Matching Items (8)
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- All Subjects: Protein
- Creators: Johnston, Carol
- Creators: Ros, Robert
- Member of: Theses and Dissertations
Description
An animal's ability to produce protein-based silk materials has evolved independently in many different arthropod lineages, satisfying various ecological necessities. However, regardless of their wide range of uses and their potential industrial and biomedical applications, advanced knowledge on the molecular structure of silk biopolymers is largely limited to those produced by spiders (order Araneae) and silkworms (order Lepidoptera). This thesis provides an in-depth molecular-level characterization of silk fibers produced by two vastly different insects: the caddisfly larvae (order Trichoptera) and the webspinner (order Embioptera).
The molecular structure of caddisfly larval silk from the species Hesperophylax consimilis was characterized using solid-state nuclear magnetic resonance (ss-NMR) and Wide Angle X-ray Diffraction (WAXD) techniques. This insect, which typically dwells in freshwater riverbeds and streams, uses silk fibers as a strong and sticky nanoadhesive material to construct cocoons and cases out available debris. Conformation-sensitive 13C chemical shifts and 31P chemical shift anisotropy (CSA) information strongly support a unique protein motif in which phosphorylated serine- rich repeats (pSX)4 complex with di- and trivalent cations to form rigid nanocrystalline β-sheets. Additionally, it is illustrated through 31P NMR and WAXD data that these nanocrystalline structures can be reversibly formed, and depend entirely on the presence of the stabilizing cations.
Nanofiber silks produced by webspinners (order Embioptera) were also studied herein. This work addresses discrepancies in the literature regarding fiber diameters and tensile properties, revealing that the nanofibers are about 100 nm in diameter, and are stronger (around 500 MPa mean ultimate stress) than previous works suggested. Fourier-transform Infrared Spectroscopy (FT-IR), NMR and WAXD results find that approximately 70% of the highly repetitive glycine- and serine-rich protein core is composed of β-sheet nanocrystalline structures. In addition, FT-IR and Gas-chromatography mass spectroscopy (GC-MS) data revealed a hydrophobic surface coating rich in long-chain lipids. The effect of this surface coating was studied with contact angle techniques; it is shown that the silk sheets are extremely hydrophobic, yet due to the microstructural and nanostructural details of the silk surface, are surprisingly adhesive to water.
The molecular structure of caddisfly larval silk from the species Hesperophylax consimilis was characterized using solid-state nuclear magnetic resonance (ss-NMR) and Wide Angle X-ray Diffraction (WAXD) techniques. This insect, which typically dwells in freshwater riverbeds and streams, uses silk fibers as a strong and sticky nanoadhesive material to construct cocoons and cases out available debris. Conformation-sensitive 13C chemical shifts and 31P chemical shift anisotropy (CSA) information strongly support a unique protein motif in which phosphorylated serine- rich repeats (pSX)4 complex with di- and trivalent cations to form rigid nanocrystalline β-sheets. Additionally, it is illustrated through 31P NMR and WAXD data that these nanocrystalline structures can be reversibly formed, and depend entirely on the presence of the stabilizing cations.
Nanofiber silks produced by webspinners (order Embioptera) were also studied herein. This work addresses discrepancies in the literature regarding fiber diameters and tensile properties, revealing that the nanofibers are about 100 nm in diameter, and are stronger (around 500 MPa mean ultimate stress) than previous works suggested. Fourier-transform Infrared Spectroscopy (FT-IR), NMR and WAXD results find that approximately 70% of the highly repetitive glycine- and serine-rich protein core is composed of β-sheet nanocrystalline structures. In addition, FT-IR and Gas-chromatography mass spectroscopy (GC-MS) data revealed a hydrophobic surface coating rich in long-chain lipids. The effect of this surface coating was studied with contact angle techniques; it is shown that the silk sheets are extremely hydrophobic, yet due to the microstructural and nanostructural details of the silk surface, are surprisingly adhesive to water.
ContributorsAddison, John Bennett (Author) / Yarger, Jeffery L (Thesis advisor) / Holland, Gregory P (Thesis advisor) / Wang, Xu (Committee member) / Ros, Robert (Committee member) / Arizona State University (Publisher)
Created2014
Description
Proteins are a fundamental unit in biology. Although proteins have been extensively studied, there is still much to investigate. The mechanism by which proteins fold into their native state, how evolution shapes structural dynamics, and the dynamic mechanisms of many diseases are not well understood. In this thesis, protein folding is explored using a multi-scale modeling method including (i) geometric constraint based simulations that efficiently search for native like topologies and (ii) reservoir replica exchange molecular dynamics, which identify the low free energy structures and refines these structures toward the native conformation. A test set of eight proteins and three ancestral steroid receptor proteins are folded to 2.7Å all-atom RMSD from their experimental crystal structures. Protein evolution and disease associated mutations (DAMs) are most commonly studied by in silico multiple sequence alignment methods. Here, however, the structural dynamics are incorporated to give insight into the evolution of three ancestral proteins and the mechanism of several diseases in human ferritin protein. The differences in conformational dynamics of these evolutionary related, functionally diverged ancestral steroid receptor proteins are investigated by obtaining the most collective motion through essential dynamics. Strikingly, this analysis shows that evolutionary diverged proteins of the same family do not share the same dynamic subspace. Rather, those sharing the same function are simultaneously clustered together and distant from those functionally diverged homologs. This dynamics analysis also identifies 77% of mutations (functional and permissive) necessary to evolve new function. In silico methods for prediction of DAMs rely on differences in evolution rate due to purifying selection and therefore the accuracy of DAM prediction decreases at fast and slow evolvable sites. Here, we investigate structural dynamics through computing the contribution of each residue to the biologically relevant fluctuations and from this define a metric: the dynamic stability index (DSI). Using DSI we study the mechanism for three diseases observed in the human ferritin protein. The T30I and R40G DAMs show a loss of dynamic stability at the C-terminus helix and nearby regulatory loop, agreeing with experimental results implicating the same regulatory loop as a cause in cataracts syndrome.
ContributorsGlembo, Tyler J (Author) / Ozkan, Sefika B (Thesis advisor) / Thorpe, Michael F (Committee member) / Ros, Robert (Committee member) / Kumar, Sudhir (Committee member) / Shumway, John (Committee member) / Arizona State University (Publisher)
Created2011
Description
Vegetarian diets can provide an abundance of nutrients when planned with care. However, research suggests that vegetarian diets may have lower protein quality than omnivore diets. Current protein recommendations assume that vegetarians obtain a majority of their protein from animal products, like dairy and eggs. Studies have shown that this assumption may not be valid. The recommended dietary allowance (RDA) may not be adequate in vegetarian populations with high protein requirements. The purpose of this study is to analyze dietary protein quality using the DIAAS (Digestible Indispensable Amino Acid Score) method in both vegetarian and omnivore endurance athletes. 38 omnivores and 22 vegetarians submitted 7-day food records which were assessed using nutrition analysis software (Food Processor, ESHA Research, Salem, OR, USA). Dietary intake data was used to calculate DIAAS and determine the amount of available dietary protein in subject diets. Dietary data was compared with the subjects’ lean body mass (obtained using DEXA scan technology), and strength (quantified using peak torque of leg extension and flexion using an isokinetic dynamometer). Statistical analyses revealed significantly higher available protein intake in the omnivore athletes (p<.001). There were significant correlations between available protein intake and strength (p=.016) and available protein intake and lean body mass (p<.001). Omnivore subjects had higher lean body mass than vegetarian subjects (p=.011). These results suggest that vegetarian athletes may benefit from higher overall protein intakes to make up for lower dietary protein quality.
ContributorsZuelke, Corinne (Author) / Johnston, Carol (Thesis advisor) / Wharton, Christopher (Christopher Mack), 1977- (Committee member) / Dixon, Kathleen (Committee member) / Arizona State University (Publisher)
Created2017
Description
Proteins continually and naturally incur evolutionary selection through mutagenesis that optimizes their fitness, which is primarily determined by their function. It is known that allosteric regulation alters a protein's conformational dynamics leading to functional changes. We have computationally introduced a mutation at a predicted regulatory site of a short, 46 residue-long, protein interaction module composed of a WW domain and corresponding polyproline ligand (PDB id: 1k9r). The dynamic flexibility index (DFI) was computed for the binding site of the wild type and mutant WW domains to quantify the mutations effect on the rigidity of the binding pocket. DFI is used as a metric to quantify the resilience of a given position to perturbation along the chain. Using steered molecular dynamics (SMD), we also measure the effect of the point mutation on allosteric regulation by approximating the binding free energy of the system calculated using Jarzynski's Equality. Calculation of the DFI shows that the overall flexibility of the protein complex increases as a result of the distal point mutation. Total change in DFI percentile of the binding site showed a 0.067 increase suggesting an allosteric, loss of function mutation. Furthermore, we see that the change in the binding free energy is greater for that of the mutated complex supporting the idea that an increase in flexibility is correlated to a decrease in proteinlig and binding affinity. We show that sequence mutation of an allosteric site affects the mechanical stability and functionality of the binding pocket.
ContributorsMarianchuk, Tegan (Author) / Ozkan, Sefika (Thesis director) / Ros, Robert (Committee member) / Barrett, The Honors College (Contributor) / Department of Physics (Contributor)
Created2018-05
Description
Objective: This research examined the impact of daily ingestions of commercial high protein nutrition bars (with or without added fiber) on 24-h energy intake and satiety
for one week among free-living young healthy adults.
Design: In a 4-week double-blind, randomized crossover trial, 21 normal and
overweight participants (Mean BMI 23.9 ± 2.7 kg/m²), free of chronic diseases, were
randomized assigned to HP (high protein: 21 g protein) or HPHF (high protein high fiber:
20g, 14 g fiber) nutrition bars. Participants were included in the trial if they meet the
criteria for non-smoking, and not taking prescribed medication for chronic diseases.
Participants were instructed to consume commercial nutrition bars daily for seven
consecutive days. Body composition was measured with a bioelectrical impedance scale
at weeks 1, 3, and 5. Dietary data was recorded by the MyFitnessPal app on Wednesday,
Friday, and Sunday of each week.
Results: The mean energy intake for the weeks HPHF bars were consumed is
significantly higher compared to baseline (1998 ± 534 vs. 1806 ± 537 respectively; p =
0.035). The mean fat mass following one week of HPHF bar consumption was
significantly higher than the baseline value (18.8 ± 6.8 vs. 18.3 ± 6.7 respectively; p =
0.023) and trended higher (18.8 ± 6.8 vs. 18.3 ± 6.7 respectively; p = 0.057) in
comparison to the value following one week of HP bar consumption. For the high
physical activity level group (n = 10), the mean energy intakes for the baseline week and
the weeks the HP and HPHF bars were consumed were 1883 ± 597 kcal, 2154 ± 712 kcal,
and 2099 ± 603 kcal respectively (p ˂ 0.04; energy intakes for both bars were
significantly different from baseline). Nutrient intakes differed significantly mirroring the
nutrient profile for each specific bar. There are significant effects after both bars on
satiety, but there were no differences between each bar.
Conclusions: Sales of nutrition bars gained rapid growth and may represent a
unique source for specific nutrients. However, ingestion of commercial high protein
nutrition bars may increase the risk of gaining fat mass and eventual body mass over
time.
ContributorsPang, Minghan (Author) / Johnston, Carol (Thesis advisor) / Shepard, Christina (Committee member) / Alexon, Christy (Committee member) / Arizona State University (Publisher)
Created2022
Description
Plant-based eaters are known to reap nutritional benefits due to their dietchoice, but it is important to evaluate dietary differences that may put them at a disadvantage compared to omnivores. Stark differences exist in daily intakes of protein between vegans and omnivores, which may lead to several risks including decreased strength and bone density. The purpose of this study was to analyze the differences in protein intake, lean mass, strength, and bone density in vegans versus omnivores in order to support the argument for an increased recommended daily allowance (RDA) for protein for plant-based eaters. Participants in this study were assigned to groups based on omnivorous (n = 25) or vegan (n = 19) dietary pattern. Nineteen matched pairs were created based on age and BMI. Data was collected at a single lab visit and included health history and physical activity readiness questionnaires, 24-hr food recall, and anthropometric measures. Bone mineral density (BMD) was measured using DEXA and strength was assessed using hand and Biodex dynamometers. Statistical analyses were conducted using independent samples t-tests and Pearson’s correlation tests to evaluate differences in body composition, bone density, strength, and dietary intake between the two groups with significance set at p.05. Differences were seen in daily calorie (p=.007), protein (p<.001), fat (p<.001), and fiber (p=.009) intake. Lean mass (p=.282) and bone density (p=.651) were not different between groups, but lower body strength was different (p=.008). There was a correlation between lower body strength and protein intake (p<.001), and lean mass was correlated with lower body strength (p<.001), grip strength (p<.001), and bone density (p<.001), but not
i
LBM (p=0.158). Correlations were also observed between BMD and lower body strength (p=.004). These data suggest that there is a significant difference between protein intake in vegans versus omnivores, which appears to have a positive association with strength. BMD also has a positive association with strength as well as lean mass. Cumulatively, the results suggest that it may be beneficial for vegans to increase daily protein intake.
ContributorsNadalet, Camila R (Author) / Johnston, Carol (Thesis advisor) / Shepard, Christina (Committee member) / Hooshmand, Shirin (Committee member) / Arizona State University (Publisher)
Created2023
Description
My research centers on the design and fabrication of biomolecule-sensing devices that combine top-down and bottom-up fabrication processes and leverage the unique advantages of each approach. This allows for the scalable creation of devices with critical dimensions and surface properties that are tailored to target molecules at the nanoscale.
My first project focuses on a new strategy for preparing solid-state nanopore sensors for DNA sequencing. Challenges for existing nanopore approaches include specificity of detection, controllability of translocation, and scalability of fabrication. In a new solid-state pore architecture, top-down fabrication of an initial electrode gap embedded in a sealed nanochannel is followed by feedback-controlled electrochemical deposition of metal to shrink the gap and define the nanopore size. The resulting structure allows for the use of an electric field to control the motion of DNA through the pore and the direct detection of a tunnel current through a DNA molecule.
My second project focuses on top-down fabrication strategies for a fixed nanogap device to explore the electronic conductance of proteins. Here, a metal-insulator-metal junction can be fabricated with top-down fabrication techniques, and the subsequent electrode surfaces can be chemically modified with molecules that bind strongly to a target protein. When proteins bind to molecules on either side of the dielectric gap, a molecular junction is formed with observed conductances on the order of nanosiemens. These devices can be used in applications such as DNA sequencing or to gain insight into fundamental questions such as the mechanism of electron transport in proteins.
My first project focuses on a new strategy for preparing solid-state nanopore sensors for DNA sequencing. Challenges for existing nanopore approaches include specificity of detection, controllability of translocation, and scalability of fabrication. In a new solid-state pore architecture, top-down fabrication of an initial electrode gap embedded in a sealed nanochannel is followed by feedback-controlled electrochemical deposition of metal to shrink the gap and define the nanopore size. The resulting structure allows for the use of an electric field to control the motion of DNA through the pore and the direct detection of a tunnel current through a DNA molecule.
My second project focuses on top-down fabrication strategies for a fixed nanogap device to explore the electronic conductance of proteins. Here, a metal-insulator-metal junction can be fabricated with top-down fabrication techniques, and the subsequent electrode surfaces can be chemically modified with molecules that bind strongly to a target protein. When proteins bind to molecules on either side of the dielectric gap, a molecular junction is formed with observed conductances on the order of nanosiemens. These devices can be used in applications such as DNA sequencing or to gain insight into fundamental questions such as the mechanism of electron transport in proteins.
ContributorsSadar, Joshua Stephen (Author) / Qing, Quan (Thesis advisor) / Lindsay, Stuart (Committee member) / Vaiana, Sara (Committee member) / Ros, Robert (Committee member) / Arizona State University (Publisher)
Created2019
Description
Background: Vegan and vegetarian diets have gained in popularity in recent years. Stated reasons for this include some possible health benefits and concerns of animal welfare. Though considered to be nutritionally adequate, questions remain over whether current protein recommendations of 0.8 g/kg/d are sufficient to maintain body processes and growth. Protein is unique in that it is the only macronutrient that contains nitrogen. Its status can be determined through nitrogen balance analysis of the urine if protein content of the diet is known. Nitrogen balance is considered the gold standard for determining protein intake requirements. A negative balance indicates a catabolic state, whereas a positive nitrogen balance is seen during anabolism. In healthy people, nitrogen equilibrium is desired under normal circumstances. This equilibrium reflects the net synthesis and breakdown of proteins. While nitrogen balance techniques have been used for decades, currently, there are no known studies measuring nitrogen balance and protein intake in strict vegans. Methods: Twenty vegan, inactive, male participants were recruited and received a 5-day eucaloric diet with a known protein content held constant at 0.8 g/kg/d. On day five, 24-hour urine was collected by participants and aliquoted for future analysis. Nitrogen content of the urine was determined through photometric assay and compared to the known nitrogen content of the diet to calculate nitrogen balance status.
Results: Mean absolute nitrogen balance (-1.38 ± 1.22 g/d, effect size = -1.13) was significantly lower than zero (equilibrium) (p < .001). Mean relative nitrogen balance (-18.60 ± 16.96 mg/kg/d, effect size = -1.10) was significantly lower than zero (p < .001). There were no correlations seen between nitrogen balance and age, years as vegan, or fat- free mass.
Conclusion: Consuming 0.8 g/kg/d of protein is insufficient to produce nitrogen balance in long-term vegans.
ContributorsBartholomae, Eric (Author) / Johnston, Carol (Thesis advisor) / Sweazea, Karen (Committee member) / Wharton, Christopher (Committee member) / Lee, Chong (Committee member) / Kressler, Jochen (Committee member) / Arizona State University (Publisher)
Created2022