao i interakcija između želatina i NaKN u kontinualnoj fazi emulzija V/U/V.</p> / <p>Microcapsules, as active substance carriers, have increasing application in different industries, especially in food and pharmaceutical industry. Incorporation of the biologically active substances inside the microcapsules allows masking of unpleasant taste and smell, protection od sensitive and volatile components.<br />The aim of this thesis is preparation of microcapsules for parallel incorporation of water and oil soluble active substances for their protection and controlled release. Microcapsules were formed from double water-oil-water emulsions (W/O/W) by coacervation method, depositing the coacervate formed in the system of two oppositely charged proteins, gelatin and sodium caseinate (NaCN), at the water/oil interface. As a model for water and oil soluble biological active substances, vitamins C and E were used.<br />First of all, interactions in the gelatin/NaCN system were investigated in detail, by using different methods (measuring of zeta potential, tensiometry, vicometry, rheological investigations). Based on these results, changes at the interface and in the bulk of the system, as well as mechanisms of coacervate formation were defined. It has been determined that at gelatin:NaCN mass ratio of 2:1 non soluble coacervate were formed.<br />Influence of the interactions in this system on properties of the W/O/W double emulsions, made by emulsification of primary water/oil (W/O) emulsions in gelatin/NaCN mixtures, at desired mass ratios of proteins, was investigated. It was concluded that interactions between proteins in continuous phase of emulsions have influence on their properties.<br />As the first step in formation of stable W/O/W emulsions is<br />obtaining stable primary W/O emulsion, possibility of using lipophilic emulsifiers, polyglycerol polyricinoleate (PGPR) and polyglycerol esters of edible fatty acids and their mixtures, for 20% W/O emulsions formation were investigated. Results of these investigations showed that composition of emulsifiers mixtures and their concentrations have an influence on adsorption layer, at the water/oil interface, formation, as well as on stability of W/O emulsion, and based on these results the most suitable system of emulsifiers were chosen.<br />After formulation of stable double W/O/W emulsions with incorporated vitamins C and E, conditions for microcapsules formation, by crosslinking of proteins complex at oil droplets with genipin, were optimized, and for their separation from dispersion spray drying method was applied. Characterization of obtained microcapsules (investigation of the surface morphology, efficiency of the vitamins C and E encapsulation, release kinetics of vitamin C under in vitro conditions) showed that concentration of crosslinking agent, as well as interaction between gelatin and NaCN, have an influence on microcapsules properties.</p>

The microstructure and mechanics of collagen and elastin protein fiber networks dictate the mechanical responses of all soft tissues and related organ systems. In this project, we endeavored to meet or exceed native tissue mechanical properties through mimicry of these extracellular matrix components with synthetic collagen fiber and elastin analogues. Significantly, these studies led to the development of a framework for the design and fabrication of protein-based soft tissue substitutes that reproduced many aspects of native biomechanics. A scalable process was developed for production of synthetic collagen microfibers at a rate of 60 m/hr. Fiber properties and ultrastructure were characterized by uniaxial mechanical testing, differential scanning calorimetry, transmission electron microscopy, and second harmonic generation analysis. In vivo responses to synthetic fibers were evaluated in a murine model. A scalable, semi-automated process was designed for the fabrication of multilamellar membranes comprised of sheets of an elastin analogue reinforced with synthetic collagen fibers. Fibers could be organized in a precisely defined three-dimensional hierarchical pattern. The structure of these fiber composites was analyzed by scanning and transmission electron microscopy, and digital volumetric imaging. The effects of fiber orientation and volume fraction on uniaxial mechanical responses were evaluated. Increased fiber volume fraction and alignment increased Young's modulus, resilience, and yield stress. Highly extensible, elastic tissues display a functionally significant transition from low to high modulus deformation at a transition point strain dictated by the crimped collagen microstructure. This response was replicated by the fabrication of dense arrays of microcrimped synthetic collagen fibers embedded in an elastin analogue. The degree of microcrimping could be varied, and generated a transition point mechanical response. Cyclic tensile deformation did not substantially alter microcrimp morphology. A series of small-diameter vascular grafts consisting of an elastin-like protein reinforced with controlled volume fractions and orientations of synthetic collagen fiber was designed and prototyped. The optimal design satisfied target properties with suture retention strength of 173 ± 4 g-f, burst strength of 1483 ± 143 mm Hg, and compliance of 5.1 ± 0.8 %/100 mm Hg.

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Mass spectrometry has enjoyed enormous popularity over the years for studying biological systems. The theme of this dissertation was to develop and use mass spectrometry based tools to solve five biologically oriented problems associated with protein architecture and extend the utility of these tools to study protein polymer conjugation. The first problem involved elucidating the false negatives of how proteins with few basic residues, forms highly charged ions in electrospray ionization mass spectrometry (ESI MS). This study showed that the unfolding of polypeptide chains in solution leads to the emergence of highly charged protein ions in ESI MS mass spectra, even if the polypeptide chains lack a sufficient number of basic sites. In the second problem, a new technique was developed that can monitor small-scale conformational transitions that triggers protein activity and inactivity using porcine pepsin as a model protein. This work allowed us to revise a commonly accepted scenario of pepsin inactivation and denaturation. The physiological relevance of an enzyme-substrate complex was probed in our third problem. We observed by ESI MS that pepsin forms a facile complex with a substrate protein, N-lobe transferrin under mildly acidic pH. The observed complex could either be a true enzyme-substrate complex or may likely results from an electrostatically driven association. Our investigation suggested that the enzyme binds nonspecifically to substrate proteins under mild acidic pH conditions. The fourth problem dealt with the investigation of conformational heterogeneity of natively unstructured proteins using a combination of spectroscopic techniques and ESI MS as tools. It was observed that four different conformations of alpha-synuclein coexist in equilibrium. One of these conformations appeared to be tightly folded. Conclusions regarding the nature of these states were made by correlating the abundance evolution of the conformers as a function of pH with earlier spectroscopic measurements. The final problem was aimed at monitoring conformational transitions in polypeptide and polymer segments of PEGylated proteins using PEGylated ubiquitin as a model system. This studies suggested that for a PEGylated protein, polypeptides maintain their folded conformation to a greater extent whiles the polymer segments are bound freely to the protein.

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While conducting pilot studies into the usefulness of fusion to TELSAM polymers as a potential protein crystallization strategy, we observed novel properties in crystals of two TELSAM–target protein fusions, as follows. (i) A TELSAM–target protein fusion can crystallize more rapidly and with greater propensity than the same target protein alone. (ii) TELSAM–target protein fusions can be crystallized at low protein concentrations. This unprecedented observation suggests a route to crystallize proteins that can only be produced in microgram amounts. (iii) The TELSAM polymers themselves need not directly contact one another in the crystal lattice in order to form well-diffracting crystals. This novel observation is important because it suggests that TELSAM may be able to crystallize target proteins too large to allow direct inter-polymer contacts. (iv) Flexible TELSAM–target protein linkers can allow target proteins to find productive binding modes against the TELSAM polymer. (v) TELSAM polymers can adjust their helical rise to allow fused target proteins to make productive crystal contacts. (vi). Fusion to TELSAM polymers can stabilize weak inter-target protein crystal contacts. We report features of these TELSAM–target protein crystal structures and outline future work needed to validate TELSAM as a crystallization chaperone and determine best practices for its use.